Patent application title: ANTIBIOTIC COMPOSITIONS AND RELATED SCREENING METHODS
Inventors:
Chi-Huey Wong (La Jolla, CA, US)
Ting-Jen Cheng (Taipei, TW)
Che Alex Ma (Taipei, TW)
Wei-Chieh Cheng (Taipei, TW)
Assignees:
Academia Sinica
IPC8 Class: AA61K31702FI
USPC Class:
514 54
Class name: Designated organic active ingredient containing (doai) carbohydrate (i.e., saccharide radical containing) doai polysaccharide
Publication date: 2015-12-03
Patent application number: 20150342973
Abstract:
Moenomycin inhibits bacterial growth by clocking the transglycosylase
activity of class A penicillin-binding proteins (PBPs), which are key
enzymes in bacterial cell wall synthesis. The binding affinities of
moenomycin A with various truncated PBPs were compared showing that the
transmembrane domain is important for moenomycin binding. Full-length
class-A PBPs from 16 bacterial species were produced, and their binding
activities showed a correlation with the antimicrobial activity of
moenomycin against Enterococcus faecalis and Staphylococcus aureus.
Moreover, a fluorescence anisotropy-based high-throughput assay was
developed and used successfully for identification of transglycosylase
inhibitors.Claims:
1. A composition comprising: an antibiotic agent for inhibiting
transglycosylase activity of bacterial class A penicillin-binding
proteins (PBPs); and pharmaceutically acceptable carrier, wherein the PBP
comprises at least a transmembrane (TM) domain and a transglycosylase
(TG) domain, and wherein the agent exhibits a higher first binding
affinity to a transmembrane (TM) domain-containing class A
penicillin-binding protein (PBP), or fragment thereof, as compared to a
second binding affinity to the PBP fragment that lacks the TM domain.
2. The composition of claim 1, wherein the binding affinity between the antibiotic agent and the class A penicillin-binding protein is determined by anisotropy measurement assay.
3. The composition of claim 2, wherein the anisotropy measurement assay PBP binding is at least one of a competition displacement assay or a direct binding assay.
4. The composition of claim 3, wherein the competition displacement assay competes a labeled moenomycin with the antibiotic agent.
5. The composition of claim 2, wherein the anisotropy measurement of PBP binding, comprises a fluorescence anisotropy (FA) assay.
6. The composition of claim 1, wherein the class A penicillin-binding protein (PBP) comprises three domains which, from N terminus to C terminus, are transmembrane, transglycosylase, and transpeptidase.
7. The composition of claim 1, wherein the antibiotic agent is selected from (Z)-5-(4-bromophenyl)-3-((5-nitrofuran-2-yl)methylene)furan-2(3H)-on- e, (Z)-1,3-diphenyl-4-(2-(thiazol-2-yl)hydrazono-1H-pyrazol-5(4H)-one, and (E)-4-(2-(5,5,8,8,-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)prop-1-e- nyl)benzoic acid.
8. The composition of claim 1, wherein the antibiotic agent inhibits bacterium growth, wherein the bacterium is Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Shigella flexneri, Haemophilus influenzae, Helicobacter pylori, Citrobacter freundii, Bordetella pertussi, Staphylococcus aureus (MRSA Mu50), Bacillus subtilis, Pseudomonas aeruginosa, Clostridium difficile, Enterococcus faecium, Enterococcus faecalis, Salmonella enterica or Neisseria gonorrhoeae.
9. The composition of claim 8, wherein the bacterium is E. faecalis or S. aureus.
10. The composition of claim 1, wherein the antibacterial agent is a small molecule.
11. The composition of claim 1, wherein the antibacterial agent has a formula selected from the group consisting of: ##STR00008## ##STR00009##
12. An antibiotic agent for inhibiting transglycosylase activity of bacterial class A penicillin-binding proteins (PBPs), wherein the PBP comprises at least a transmembrane (TM) domain and a transglycosylase (TG) domain, wherein the agent exhibits a higher first binding affinity to a transmembrane (TM) domain-containing class A penicillin-binding protein (PBP), or fragment thereof, as compared to a second binding affinity to the PBP fragment that lacks the TM domain and wherein the antibiotic agent has a formula selected from the group consisting of: ##STR00010## ##STR00011##
13. A method for inhibiting bacterial growth in a subject, the method comprising treating a subject having a bacterial infection or susceptible to a bacterial infection with a pharmaceutical composition comprising: an antibiotic agent for inhibiting transglycosylase activity of bacterial class A penicillin-binding proteins (PBPs); and pharmaceutically acceptable carrier, wherein the PBP comprises at least a transmembrane (TM) domain and a transglycosylase (TG) domain, and wherein the agent exhibits a higher first binding affinity to a transmembrane (TM) domain-containing class A penicillin-binding protein (PBP), or fragment thereof, as compared to a second binding affinity to the PBP fragment that lacks the TM domain.
14. The method of claim 13, wherein the class A penicillin-binding protein (PBP) comprises three domains which, from N terminus to C terminus, are transmembrane, transglycosylase, and transpeptidase.
15. The method of claim 13, wherein the antibiotic agent is selected from (Z)-5-(4-bromophenyl)-3-((5-nitrofuran-2-yl)methylene)furan-2(3H)-one, (Z)-1,3-diphenyl-4-(2-(thiazol-2-yl)hydrazono-1H-pyrazol-5(4H)-one, and (E)-4-(2-(5,5,8,8,-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)prop-1-e- nyl)benzoic acid.
16. The method of claim 13, wherein the antibiotic agent inhibits bacterium growth, wherein the bacterium is Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Shigella flexneri, Haemophilus influenzae, Helicobacter pylori, Citrobacter freundii, Bordetella pertussi, Staphylococcus aureus (MRSA Mu50), Bacillus subtilis, Pseudomonas aeruginosa, Clostridium difficile, Enterococcus faecium, Enterococcus faecalis, Salmonella enterica or Neisseria gonorrhoeae.
17. The method of claim 13, wherein the antibiotic agent inhibits bacterial growth, wherein the bacteria are gram-positive bacteria.
18. The method of claim 16, wherein the bacterium is E. faecalis or S. aureus.
19. The method of claim 13, wherein the antibiotic agent has a formula selected from the group consisting of: ##STR00012## ##STR00013##
Description:
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application Ser. No. 12/354,717 filed Jan. 15, 2009 and issued as U.S. Pat. No. 8,916,540 which claims the benefit of and priority to U.S. Provisional Application Ser. No. 61/021,279, filed Jan. 15, 2008, Taiwan Application Serial No. 097125469, filed Jul. 4, 2008, and Japan Application Serial No. 2008-178187, filed Jul. 7, 2008, and the contents of each are incorporated by reference herein in their entirety.
BACKGROUND
[0002] This disclosure relates to methods for expressing and purifying full-length class A penicillin-binding protein from bacteria. This disclosure also relates to high-throughput screening methods for an antibiotic.
SUMMARY
[0003] According to a feature of the present disclosure, a method is disclosed comprising obtaining a candidate for screening; carrying out an anisotropy measurement assay with a class A penicillin-binding protein comprising at least a transmembrane and a transglycosylase domains; and determining the effectiveness of the candidate as a transglycosylase inhibitor.
[0004] According to a feature of the present disclosure, a high throughput device is contemplated, having multiple versions of a class A penicillin-binding protein comprising at least transmembrane and a transglycosylase domain for determining the effectiveness of a candidate agent as a transglycosylation inhibitor. The assays may be carried out as either competition displacement assays or direct binding assays or both.
[0005] According to a feature of the present disclosure, a method is disclosed comprising amplifying DNA sequence of full-length penicillin-binding protein from bacterial genomic DNA; cloning the DNA sequence into a vector, expressing the DNA sequence in a host cell to obtain a full-length penicillin-binding protein, solubilizing the protein with a detergent, and purifying the protein.
[0006] According to a feature of the present disclosure, a composition is disclosed comprising an agent having the formula selected from the group consisting of:
##STR00001## ##STR00002##
and a pharmaceutically acceptable carrier.
[0007] According to a feature of the present disclosure, a method is disclosed comprising treating a subject having a bacterial infection or susceptible for a bacterial infection with a pharmaceutical composition comprising an agent having the formula selected from the group consisting of:
##STR00003## ##STR00004##
and a pharmaceutically acceptable carrier.
[0008] According to implementations of the present disclosure, the antibiotic is a transglycosylase inhibitor, and, preferably, it is (Z)-5-(4-bromophenyl)-3-((5-nitrofuran-2-yl)methylene)furan-2(3H)-one; (Z)-1,3-diphenyl-4-(2-(thiazol-2-yl)hydrazono-1H-pyrazol-5(4H)-one; (E)-4-(2-(5,5,8,8,-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)prop-1-e- nyl)benzoic acid.
[0009] According to implementations of the present disclosure, the protein comprises three domains which, from N terminus to C terminus, are transmembrane, transglycosylase and transpeptidase.
[0010] According to implementations of the present disclosure, the moenomycin used as fluorescent probe binds the penicillin-binding protein when fluorescence-anisotropy-based assay is carried out.
[0011] According to implementations of the present disclosure, the hits screened by the method of present disclosure, which are antibiotics, inhibit the growth bacterium which is Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Shigella flexneri, Haemophilus influenzae, Helicobacter pylori, Citrobacter freundii, Bordetella pertussi, Staphylococcus aureus (MRSA Mu50), Bacillus subtilis, Pseudomonas aeruginosa, Clostridium difficile, Enterococcus faecium, Enterococcus faecalis, Salmonella enterica or Neisseria gonorrhoeae.
[0012] According to implementations of the present disclosure, the bacterium is E. faecalis or S. aureus.
[0013] According to implementations of the present disclosure, a method is provided for expressing and purifying full-length class A penicillin-binding protein from a bacterium comprising: (a) amplifying DNA sequence of full-length penicillin-binding protein from bacterial genomic DNA; (b) cloning the DNA sequence into a vector; (c) expressing the DNA sequence in a host cell to obtain a full-length penicillin-binding protein; (d) solubilizing the protein with a detergent; and (e) purifying the protein; wherein the detergent is n-decyl-β-D-maltopyranoside, n-undecyl-β-D-maltopyranoside, n-dodecyl-β-D-maltopyranoside, n-octyl-β-D-glucopyranoside, n-nonyl-β-D-glucopyranoside, n-tetradecylphosphocholine, n-dodedyl-N,N-dimethylamine-N-oxide, 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane sulfonate or α-[4-(1,1,3,3-Tetramethylbutyl)phenyl]-w-hydroxy-poly(oxy-1,2-ethan- ediyl).
[0014] According to implementations of the present disclosure, the bacterium is Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Shigella flexneri, Haemophilus influenzae, Helicobacter pylori, Citrobacter freundii, Bordetella pertussi, Staphylococcus aureus (MRSA Mu50), Bacillus subtilis, Pseudomonas aeruginosa, Clostridium difficile, Enterococcus faecium, Enterococcus faecalis, Salmonella enterica or Neisseria gonorrhoeae.
[0015] According to implementations of the present disclosure, the detergent used for purifying full length class A penicillin-binding protein is n-dodecyl-β-D-maltopyranoside.
[0016] According to implementations of the present disclosure, the full length protein comprises three domains, which, from N terminus to C terminus, are transmembrane, transglycosylase and transpeptidase.
DRAWINGS
[0017] The above-mentioned features and objects of the present disclosure will become more apparent with reference to the following description taken in conjunction with the accompanying drawings wherein like reference numerals denote like elements and in which:
[0018] FIGS. 1A-1B show inhibition of transglycosylase by moenomycin and binding affinities of truncated PBP variants, according to implementations of the present disclosure. FIG. 1A shows Moenomycin A (1) inhibiting the transglycosylation step in the bacterial cell wall synthesis. FIG. 1B is a schematic representation of the PBP variants used for moenomycin binding studies.
[0019] FIG. 2 shows sequence alignment of full-length bi-functional PBPs from 16 bacterial strains (Seq. ID Nos. 1-16), according to implementations of the present disclosure.
[0020] FIG. 3 shows the SDS-PAGE analysis of purified protein of class A PBPs from sixteen bacterial species, according to implementations of the present disclosure.
[0021] FIGS. 4A-4D show the design of a novel fluorescence anisotropy assay for transglycosylase, according to implementations of the present disclosure. FIG. 4A shows the chemical structures of the modified moenomycin A (2), and FIG. 4B shows the chemical structures of the fluorescein-labeled moenomycin, F-Moe (3). FIG. 4C shows the SPR analysis of the binding activity of moenomycin A to E. coli PBP1b, and FIG. 4D shows the SPR analysis of the binding activity of F-Moe to E. coli PBP1b.
[0022] FIGS. 5A-5C show the development of high-throughput FA assay for transglycosylase. FIG. 5A shows the concentration-dependent changes in fluorescence anisotropy observed when E. coli PBP1b bound to F-Moe. FIG. 5B shows the improved FA assay with the Helicobacter pylori PBP1a. FIG. 5C shows the displacement of the PBP1a bound F-Moe complex by unlabeled moenomycin at various concentrations.
[0023] FIG. 6 illustrates chemical structures of moenomycin derivatives (4 and 5), according to implementations of the present disclosure; and
[0024] FIGS. 7A-7B illustrate screening for small molecules as TG inhibitors using the class A PBPs, according to implementations of the present disclosure. FIG. 7A shows the scheme of HTS for TG inhibitors using FA assay. FIG. 7B shows chemical structures of the HTS hits (compounds 6-8).
DETAILED DESCRIPTION
[0025] All scientific terms are to be given their ordinary meanings as understood by those of skill in the art, unless an alternate meaning is set forth below. In case of conflict, the definitions set forth in this specification shall control.
[0026] As used in this application, the term "host cell" means a cell that may be used to express a particular DNA molecule, vector, or plasmid other than the naturally occurring segments of the host cell's chromosomal DNA.
[0027] As used in this application, the term "vector" means a DNA molecule that is at least originally separate from the chromosomal DNA of a host cell, and may include plasmids.
[0028] As used in this application, the term "detergent" means any agent that causes a substance to become soluble or more soluble in a solution.
[0029] Many common bacterial pathogens such as Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis have become multidrug-resistant and emerged as a public health concern, demanding an unmet medical need for novel antibiotics.
[0030] The bacterial cell wall or peptidoglycan synthetic pathway has been targeted for the development of antibacterial agents. The synthesis of peptidoglycan consists of several steps including the synthesis of lipid I and lipid II followed by the final transglycosylation and transpeptidation of lipid II to form peptidoglycan. Many of the current antibiotics are β-lactam derivatives that target the transpeptidation. No medicines are yet developed to inhibit the transglycosylation process. The only known potent inhibitors for transglycosylase (TG) are moenomycin complexes (flavomycin), including moenomycin A (FIG. 1A, Moe A, 1), A12, C1, C3 and C4. Among them, Moe A is the most abundant agent in its family. FIG. 1 shows inhibition of transglycosylase by moenomycin and binding affinities of truncated PBP variants. FIG. 1(A) shows Moenomycin A (1) inhibiting the transglycosylation step in the bacterial cell wall synthesis. The unique antibacterial properties have prompted chemists to synthesize moenomycin fragments and derivatives. The first total synthesis of moenomycin A was achieved recently by Kahne and co-workers. Its biosynthesis pathway has also been elucidated. However, due to poor bioavailability, flavomycin is only employed as an antibiotic growth promoter in animal feeds.
[0031] Transglycosylation is mainly catalyzed by bi-functional class A penicillin-binding proteins (PBPs). PBPs are the major enzymes responsible for the last-step cell-wall formation by polymerization of N-acetylglucosamine-N-acetylmuramyl-pentapeptide, and cross-linking between penta-peptide, and thus have been considered as one of the important targets for antibiotics discovery. These membrane-anchored enzymes consist of three distinct protein domains, i.e., transmembrane (TM), transglycosylase (TG) and transpeptidase (TP) domains from their amino to carboxyl termini. The recent X-ray crystal structures of the PBP2 extracellular domain from S. aureus and the transglycosylase domain of PBP1a from A. aeolicus have provided invaluable structural insight into the plausible mechanism of cell-wall peptidoglycan polymerization, albeit lacking the information regarding the role of the transmembrane domain of PBPs in catalysis. The transmembrane domain of PBPs has been speculated to interact with the lipid moiety of moenomycin or lipid II.
[0032] The characterization of class A PBPs and the identification of TG inhibitors require functional PBP and lipid II as the substrate for the polymerization. The limited availability of lipid II has hampered the enzymatic study as well as the development of inhibitors. To get around the lipid II deficiency, a majority of screening methods to search for TG inhibitors rely mainly on moenomycin. Based on moenomycin activity, low-throughput inhibitor screening methods using SPR assays or radioactive assays have been developed. A TG activity assay platform that is amenable to high-throughput screening is thus desirable for inhibitor identification.
[0033] According to implementations, a high throughput device is contemplated. According to implementations, the high throughput device comprises a platform having multiple versions of a class A penicillin-binding protein comprising at least transmembrane and a transglycosylase domain for determining the effectiveness of a candidate agent as a transglycosylation inhibitor. FA assays may then be carried out on the platform, for example either competition displacement assays or direct binding assays or both, and a determination made of the effectiveness of the candidate agent determined.
[0034] According to implementations, a method is disclosed comprising obtaining a candidate for screening; carrying out an anisotropy measurement assay, such as fluorescent anisotropy, with a class A penicillin-binding protein comprising at least a transmembrane and a transglycosylase domains; and determining the effectiveness of the candidate as a transglycosylase inhibitor.
[0035] According to implementations, a method is provided for amplifying DNA sequence of full-length penicillin-binding protein from bacterial genomic DNA; cloning the DNA sequence into a vector, expressing the DNA sequence in a host cell to obtain a full-length penicillin-binding protein, solubilizing the protein with a detergent, and purifying the protein.
[0036] According to similar implementations of the present disclosure, a high-throughput screening method is provided for identification of TG inhibitors and antibiotic development. The method may comprise: (a) providing a candidate for screening; (b) carrying out fluorescence anisotropy measurement with fluorescent moenomycin and class A penicillin-binding protein comprising transmembrane and transglycosylase domains; (c) calculating fluorescence anisotropy value and KD value; (d) calculating KI, and IC50 value of an transglycosylase inhibitor; and (e) determining minimal inhibitory concentration of the inhibitor; wherein KD is the dissociation constant between the moenomycin and the protein; KI, is the dissociation constant between the inhibitor and the protein; IC50 is inhibitory concentration needed to inhibit bacterial growth by half; minimal inhibitory concentration is the minimal concentration of the inhibitor that prevents bacterial growth.
[0037] A high-throughput screening method according to implementations of the present disclosure may be used to determine the protein binding affinity for a candidate inhibitor or compound. Where a protein may be known or suspected to play a role in the activity of a target cell, the binding affinity of the candidate may correspond to the effectiveness of the candidate as an inhibitor of the protein with regard to the known or suspected cell activity.
[0038] An FA-based PBP binding assay according to implementations of the present disclosure has been demonstrated as a powerful HTS assay for the discovery of novel antibacterial agents. An FA-based assay according to implementations of the present disclosure can be used as a robust primary screening to quickly identify the potential hits from large antibiotic compound libraries. Furthermore, an efficient and economical TG assay in an HTS format according to implementations of the present disclosure may facilitate the identification of new TG inhibitors. The selected hits may be further screened using antibacterial assay or lipid II polymerizing activity analysis to identify leads.
[0039] According to implementations of the present disclosure, a method is provided for expressing and purifying full-length class A penicillin-binding protein from a bacterium comprising: (a) amplifying DNA sequence of full-length penicillin-binding protein from bacterial genomic DNA; (b) cloning the DNA sequence into a vector; (c) expressing the DNA sequence in a host cell to obtain a full-length penicillin-binding protein; (d) solubilizing the protein with a detergent; and (e) purifying the protein.
[0040] A method for expressing and purifying proteins according to implementations of the present disclosure may be applied to obtain a variety of proteins that are capable of being expressed in a host cell. Such proteins may correspond to proteins naturally generated within living organisms and may include proteins known or suspected to play a role in the activity of a cell of the living organism.
[0041] Proteins obtained by implementations of the disclosed method may be used for binding experiments for screening purposes. For example, proteins obtained may be used to evaluate the binding activity of candidate compounds to determine the effectiveness of the compound as an inhibitor of the protein obtained.
[0042] PBPs may be obtained by implementations of the disclosed method, including PBP variants including various combinations of transmembrane (TM), transglycosylase (TG) and transpeptidase (TP) domains. For example, the following variants of domain combinations may be obtained: (i) full-length protein containing the TM, TG, and TP domains (TM+TG+TP), (ii) TG+TP, (iii) TM+TG, (iv) TG alone, and (v) TP alone (see FIG. 1B).
[0043] A method for expressing and purifying full-length class A penicillin-binding protein from a bacterium according to implementations of the present disclosure may be used to obtain the PBPs from different species with purity greater than 80%. Implementations of the method may include selection of a DNA sequence from bacterial genomic DNA that is known to be active in certain activities of the corresponding bacterium.
[0044] Pharmaceutical compositions are also contemplated. The methods of the present disclosure allow for the rapid determination of agents that are useful as antibiotics. For example, as disclosed in more detail in Example 9 and 10, compounds 2 through 10 were tested for antibiotic activity. Moenomycin analogs 2 and 4, as well as small molecules 6-8, were confirmed to have both antibacterial and TG binding inhibition activities (see Table 2).
[0045] As disclosed above, pharmaceutical preparations are herein disclosed having antibiotic or TG binding inhibition activities. According to implementations, one or more of the following agents for a pharmaceutical comprised of a pharmaceutical carrier and one of the following agents:
##STR00005## ##STR00006##
and administered to a subject in need of an antibiotic due to bacterial infection or as a preventative measure.
Pharmaceutical Compositions
[0046] The instant disclosure also provides pharmaceutical compositions. In some implementations, the pharmaceutical compositions comprise agents, namely moenomycin analogs and small molecules (TG Inhibitors) shown to have antibiotic activity via inhibition of TG binding. In such pharmaceutical compositions, the TG Inhibitors form the "active compound" or "agent." According to implementations, the pharmaceutical compositions are administered to a subject to in need of anti-bacterial therapy, including gram-negative bacteria. According to other implementations, the pharmaceutical compositions are administered to a subject having a bacterial infection to inhibit the transgylcosylation process during the synthesis of bacterial cell wall.
[0047] In addition to active compound, the pharmaceutical compositions preferably comprise at least one pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
[0048] Subject as used herein refers to humans and non-human primates (e.g., guerilla, macaque, marmoset), livestock animals (e.g., sheep, cow, horse, donkey, pig), companion animals (e.g., dog, cat), laboratory test animals (e.g., mouse, rabbit, rat, guinea pig, hamster), captive wild animals (e.g., fox, deer) and any other organisms who can benefit from the agents of the present disclosure. There is no limitation on the type of animal that could benefit from the presently described agents. Human subjects are expressly contemplated. A subject regardless of whether it is a human or non-human organism may be referred to as a patient, individual, animal, host, or recipient.
[0049] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
[0050] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
[0051] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
[0052] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Other delivery methods and devices common in the art, including mechanically actuated atomizing-like devices are expressly contemplated.
[0053] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For epidermal, dermal, or transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
[0054] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
[0055] In one implementation, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to cell-specific antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811, incorporated by reference herein.
[0056] It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
[0057] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
[0058] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in subjects. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in subjects. Levels in plasma can be measured, for example, by high performance liquid chromatography.
[0059] As defined herein, a therapeutically effective amount of an active compound of the disclosure may range, for examples, from about 0.001 to 30 mg/kg body weight, about 0.01 to 25 mg/kg body weight, about 0.1 to 20 mg/kg body weight, or about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. Without limitation, the active compound can be administered between one time per week and three or more times per day, for between about 1 to 10 weeks, for example between 2 to 8 weeks, between about 3 to 7 weeks, or for about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a pharmaceutical composition of the disclosure can include a single treatment or, preferably, can include a series of treatments.
[0060] For those agents determined to be transglycosylation inhibitors or antibiotics, further testing may then be performed for the candidate agent to determine agents that are both good transglycosylation inhibitors and also having reasonably bioavailability.
EXAMPLES
[0061] The present disclosure and its implementations may be further illustrated by the following examples.
Example 1
Domain Requirement of Moenomycin Binding to Class A Penicillin-Binding Proteins (Bi-Functional Transglycosylase)
[0062] Protein constructs containing different domains of PBP1b from E. coli were expressed and purified with suitable detergents for moenomycin binding activities using Surface Plasmon Resonance (SPR). Five E. coli PBP1b variants were expressed and purified: 1) full-length protein containing transmembrane, transglycosylase and transpeptidase domains (TM+TG+TP), 2) TG+TP, 3) TM+TG, 4) TG, and 5) TP (FIG. 1B).
[0063] FIG. 1 shows inhibition of transglycosylase by moenomycin and binding affinities of truncated PBP variants. FIG. 1(B) is a schematic representation of the PBP variants used for moenomycin binding studies. The full-length PBP (TM+TG+TP) contains amino acid residues 1-844 of the E. coli PBP1b P02919. TG+TP (residues 88-844), TM+TG (residues 1-409), TG (residues 195-409), and TP (residues 444-736) are truncated variants with the part of the full-length PBP1b. The moenomycin binding constant (KD) for these variants were determined using SPR.
[0064] After immobilization of the target proteins on the sensor chip, different concentrations of moenomycin were passed through the surface and the binding affinities were determined. As shown in FIG. 1B, the moenomycin binding activity of the PBP without the transmembrane domain (TG+TP) was about 5-fold lower in comparison to that of the full-length PBP1b. On the other hand, the binding affinity of TM+TG is similar to that of the full-length protein. The results suggested that the transmembrane domain may play an important role in moenomycin binding.
[0065] The moenomycin binding affinity of the full-length was determined as 4.4×10-7 M, similar to the inhibition potency for the TG reaction. Using a TP variant as a control protein, it was confirmed that the determined binding affinities of moenomycin to the PBP1b variants is significant and specific. Binding studies using truncated PBP1b variants further showed that the binding affinities of the TM+TG variant is similar to that of the full-length protein. The role of the TM domain in Moe A binding was shown in the observation that the binding capacity of the TG+TP variant decreased five-fold compared with that of full-length PBP1b (FIG. 1B).
Example 2
[0066] Correlation of PBP Binding Activities and Minimal Inhibitory Concentration (MIC) of Moenomycin
[0067] Since the transmembrane domain contributes significantly to moenomycin binding, full-length PBPs were prepared from 16 Gram-negative and -positive bacteria (Table 1) for moenomycin binding studies. The genes of Class A PBPs were identified using NCBI database and all of them were confirmed to have TG and TP motifs (FIG. 2).
[0068] FIG. 2 shows sequence alignment of full-length bi-functional PBPs from 16 bacterial strains (Seq. ID Nos. 1-16). Each section A-L of FIG. 2 contains 16 rows, with each row corresponding to, in order, Seq. ID Nos. 1-16, respectively. The order of the aligned sequences, in rows, is identical to the order given in Table 1 and FIG. 3.
[0069] Each row of FIG. 2 is labeled with the following: "Bpe" (row 1) indicates PBP1b from Bordetella pertussis (Seq. ID No. 1); "Cfr" (row 2) indicates PBP1b from Citrobacter freundii (Seq. ID No. 2); "Eco" (row 3) indicates PBP1b from Escherichia coli (Seq. ID No. 3); "Hin" (row 4) indicates PBP1b from Haemophilus influenzae (Seq. ID No. 4); "Hpy" (row 5) indicates PBP1a from Helicobacter pylori (Seq. ID No. 5); "Kpn" (row 6) indicates PBP1b from Klebsiella pneumoniae (Seq. ID No. 6); "Ngo" (row 7) indicates PBP1 from Neisseria gonorrhoeae (Seq. ID No. 7); "Pae" (row 8) indicates PBP1b from Pseudomonas aeruginosa (Seq. ID No. 8); "Sen" (row 9) indicates PBP1b from Salmonella enterica (Seq. ID No. 9); "Sfl" (row 10) indicates PBP2 from Shigella flexneri (Seq. ID No. 10); "Bsu" (row 11) indicates PBP1a/1 b from Bacillus subtilis (Seq. ID No. 11); "Cdi" (row 12) indicates PBP from Clostridium difficile (Seq. ID No. 12); "Efa" (row 13) indicates PBP2a from Enterococcus faecalis (Seq. ID No. 13); "Efc" (row 14) indicates PBP1 from Enterococcus faecium (Seq. ID No. 14); "Sau" (row 15) indicates PBP2 from Staphylococcus aureus (Seq. ID No. 15); "Spn" (row 16) indicates PBP1b from Streptococcus pneumoniae (Seq. ID No. 16).
[0070] Each of the 16 rows of FIG. 2 represents a sequence that continues through each of sections A-L of FIG. 2. Each sequence contains a Transmembrane (TM) domain, a Transglycosylase (TG) domain, and a Transpeptidase (TP) domain. Each domain is represented by gray background shading, with the type of domain ("Transmembrane (TM)", "Transglycosylase (TG)", or "Transpeptidase (TP)") identified by a label at the beginning of each domain (section of gray shading). Conserved residues are represented by boxes of thin black lines that encompass one or more columns. Where a set of conserved residues (i.e., conserved residues in a given column) contains some similarities among the 16 sequences, the text is shown in black or dark gray. Where a set of conserved residues contains identical entries across the 16 sequences, the column is represented with a black background and light text. Note that such columns represented with black background and light text indicate sets of conserved residues.
[0071] The sequences of FIG. 2 have been distributed such that beginning and end of each domain and the sets of conserved residues across the 16 sequences are aligned along vertical columns. To accomplish this, "gaps" have been provided, each represented by a ("•"). Such "gaps" do not represent entries in the structural continuity of each sequence or characteristics thereof, but rather are provided only to accomplish the alignment described herein. Accordingly, the numbering of sequence entries (excluding gaps) is specific to each sequence. The number corresponding to the first entry in each row of FIGS. 2A, 2C, 2E, 2G, 21, and 2K is given to the left of each said first entry. The dots above the top row of each section are provided to indicate the numbering of the first sequence ("Bpe"), occurring at intervals of 10 entries with respect to sequence "Bpe".
[0072] Stars above the top rows of FIG. 2 indicate the motif for TG: EDxxFxxHxxG, GxSTxTQQ, RKxxE, KxxILxxYxN, and RRxxVL, and the motif for TP: SxxK, SxN, and KTG. Underlined stars indicate catalytic residues. The 16 full-length PBP sequences used in this study were aligned with program Jalview and the figure was prepared with ESPript.
[0073] The target genes were amplified from respective genomic DNA from each individual bacterial species, and cloned into expression vectors for recombinant protein production using E. coli host. The enzymatic activities of the purified proteins were confirmed by lipid II polymerization (data not shown) and moenomycin binding were measured with SPR. As shown in Table 1, varied steady-state affinity (KD) values were found among PBPs from different species. Nonetheless, all the measured KD values fell into the range of 10-7 M, close to the reported inhibition concentration for the transglycosylation process. Among the Gram-positive bacteria tested, the KD values of PBP from E. faecalis and S. aureus correlate with the MIC values, suggesting that the moenomycin binding site of PBP may be a good target for development of antibiotics against these species.
TABLE-US-00001 TABLE 1 Correlation of antimicrobial activity and PBP binding affinity of moenomycin for 16 bacterial strains Reported values.sup.† SPR. FA. Species MIC, μM* MIC, μM Citation Gene sequences ID.sup..dagger-dbl. KD, nM.sup.§ KD, nM.sup. Gram-negative Bordetella pertussis 0.011-0.021 -- PBP1a (NP_882163) 1,270 594 ± 230 Citrobacter freudii -- 352 28 PBP1b (CAA90232) 728 -- Escherichia coli 80 55-110 28 PBP1b (NP_414691) 440 54 ± 17 Haemophilus influenzae -- -- PBP1b (AAX88775) 966 174 ± 13 Helicobacter pylori -- 1.3 29 PBP1a (NP_207392) 334 25 ± 14 Klebsiella pneumoniae 80 13.75-27.5 28 PBP1b (NTUH-2044) 819 78 ± 24 Neisseria gonorrhoeae -- 1.69 28 PBP1 (YP_207272) 450 47 ± 26 Pseudomonas aeruginosa 40 55-110 28 PBP1b (YP_793163) 866 42 ± 29 Salmonella enterica 160 13.75-110 28 PBP1b (YP_149541) 561 138 ± 113 Shigella flexneri 40 27.5-110 28 PBP2 (YP688236) 290 -- Gram-positive Bacillus subtilis 0.3-0.6 0.07-0.43 30 PBP1a/1b (NP_390113) 1,690 197 ± 80 Clostridium difficile >160 220-440 30 PBP (CAJ67615) 582 37 ± 33 Enterococcous faecalis 0.075-0.3 0.1 31 PBP2a (NP_814430) 619 276 ± 14 Enterococcous faeclum -- >40.5 10 PBP1 (EAN08787) 94 56 ± 11 Straphylococcus aureus 0.075 0.02-0.12 10 PBP2 (NP_371974) 393 30 ± 29 Streptococcus pneumoniae 0.625 -- PBP1b (NP_359500) 900 38 ± 24 *MIC of moenomycin against different bacterial species. .sup.†MIC values of moenomycin from the literature. .sup..dagger-dbl.Sequence ID of class A PBP genes from individual bacterial strains. Homologs of class A PBPs were identified by using BLAST against the NCBI database. .sup.§KD of PBP homologs and moenomycin using SPR. Average values are shown. .sup. KD of PBP homologs and moenomycin using our FA-based assay.
Example 3
Expression and Purification of E. coli PBP1b Variants
[0074] Full-length PBP1b from E. coli (Swiss-Prot accession number P02919) was amplified from genomic DNA, cloned into the pET vectors (EMD Sciences, San Diego, Calif.), and expressed with an N-terminal (His)6 tag. BL21(DE3) E. coli host cells were grown at 37° C. until OD at 600 nm reached 0.6 and protein expression was induced with 1 mM IPTG for 3 hours. Cell pellets were resuspended in 20 mM Tris at pH 8.0, 300 mM NaCl and broken by Microfluidizer (Microfluidics, Newton, Mass., USA). The solubilization and purification of the recombinant full-length membrane proteins were tested with various detergents (Anatrace, Maumee, Ohio, USA), including n-decyl-β-D-maltopyranoside, n-undecyl-β-D-maltopyranoside, n-dodecyl-β-D-maltopyranoside, n-octyl-β-D-glucopyranoside, n-nonyl-β-D-glucopyranoside, n-tetradecylphosphocholine, n-dodedyl-N,N-dimethylamine-N-oxide, CHAPS (3[(3-Cholamidopropyl)-dimethylammonio]-1-propane sulfonate) and Triton X-100 (α-[4-(1,1,3,3-Tetramethylbutyl)phenyl]-w-hydroxy-poly(oxy-1,- -2-ethanediyl)). Among them, DDM (n-dodecyl-β-D-maltopyranoside) was found to solubilize the full-length PBP1b and retain its moenomycin binding activities. Therefore, full-length PBP1b was solubilized with 20 mM DDM and purified by Nickel chelation chromatography following manufacturer's instruction in the presence of 1 mM DDM.
[0075] The domains of PBP1b are defined as: transmembrane (TM, residues 64-87), transglycosylase (TG, residues 195-409) and transpeptidase (TP, residues 444-736). Nucleotide primers were designed to amplify respective variants containing TG+TP (residues 88-844), TM+TG (residues 1-409), TG (residues 195-409), and TP (residues 444-736). The amplified products were cloned into pET vectors for expression and purification. The procedure to obtain the variant TM+TG was as described for full-length protein preparations. For the variants TG+TP, TG and TP, 13 mM n-tetradecylphosphocholine (Anatrace) was used for protein extraction and 0.25 mM for Nickel chelation chromatography. The detergent was exchanged to 1 mM DDM and concentrated using Amicon Ultra filter units (Millipore, Billerica, Mass., USA).
Example 4
Expression and Purification of Recombinant Full-Length Bi-Functional PBPs from 16 Bacterial Strains
[0076] Homologs of full-length bi-functional PBPs were identified with BLAST against NCBI database using PBP1b protein sequence from E. coli (Swiss-Prot accession number P02919). To confirm the presence of the transglycosylase motifs and active sites, the sequences were aligned using program Jalview (4) with MUSCLE multiple alignment algorithm (5), and the alignment output (FIG. 2) were prepared with ESPript (6). The following residues are omitted in the alignment for clarity, Bpe (B. pertussis): 354-443, Ngo (N. gonorrhoeae): 331-416, Bsu (B. subtilis): 615-643, 735-787, 797-835 and 897-914, and Cdi (C. difficile): 267-322 and 804-842.
[0077] FIG. 3 shows the SDS-PAGE analysis of purified protein of class A PBPs from sixteen bacterial species. Each lane contains 5 μg of protein and M denotes molecular weight markers in kDa. 1: PBP1a from Bordetella pertussis (Seq. ID No. 1), 2: PBP1b from Citrobacter freundii (Seq. ID No. 2), 3: PBP1b from Escherichia coli (Seq. ID No. 3), 4: PBP1b from Haemophilus influenzae (Seq. ID No. 4), 5: PBP1a from Helicobacter pylori (Seq. ID No. 5), 6: PBP1b from Klebsiella pneumoniae (Seq. ID No. 6), 7: PBP1 from Neisseria gonorrhoeae (Seq. ID No. 7), 8: PBP1b from Pseudomonas aeruginosa (Seq. ID No. 8), 9: PBP1b from Salmonella enterica (Seq. ID No. 9), 10: PBP2 from Shigella flexneri (Seq. ID No. 10), 11: PBP1a/1b from Bacillus subtilis (Seq. ID No. 11), 12: PBP from Clostridium difficile (Seq. ID No. 12), 13: PBP2a from Enterococcus faecalis (Seq. ID No. 13), 14: PBP1 from Enterococcus faecium (Seq. ID No. 14), 15: PBP2 from Staphylococcus aureus (Seq. ID No. 15), 16: PBP1b from Streptococcus pneumoniae (Seq. ID No. 16).
[0078] For expression of individual PBPs, genomic DNAs were purchased from American Type Culture Collection (ATCC, Manassas, Va., USA). The recombinant proteins of the full-length genes from 16 bacteria, including Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Shigella flexneri, Haemophilus influenzae, Helicobacter pylori, Citrobacter freundii, Bordetella pertussi, Staphylococcus aureus (MRSA Mu50), Bacillus subtilis, Pseudomonas aeruginosa, Clostridium difficile, Enterococcus faecium, Enterococcus faecalis, Salmonella enterica and Neisseria gonorrhoeae, were prepared using the same protocols as described for the full-length E. coli PBP1b. Most of them can be obtained with purity greater than 80% (FIG. 3) and used for binding experiments.
Example 5
Surface Plasmon Resonance Detection
[0079] Purified PBP and its variants were immobilized onto CM3 chip (GE healthcare, Uppsala, Sweden) to about 1,500˜2,000 RU via amine-coupling method. The chips were then passed over with different concentrations of moenomycin A (0-2000 nM). Immobilization and data collection were performed with BIAcore T100 (GE Healthcare, Uppsala, Sweden) at 25° C.
Example 6
Fluorescence Anisotropy Measurements
[0080] Fluorescence anisotropy measurements were carried out in triplicates in the wells of 384-well plates using a laser fluorimetry equipped with a 488 nm laser (IsoCyte, Blueshift Biotech, Inc., Sunnyvale, Calif., USA). Various buffers, salts, pH values, and divalent cations (Ca++, Mg++, Co++) were optimized for fluorescence anisotropy measurements. KD and KI, determinations were carried out in 100 mM NaCl, 10 mM Tris, pH 8.0. The scanning focus was above the plate bottom to avoid detection interferences. Data analysis was performed with the proprietary software, BlueImage (Blueshift Biotech Inc). Fluorescence anisotropy values (A) were calculated using the equation: A=(I.sub.|-G*I.sub.∥)/(I.sub.|+2G*I.sub.∥), where I.sub.∥ is the fluorescence intensity of emitted light parallel to excitation, I.sub.∥ is the fluorescence intensity of emitted light perpendicular to excitation, and G is the gating factor that corrects for instrument bias. The G factor is experimentally determined for each run using the probe-only well as the basal an isotropy.
[0081] FIG. 4 shows the design of a novel fluorescence anisotropy assay for transglycosylase, according to implementations of the present disclosure.
[0082] Based on the conclusion that the full-length proteins shall be used for moenomycin binding studies, a fluorescence anisotropy (FA)-based assay was thus designed to monitor the binding affinities of small molecules towards TG. Previous studies on the complex crystal structure and the structure-activity relationship suggested that the modification of compound 1 to 2 (FIG. 4A) will not dramatically reduce the binding and antibacterial activities. FIG. 4(A) shows the chemical structures of the modified moenomycin A (2). Furthermore, the amino moiety in 2 allows the conjugation with any fluorophore as a moenomycin-based probe for binding studies. Indeed, compound 2 was readily linked with a fluorescein (6-carboxyfluoresein N-hydroxysuccinimide ester) under basic conditions to prepare the fluorescent probe 3 (F-Moe, FIG. 4B). FIG. 4(B) shows the chemical structures of the fluorescein-labeled moenomycin, F-Moe (3). One major concern about the fluorescent probe used in the FA assay is the probe itself; either the fluorophore or the structure modification, may interfere with the binding between the targeted protein and the small molecule. Therefore, the PBP binding affinities of Moe A and the fluorescent probe were compared using SPR. The determined steady-state affinity (KD) values are similar for Moe A and F-Moe (4.4×10-7 vs. 5.2×10-7 M) (FIGS. 4C and 4D).
[0083] FIG. 4(C) shows the SPR analysis of the binding activity of moenomycin A to E. coli PBP1b, and FIG. 4(D) shows the SPR analysis of the binding activity of F-Moe to E. coli PBP1b. Responses for moenomycin binding to immobilized E. coli PBP1b were shown. The data were analyzed using steady-state affinity and fitted to a 1:1 interaction model, as shown in the inset graph. The KD values deduced from the intercepts of X-axis and the dotted lines, are 4.4×10-7 M and 5.161×10-7 M for Moe A and F-Moe, respectively.
[0084] The result confirms that F-Moe is a valid fluorescence probe useful for the FA-based PBP binding assay.
[0085] FIG. 5 shows the development of high-throughput FA assay for transglycosylase. FIG. 5(A) shows the concentration-dependent changes in fluorescence anisotropy observed when E. coli PBP1b bound to F-Moe. FIG. 5(B) shows the improved FA assay with the Helicobacter pylori PBP1a. The concentration-dependent fluorescence anisotropy changes were performed similarly using the H. pylori PBP1a. The maximum anisotropy value was 0.2. FIG. 5(C) shows the displacement of the PBP1a bound F-Moe complex by unlabeled moenomycin at various concentrations. The changes in fluorescence anisotropy is defined as [(Aobs-Amin)/(Amax-Amin)×100%]. The KD and IC50 value was calculated as described in example 2.
[0086] The anisotropy of F-Moe increased significantly by incubation with E. coli PBP1b, supposedly due to the formation of F-Moe-PBP1b complex (FIG. 5A). In contrast, the anisotropy of F-Moe was unchanged when incubated with bovine serum albumin, up to 100 μM (data not shown). The protein concentrations were further titrated down to establish the dose-dependency of the anisotropy changes. The deduced dissociation constant (KD) for F-Moe was in the sub-micromolar range (54±17 nM) for E. coli PBP1b (FIG. 5A). However, the signal-to-noise ratio of FA assay using the E. coli PBP1b is of limited satisfaction with the maximum anisotropy equal to 0.120. Different class A PBPs were screened to improve the assay. Of all homologs tested, the FA assay using Helicobacter pylori PBP1a produced the best signal-to-noise ratio with KD of 25±14 nM, and with the anisotropy increases from 0.018 to 0.2 upon binding to F-Moe (FIG. 5B). For the development of an assay for inhibitor screening, F-Moe was pre-incubated with H. pylori PBP1a and then competed with unlabeled Moe A at various concentrations. A decrease in anisotropy was observed as the concentration of Moe A increased, resulting in the KI, (inhibition constant) and IC50 values as 0.47±0.10 μM and 0.36 μM, respectively (FIG. 5C). The result validates the FA assay to screen for inhibitors that displace the probe competitively from the moenomycin binding pocket of PBP.
Example 7
Determination of KD Values from Direct Binding Assay
[0087] For direct binding assay, the initial condition contained 40 μl of 100 nM F-Moe in 10 mM Tris, pH 8.0, 100 mM NaCl. After a small volume of PBP stock was added, the anisotropy change was measured after a 5-minute equilibration. We assume that the direct titration follows a simple binding equilibrium between Ligand (L) and Receptor (R) (R+LSKD/RLS with KD=RLS/RLS). The data was fitted to the [Eq. 1] using Prism (GraphPad Software, San Diego, Calif.) for the determination of KD (3):
[(Aobs-Amin)/(Amax-Amin)=(KD+LST+RT)-- [(KD+LST+RT)2-4LSTRT]1/2 [Eq. 1]
where Aobs is the observed anisotropy; Amin is the minimum anisotropy; Amax is the maximum anisotropy; LST is the total concentration of F-Moe, RT is the total concentration of PBP, and KD is the dissociation constant between the ligand (F-Moe) and the receptor (PBP). The reported value is the average of four different runs.
Example 8
Determination of KI, and IC50 Values from Competitive Displacement Assay
[0088] For displacement assay, the initial condition contained 40 μl of 100 nM F-Moe, 10 μg/ml H. pylori PBP1a in 10 mM Tris, pH 8.0, 100 mM NaCl. Aliquots of compound stock solution were added and the anisotropy was monitored after 5 minutes of equilibration. The data from the displacement assay was used to calculate the inhibition constant (KI) and IC50 value of an inhibitor using the complete competitive binding model
##STR00007##
The KI value was determined by plotting the total inhibitor concentration (C) versus the molar ratio of bound/free F-Moe and fitting the resulting curve to [Eq. 2],
C=[(KI/KD*X)+1][RT-(LST*X)/(X+1)-KD*X [Eq. 2]
[0089] X=Bound/Free=(Aobs-Amin)/(Amax-Amin)
where Aobs is the observed anisotropy at a particular concentration of a compound (C), Amin is the minimum anisotropy, Amax is the maximum anisotropy, LST is the total concentration of F-Moe, RT is the total concentration of PBP1a, KD is the dissociation constant between the ligand (F-Moe) and the receptor (PBP) and KI is the dissociation constant between the inhibitor and PBP1a. The reported value is the average of four different runs.
[0090] For IC50 determination, the fraction of bound fluorescent probe is plotted versus the log value of the competitor concentration (C). Fitting the resulting sigmoidal curve using [Eq. 3] would result in an IC50 value,
(Aobs-Amin)/(Amax-Amin)-1/(1+10((C-log(IC50))*H- ill slope) [Eq. 3]
where Aobs is the observed anisotropy at a particular concentration of a compound (C), Amin is the minimum anisotropy, Amax is the maximum anisotropy.
Example 9
High-Throughput Screening for Transglycosylase Inhibitors
[0091] The FA assay was used to screen against 50,000 purchased small molecules (ChemBridge Inc., San Diego, Calif., USA) and 7,000 from proprietary collections. The compounds were transferred to 96-well plates (Freedom Evo, Tecan Schweiz AG, Mannedorf, Switzerland) and then to 384-well plates using a multi-dispenser (Labcyte, Sunnyvale, Calif., USA) to prepare the compound plates for screening. The H. pylori PBP1a (10 μg/ml) in 100 nM F-Moe, 10 mM Tris, 100 mM NaCl, pH 8.0 at a final volume of 40 μl was added to 384-well plates (Freedom Evo 150, Tecan). One μL of 2 mM compound stocks were added to wells using a multi-dispenser (Labcyte). The last two columns of every plate were controls with 10 μM moenomycin and 2.5% DMSO, respectively. After a 30-minute incubation, changes in fluorescence anisotropy were determined with Isocyte (Blueshift Biotech Inc). Hits that showed greater than 75% reduction compared to the control anisotropy values were selected for further confirmation.
[0092] Several moenomycin analogues (2, 4, and 5) were prepared to evaluate the reliability of the FA-based assay. As shown in Table 2, the differential binding affinities (the IC50 values) of these moenomycin analogues by the FA-based assay are comparable with the published reports. The modification of moenomycin A to 2 resulted in a little decrease in TG binding affinities. Dimerization of 2 via an eight-carbon spacer produced 4 with a two-fold increase over 2 in both PBP binding and the antimicrobial potencies, although 4 is still less potent than Moe A (1). More significantly, compound 5 without the C25 lipid moiety could hardly displace the F-Moe probe 3, indicating that the hydrophobic part of moenomycin is crucial to the binding to the full-length PBP1b. It is noteworthy that the order in inhibition potency is in agreement with that of MIC values (Table 2).
TABLE-US-00002 TABLE 2 Inhibition of TG activity and the antibacterial determinations of selected hits MIC, μM.sup..dagger-dbl. B. subtilis E. faecalis S. aureus S. pneumoniae Compound* IC50, μM.sup.† (ATCC23857) (ATCC29212) (ATCC29213) (ATCC49619) 1 0.36 0.33 0.04 <0.01 0.33 2 2.10 2.50 10.0 1.25 20.0 4 0.92 1.25 5.0 0.625 20.0 5 125.00 -- -- -- -- 6 34.00 0.25 0.25 1.0 4.0 7 3.70 0.25 1.0 4.0 -- 8 9.30 4.0 >4.0 >4.0 >4.0 *Compounds 2, 4, and 5 are moenomycin derivatives. Compounds 6-8 were HTS hits. .sup.†IC50 values were determined by using the fluorescence anisotropy assay shown in FIG. 5A. .sup..dagger-dbl.The MICs of moenomycin against different bacterial species were determined as described in Materials and Methods. ATCC, American Type Culture Collection.
[0093] The FA assay was used to screen a collection of 57,000 small molecules along with Moe A derivatives (FIG. 6) at 50 μM. FIG. 6 illustrates chemical structures of moenomycin derivatives (4 and 5), according to implementations of the present disclosure.
[0094] High controls are assays with 1 μM of moenomycin and low controls are assays with 2.5% DMSO. Other proteins, such as bovine serum albumin, were also included as a control to confirm that the anisotropy increase is not from non-specific binding.
[0095] FIG. 7 illustrates screening for small molecules as TG inhibitors using the class A PBPs, according to implementations of the present disclosure. FIG. 7(A) shows the scheme of HTS for TG inhibitors using FA assay. Protein structure graphics were created from PDB ID 2OLV (12). 7(B) shows chemical structures of the HTS hits (compounds 6-8).
[0096] Z' value, a statistical parameter ranging from 0 to 1 to evaluate the robustness of high-throughput screening, was determined as 0.895 from more than 100 independent experiments. Eleven possible hits that showed at least 75% inhibition in the screening were selected for additional studies involving antimicrobial assays and IC50 values determinations for PBP binding (FIG. 7A). Among which, two moenomycin analogues (2 and 4) and three small molecules (compounds 6, 7 and 8) were confirmed to have both antibacterial and the TG binding inhibition activities (Table 2). Respectively, compounds 6, 7 and 8 are (Z)-5-(4-bromophenyl)-3-((5-nitrofuran-2-yl)methylene)furan-2(3H)-one(Z)-- 1,3-diphenyl-4-(2-(thiazol-2-yl)hydrazono-1H-pyrazol-5(4H)-one; (E)-4-(2-(5,5,8,8,-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)prop-1-e- nyl)benzoic acid.
[0097] Using the FA-based assay, a hit rate of 0.02% was achieved with the Z' value equal to 0.895.
Example 10
Determination of Minimal Inhibitory Concentration (MIC)
[0098] The minimal inhibitory concentration (MIC) of tested compounds was determined following the NCCLS standard. The experiments were conducted in 96-well microtiter plates using two-fold dilutions in Muller-Hilton broth with (Streptococcus pneumonia) or without blood (Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus). Exponentially growing cells at 5×105 cells/ml were incubated with test compounds at various concentrations. After an 18 h to 24 h incubation at 37° C., MIC was determined as the minimal concentration of the compound that prevents bacterial growth.
[0099] While the apparatus and method have been described in terms of what are presently considered to be the most practical and preferred implementations, it is to be understood that the disclosure need not be limited to the disclosed implementations. It is intended to cover various modifications and similar arrangements included within the spirit and scope of the claims, the scope of which should be accorded the broadest interpretation so as to encompass all such modifications and similar structures. The present disclosure includes any and all implementations of the following claims.
Sequence CWU
1
1
151814PRTBordetella
pertussisTRANSMEMBRANE(19)..(42)DOMAIN(70)..(289)DOMAIN(317)..(716) 1Met
Ser Lys Arg Gln Asn Ser Thr Lys Gln Asp Lys Pro Ala Lys Ser 1
5 10 15 Gly Ser Ala Ile Leu Arg
Phe Phe Leu Lys Ala Gly Ile Phe Phe Gly 20
25 30 Gly Leu Ala Leu Cys Gly Val Leu Leu Ala
Gly Met Ala Leu Ala Leu 35 40
45 Ala Trp Pro Asn Leu Pro Asp Leu His Ala Met Thr Asp Tyr
Arg Pro 50 55 60
Arg Val Pro Leu Arg Val Tyr Thr Ala Asp Arg Val Leu Ile Gly Glu 65
70 75 80 Phe Gly Glu Glu Arg
Arg Asn Val Leu Arg Phe Asn Glu Ile Pro Asp 85
90 95 Val Met Lys Ser Ala Val Leu Ala Ala Glu
Asp Asp Arg Phe Tyr Gln 100 105
110 His Gly Gly Ile Asp Trp Met Gly Val Ile Arg Ala Gly Leu Thr
Asn 115 120 125 Leu
Ile Ser Met Ser Lys Ser Gln Gly Ala Ser Thr Ile Thr Met Gln 130
135 140 Val Ala Arg Asn Phe Tyr
Leu Ser Ser Glu Lys Thr Tyr Ser Arg Lys 145 150
155 160 Phe Tyr Glu Leu Leu Leu Thr Phe Lys Ile Glu
Ser Gln Leu Thr Lys 165 170
175 Asp Gln Ile Leu Glu Leu Tyr Met Asn Gln Ile Tyr Leu Gly His Arg
180 185 190 Ala Tyr
Gly Phe Ala Ala Ala Ser Arg Thr Tyr Phe Gly Lys Pro Leu 195
200 205 Ser Gln Val Thr Pro Ser Glu
Ala Ala Met Leu Ala Gly Ile Pro Lys 210 215
220 Ala Pro Ser Arg Phe Asn Pro Ile Ser Asn Arg Pro
Arg Ala Glu Leu 225 230 235
240 Arg Gln Arg Tyr Val Leu Gly Arg Met Tyr Ser Leu Gly Tyr Leu Thr
245 250 255 Glu Pro Glu
Tyr Lys Glu Ala Met Ala Gln Pro Ile Val Ile Lys Ser 260
265 270 Ala Glu Gly Thr Pro Ala Gly Gly
Tyr Ala Ile His Gly Glu Tyr Val 275 280
285 Ala Glu Leu Ala Arg Gln Leu Leu Tyr Asn Val Tyr Gln
Asp Asn Leu 290 295 300
Tyr Ser Arg Gly Ile Asn Ile Tyr Thr Thr Val Gln Ser Lys Asp Gln 305
310 315 320 Glu Ser Ala Tyr
Arg Ala Val Arg Asp Gly Val Leu Glu Tyr Thr Arg 325
330 335 Arg Ala Pro Tyr Pro Gly Pro Glu Glu
Gln Leu Asp Met Pro Ala Gly 340 345
350 Val Glu Asn Asp Pro Gln Ala Leu Asp Glu Phe Leu Asp Gly
Val Phe 355 360 365
Asp Lys Phe Ser Asp Ser Gly Asp Leu Leu Thr Ala Val Val Leu Ser 370
375 380 Ala Ser Pro Thr Glu
Ile Lys Leu Val Arg Ser Ser Arg Glu Val Ile 385 390
395 400 Ser Ile Thr Asp Lys Lys Ala Leu Gly Val
Val Ala Arg Ala Leu Thr 405 410
415 Asp Lys Ala Lys Pro Glu Met Arg Leu Lys Arg Gly Ser Val Val
Tyr 420 425 430 Ile
His Lys Tyr Asn Asp Asn Trp Glu Val Ile Asn Met Pro Ala Val 435
440 445 Gln Ala Ala Phe Val Ala
Leu Ser Pro Gln Asp Gly Ala Ile Arg Ala 450 455
460 Met Val Gly Gly Phe Asp Phe Tyr Arg Gly Asn
Phe Asn Arg Val Thr 465 470 475
480 Gln Ala Trp Arg Gln Pro Gly Ser Asn Ile Lys Pro Phe Ile Tyr Ala
485 490 495 Ala Ser
Leu Glu Arg Gly Leu Thr Pro Ala Thr Gln Ile Ser Asp Gln 500
505 510 Pro Phe Glu Leu Ser Ala Ala
Gln Thr Gly Ser Lys Ala Trp His Pro 515 520
525 Lys Asn Tyr Gly Asn Gln Tyr Glu Pro Met Leu Thr
Met Arg Gln Gly 530 535 540
Leu Tyr Lys Ser Lys Asn Met Val Ser Ile Arg Ile Leu Gln Ala Ile 545
550 555 560 Gly Pro Gln
Tyr Ala Gln Asp Tyr Leu Thr Arg Phe Gly Phe Asp Lys 565
570 575 Ala Arg Gln Pro Ala Val Leu Pro
Leu Ala Leu Gly Ala Gly Ser Val 580 585
590 Thr Pro Leu Gln Leu Ala Gly Ala Tyr Ala Val Phe Ala
Asn Gly Gly 595 600 605
Tyr Arg Ile Thr Pro Tyr Leu Ile Asp Arg Val Thr Asp Ser Ser Gly 610
615 620 Lys Val Leu Met
Gln Ser Arg Pro Val Ile Ala Gly Asp Ala Ala Ala 625 630
635 640 Arg Ala Ile Asp Ala Arg Thr Ala Phe
Val Met Asp Asp Met Leu Arg 645 650
655 Gly Val Ala Thr Ser Gly Thr Ala Ala Arg Ala Arg Ala Thr
Leu Lys 660 665 670
Arg Ser Asp Val Ala Gly Lys Thr Gly Thr Thr Asn Glu Ser Val Asp
675 680 685 Ala Trp Phe Ser
Gly Tyr Thr Pro Ser Leu Val Ala Thr Ala Trp Leu 690
695 700 Gly Phe Asp Gln Pro Lys Ser Leu
Gly Ser Arg Glu Thr Gly Gly Gly 705 710
715 720 Val Ala Met Pro Ile Trp Leu Asp Tyr Met Lys Asp
Ala Leu Lys Gly 725 730
735 Val Pro Glu Glu Lys Gln Arg Pro Arg Pro Asp Gly Leu Leu Val Glu
740 745 750 Asn Gly Glu
Leu Tyr Phe Ser Glu Phe Pro Pro Gly Gln Ala Val Ala 755
760 765 Arg Leu Gly Leu Pro Gln Ala Gly
Asp Ala Leu Gly Asp Phe Leu Asn 770 775
780 Gly Leu Thr Gly Gly Asn Asp Asn Ser Ile Arg Val Ala
Pro Gly Val 785 790 795
800 Gly Thr Gln Gly Ser Gln Pro Trp Ser Gln Asn Ile Pro Phe
805 810 2846PRTCitrobacter
freundiiTRANSMEMBRANE(64)..(86)DOMAIN(194)..(410)DOMAIN(444)..(732) 2Met
Ala Gly Asn Asp Arg Glu Pro Ile Gly Arg Lys Gly Lys Pro Ser 1
5 10 15 Arg Pro Val Lys Gln Lys
Val Ser Arg Arg Gln Leu Arg Asp Glu Glu 20
25 30 Tyr Asp Asp Asp Tyr Asp Asp Asp Asp Tyr
Glu Asp Glu Glu Pro Met 35 40
45 Pro Arg Lys Gly Lys Gly Lys Gly Arg Lys Pro Arg Gly Lys
Arg Gly 50 55 60
Trp Phe Trp Leu Leu Val Lys Leu Ser Ile Val Phe Leu Val Leu Ile 65
70 75 80 Ala Ile Tyr Gly Val
Tyr Leu Asp Gln Lys Ile Arg Ser Arg Ile Asp 85
90 95 Gly Lys Val Trp Gln Leu Pro Ala Ala Val
Tyr Gly Arg Met Val Asn 100 105
110 Leu Glu Pro Glu Met Pro Ile Gly Lys Asn Glu Met Val Glu Leu
Leu 115 120 125 Glu
Ala Thr Gln Tyr Arg Gln Val Thr Lys Met Thr Arg Pro Gly Glu 130
135 140 Phe Thr Val Gln Ala Lys
Ser Ile Glu Met Ile Arg Arg Pro Phe Asp 145 150
155 160 Phe Pro Asp Ser Lys Glu Gly Gln Val Arg Ala
Arg Leu Thr Phe Asp 165 170
175 Gly Asp His Leu Asp Thr Ile Glu Asn Met Asp Asn Asn Arg Gln Phe
180 185 190 Gly Phe
Phe Arg Leu Asp Pro Arg Leu Ile Thr Met Leu Ser Ser Pro 195
200 205 Asn Gly Glu Gln Arg Leu Phe
Val Pro Arg Ser Gly Phe Pro Asp Leu 210 215
220 Leu Val Asp Thr Leu Leu Ala Thr Glu Asp Arg His
Phe Tyr Glu His 225 230 235
240 Asp Gly Ile Ser Leu Tyr Ser Ile Gly Arg Ala Val Leu Ala Asn Leu
245 250 255 Thr Ala Gly
Arg Thr Val Gln Gly Ala Ser Thr Leu Thr Gln Gln Leu 260
265 270 Val Lys Asn Leu Phe Leu Ser Ser
Glu Arg Ser Tyr Trp Arg Lys Ala 275 280
285 Asn Glu Ala Tyr Met Ala Leu Ile Met Asp Ala Gly Tyr
Ser Lys Asp 290 295 300
Arg Ile Leu Glu Leu Tyr Met Asn Glu Val Tyr Leu Gly Gln Ser Gly 305
310 315 320 Asp Asn Glu Ile
Arg Gly Phe Pro Leu Ala Ser Leu Tyr Tyr Phe Gly 325
330 335 Arg Pro Val Glu Glu Leu Ser Leu Asp
Gln Gln Ala Leu Leu Val Gly 340 345
350 Met Val Lys Gly Ala Ser Ile Tyr Asn Pro Trp Arg Asn Pro
Lys Leu 355 360 365
Ala Leu Glu Arg Arg Asn Leu Val Leu Arg Leu Leu Gln Gln Gln Gln 370
375 380 Ile Ile Asp Gln Asp
Leu Tyr Asp Met Leu Ser Ala Arg Pro Leu Gly 385 390
395 400 Val Gln Pro Arg Gly Gly Val Ile Ser Pro
Gln Pro Ala Phe Met Gln 405 410
415 Met Val Arg Gln Glu Leu Gln Ala Lys Leu Gly Asp Lys Val Lys
Asp 420 425 430 Leu
Ser Gly Val Lys Ile Phe Thr Thr Phe Asp Ser Val Ala Gln Asp 435
440 445 Ala Ala Glu Lys Ala Ala
Val Glu Gly Ile Pro Val Leu Lys Lys Gln 450 455
460 Arg Lys Leu Ser Asp Leu Glu Thr Ala Ile Val
Val Val Asp Arg Phe 465 470 475
480 Ser Gly Glu Val Arg Ala Met Val Gly Gly Ala Glu Pro Gln Phe Ala
485 490 495 Gly Tyr
Asn Arg Ala Met Gln Ala Arg Arg Ser Ile Gly Ser Leu Ala 500
505 510 Lys Pro Ala Thr Tyr Leu Thr
Ala Leu Ser Gln Pro Lys Leu Tyr Arg 515 520
525 Leu Asn Thr Trp Ile Arg His Ala Pro Ile Ala Leu
Arg Gln Pro Asn 530 535 540
Gly Gln Val Trp Ser Pro Gln Asn Asp Asp Arg Arg Tyr Ser Glu Thr 545
550 555 560 Gly Lys Val
Met Leu Val Asp Ala Leu Thr Arg Ser Met Asn Val Pro 565
570 575 Thr Val Asn Leu Gly Met Ala Leu
Gly Leu Pro Ala Val Thr Asp Thr 580 585
590 Trp Leu Lys Leu Gly Ala Pro Lys Asp Gln Leu Asn Pro
Val Pro Ala 595 600 605
Met Leu Leu Gly Ala Leu Asn Leu Thr Pro Ile Glu Val Ala Gln Ala 610
615 620 Phe Gln Thr Ile
Ala Ser Gly Gly Asn Arg Ala Pro Leu Ser Ala Leu 625 630
635 640 Arg Ser Val Ile Ala Glu Asp Gly Thr
Val Leu Tyr Gln Ser Phe Pro 645 650
655 Gln Ala Glu Arg Ser Val Pro Ala Gln Ala Ala Tyr Met Thr
Leu Trp 660 665 670
Thr Met Gln Gln Val Val Gln Arg Gly Thr Gly Arg Gln Leu Gly Ala
675 680 685 Lys Tyr Pro Gly
Leu His Leu Ala Gly Lys Thr Gly Thr Thr Asn Asn 690
695 700 Asn Val Asp Thr Trp Phe Ala Gly
Ile Asp Gly Ser Gln Val Thr Ile 705 710
715 720 Thr Trp Val Gly Arg Asp Asn Asn Gln Pro Thr Lys
Leu Tyr Gly Ala 725 730
735 Ser Gly Ala Met Ser Ile Tyr Gln Arg Tyr Leu Ala Asn Gln Thr Pro
740 745 750 Thr Pro Leu
Val Leu Thr Pro Pro Glu Asp Val Val Asp Met Gly Val 755
760 765 Asp Tyr Asp Gly Asn Phe Val Cys
Ser Gly Gly Met Arg Thr Leu Pro 770 775
780 Val Trp Thr Thr Asp Pro Asp Ser Leu Cys Gln Gln Gly
Glu Met Met 785 790 795
800 Gln Gln Pro Thr Gly Asn Pro Phe Asp Gln Ser Thr Pro Gln Gln Gln
805 810 815 Pro Gln Gln Gln
Gln Gln Gln Pro Ala Gln Gln Glu Lys Lys Asp Ser 820
825 830 Asp Gly Val Ala Gly Trp Ile Lys Asp
Met Phe Gly Ser Asn 835 840 845
3844PRTEscherichia
coliTRANSMEMBRANE(64)..(87)DOMAIN(194)..(410)DOMAIN(444)..(732) 3Met Ala
Gly Asn Asp Arg Glu Pro Ile Gly Arg Lys Gly Lys Pro Thr 1 5
10 15 Arg Pro Val Lys Gln Lys Val
Ser Arg Arg Arg Tyr Glu Asp Asp Asp 20 25
30 Asp Tyr Asp Asp Tyr Asp Asp Tyr Glu Asp Glu Glu
Pro Met Pro Arg 35 40 45
Lys Gly Lys Gly Lys Gly Lys Gly Arg Lys Pro Arg Gly Lys Arg Gly
50 55 60 Trp Leu Trp
Leu Leu Leu Lys Leu Ala Ile Val Phe Ala Val Leu Ile 65
70 75 80 Ala Ile Tyr Gly Val Tyr Leu
Asp Gln Lys Ile Arg Ser Arg Ile Asp 85
90 95 Gly Lys Val Trp Gln Leu Pro Ala Ala Val Tyr
Gly Arg Met Val Asn 100 105
110 Leu Glu Pro Asp Met Thr Ile Ser Lys Asn Glu Met Val Lys Leu
Leu 115 120 125 Glu
Ala Thr Gln Tyr Arg Gln Val Ser Lys Met Thr Arg Pro Gly Glu 130
135 140 Phe Thr Val Gln Ala Asn
Ser Ile Glu Met Ile Arg Arg Pro Phe Asp 145 150
155 160 Phe Pro Asp Ser Lys Glu Gly Gln Val Arg Ala
Arg Leu Thr Phe Asp 165 170
175 Gly Asp His Leu Ala Thr Ile Val Asn Met Glu Asn Asn Arg Gln Phe
180 185 190 Gly Phe
Phe Arg Leu Asp Pro Arg Leu Ile Thr Met Ile Ser Ser Pro 195
200 205 Asn Gly Glu Gln Arg Leu Phe
Val Pro Arg Ser Gly Phe Pro Asp Leu 210 215
220 Leu Val Asp Thr Leu Leu Ala Thr Glu Asp Arg His
Phe Tyr Glu His 225 230 235
240 Asp Gly Ile Ser Leu Tyr Ser Ile Gly Arg Ala Val Leu Ala Asn Leu
245 250 255 Thr Ala Gly
Arg Thr Val Gln Gly Ala Ser Thr Leu Thr Gln Gln Leu 260
265 270 Val Lys Asn Leu Phe Leu Ser Ser
Glu Arg Ser Tyr Trp Arg Lys Ala 275 280
285 Asn Glu Ala Tyr Met Ala Leu Ile Met Asp Ala Arg Tyr
Ser Lys Asp 290 295 300
Arg Ile Leu Glu Leu Tyr Met Asn Glu Val Tyr Leu Gly Gln Ser Gly 305
310 315 320 Asp Asn Glu Ile
Arg Gly Phe Pro Leu Ala Ser Leu Tyr Tyr Phe Gly 325
330 335 Arg Pro Val Glu Glu Leu Ser Leu Asp
Gln Gln Ala Leu Leu Val Gly 340 345
350 Met Val Lys Gly Ala Ser Ile Tyr Asn Pro Trp Arg Asn Pro
Lys Leu 355 360 365
Ala Leu Glu Arg Arg Asn Leu Val Leu Arg Leu Leu Gln Gln Gln Gln 370
375 380 Ile Ile Asp Gln Glu
Leu Tyr Asp Met Leu Ser Ala Arg Pro Leu Gly 385 390
395 400 Val Gln Pro Arg Gly Gly Val Ile Ser Pro
Gln Pro Ala Phe Met Gln 405 410
415 Leu Val Arg Gln Glu Leu Gln Ala Lys Leu Gly Asp Lys Val Lys
Asp 420 425 430 Leu
Ser Gly Val Lys Ile Phe Thr Thr Phe Asp Ser Val Ala Gln Asp 435
440 445 Ala Ala Glu Lys Ala Ala
Val Glu Gly Ile Pro Ala Leu Lys Lys Gln 450 455
460 Arg Lys Leu Ser Asp Leu Glu Thr Ala Ile Val
Val Val Asp Arg Phe 465 470 475
480 Ser Gly Glu Val Arg Ala Met Val Gly Gly Ser Glu Pro Gln Phe Ala
485 490 495 Gly Tyr
Asn Arg Ala Met Gln Ala Arg Arg Ser Ile Gly Ser Leu Ala 500
505 510 Lys Pro Ala Thr Tyr Leu Thr
Ala Leu Ser Gln Pro Lys Ile Tyr Arg 515 520
525 Leu Asn Thr Trp Ile Ala Asp Ala Pro Ile Ala Leu
Arg Gln Pro Asn 530 535 540
Gly Gln Val Trp Ser Pro Gln Asn Asp Asp Arg Arg Tyr Ser Glu Ser 545
550 555 560 Gly Arg Val
Met Leu Val Asp Ala Leu Thr Arg Ser Met Asn Val Pro 565
570 575 Thr Val Asn Leu Gly Met Ala Leu
Gly Leu Pro Ala Val Thr Glu Thr 580 585
590 Trp Ile Lys Leu Gly Val Pro Lys Asp Gln Leu His Pro
Val Pro Ala 595 600 605
Met Leu Leu Gly Ala Leu Asn Leu Thr Pro Ile Glu Val Ala Gln Ala 610
615 620 Phe Gln Thr Ile
Ala Ser Gly Gly Asn Arg Ala Pro Leu Ser Ala Leu 625 630
635 640 Arg Ser Val Ile Ala Glu Asp Gly Lys
Val Leu Tyr Gln Ser Phe Pro 645 650
655 Gln Ala Glu Arg Ala Val Pro Ala Gln Ala Ala Tyr Leu Thr
Leu Trp 660 665 670
Thr Met Gln Gln Val Val Gln Arg Gly Thr Gly Arg Gln Leu Gly Ala
675 680 685 Lys Tyr Pro Asn
Leu His Leu Ala Gly Lys Thr Gly Thr Thr Asn Asn 690
695 700 Asn Val Asp Thr Trp Phe Ala Gly
Ile Asp Gly Ser Thr Val Thr Ile 705 710
715 720 Thr Trp Val Gly Arg Asp Asn Asn Gln Pro Thr Lys
Leu Tyr Gly Ala 725 730
735 Ser Gly Ala Met Ser Ile Tyr Gln Arg Tyr Leu Ala Asn Gln Thr Pro
740 745 750 Thr Pro Leu
Asn Leu Val Pro Pro Glu Asp Ile Ala Asp Met Gly Val 755
760 765 Asp Tyr Asp Gly Asn Phe Val Cys
Ser Gly Gly Met Arg Ile Leu Pro 770 775
780 Val Trp Thr Ser Asp Pro Gln Ser Leu Cys Gln Gln Ser
Glu Met Gln 785 790 795
800 Gln Gln Pro Ser Gly Asn Pro Phe Asp Gln Ser Ser Gln Pro Gln Gln
805 810 815 Gln Pro Gln Gln
Gln Pro Ala Gln Gln Glu Gln Lys Asp Ser Asp Gly 820
825 830 Val Ala Gly Trp Ile Lys Asp Met Phe
Gly Ser Asn 835 840
4781PRTHaemophilus
influenzaeTRANSMEMBRANE(20)..(44)DOMAIN(150)..(364)DOMAIN(400)..(686)
4Met Thr Ser Gln His Ser Ala Lys Lys Ser Ser Lys Asn Thr Pro Lys 1
5 10 15 Asn Asn Arg Thr
Phe Lys Gly Phe Leu Leu Lys Phe Ser Phe Thr Ala 20
25 30 Leu Val Leu Thr Ile Phe Tyr Gly Gly
Tyr Leu Asp Trp Gln Ile Arg 35 40
45 Ser Lys Met Asp Gly Gln Ile Trp His Leu Pro Ala Glu Val
Tyr Ser 50 55 60
Arg Leu Glu Ser Val Lys Ile Ala Asp Asn Leu Ala Phe Asp Glu Val 65
70 75 80 Ile Gln Ile Leu Leu
Asp Asn Glu Tyr Arg Gln Thr Thr Met Val Ala 85
90 95 Ala Pro Gly Asp Phe Lys Leu Glu Asp Asp
Ser Ile Val Ile Leu Arg 100 105
110 Arg Ala Phe Pro Phe Pro Asp Lys Ala Glu Pro Gln Arg Val Leu
Arg 115 120 125 Leu
Arg Phe Ser His Asn Lys Leu Ser Arg Ile Glu Asp Leu Val Thr 130
135 140 Val Lys Thr Val Asp Glu
Phe Arg Leu Ala Pro Lys Leu Ile Ala Met 145 150
155 160 Leu Gln Ser Asp Asn Glu Asp Arg Leu Ala Ile
Pro Leu Gln Asn Tyr 165 170
175 Pro Arg Leu Leu Ile Asp Thr Leu Ile Leu Thr Glu Asp Arg Arg Phe
180 185 190 Tyr Glu
His Asn Gly Ile Asn Pro Val Gly Ile Leu Arg Ala Leu Ile 195
200 205 Ala Asn Ile Arg Ala Gly Gln
Thr Val Gln Gly Gly Ser Thr Leu Thr 210 215
220 Gln Gln Leu Val Lys Asn Leu Phe Leu Ser Arg Glu
Arg Thr Ile Thr 225 230 235
240 Arg Lys Ala Asn Glu Ala Leu Met Ser Leu Val Leu Asp Trp Arg Tyr
245 250 255 Asp Lys Asn
Arg Ile Leu Glu Thr Tyr Leu Asn Glu Ile Tyr Leu Gly 260
265 270 Gln Asn Gly Asp Thr Gln Ile His
Gly Phe Glu Leu Ala Ser Gln Phe 275 280
285 Tyr Phe Gly Arg Ser Ile Arg Glu Ile Ser Leu Asp Gln
Ile Ala Leu 290 295 300
Leu Val Gly Met Val Lys Gly Pro Ser Leu Tyr Asn Pro Trp Arg Asn 305
310 315 320 Pro Gln Asn Ala
Leu Glu Arg Arg Asn Ile Val Leu Arg Leu Met Leu 325
330 335 Glu His Lys Met Ile Gly Asp Glu Leu
Tyr Gln Leu Leu Ser Gln Arg 340 345
350 Pro Leu Gly Val Gln Lys Lys Gly Gln Ile Ser Arg Lys Tyr
Pro Ala 355 360 365
Phe Ile Gln Thr Leu Gln Ala Asp Leu Arg Arg Glu Leu Gly Glu His 370
375 380 Lys Ile Ser Ser Leu
Leu Gly Ala Arg Ile Phe Ser Thr Met Asp Leu 385 390
395 400 Lys Gln Gln Ala Gln Ala Glu Asn Ala Val
Val Asn Thr Val Ser Gln 405 410
415 Leu Gln Leu Lys Met Lys Asn Pro His Leu Glu Gly Ala Met Ile
Ile 420 425 430 Thr
Asp Tyr Arg Thr Gly Glu Ile Arg Ala Val Val Gly Gly Leu Gln 435
440 445 Thr Gln Tyr Ala Gly Phe
Asn Arg Ala Leu Met Ala Lys Arg Gln Ile 450 455
460 Gly Ser Leu Val Lys Pro Ser Ile Tyr Leu Thr
Ala Leu Ser Asn Pro 465 470 475
480 Glu Gln Phe Arg Leu Asn Thr Pro Ile Asn Asn Gln Pro Ile Thr Ile
485 490 495 Asn Val
Lys Gly Ser Pro Pro Trp Gln Pro Arg Asn Tyr Asp Lys Lys 500
505 510 Tyr Ser Asp Ser Val Met Leu
Met Asp Ala Leu Ala Arg Ser Leu Asn 515 520
525 Ile Pro Thr Val Asn Ile Gly Met Lys Val Gly Leu
Ser Lys Val Ile 530 535 540
Asp Thr Gln Lys Ala Met Gly Trp Asp Asn Val Glu Ile Pro Lys Val 545
550 555 560 Pro Ala Met
Leu Leu Gly Ser Tyr Ser Ile Ser Pro Tyr Asp Val Thr 565
570 575 Lys Leu Tyr Gln Thr Leu Ala Asn
Gln Gly Gly Arg Ile Ala Leu Thr 580 585
590 Thr Val Asp Ser Ile Ala Asp Arg Gln Gly Asn Leu Ile
Phe Gln His 595 600 605
Asp Lys Ser Ala Lys Gln Val Val Pro Gln Glu Ala Ala Phe Gln Thr 610
615 620 Leu Phe Ala Met
Gln Gln Thr Val Glu Arg Gly Thr Ala Arg Ser Leu 625 630
635 640 Gln Lys Asp Tyr Ala Asp Leu His Leu
Ala Gly Lys Thr Gly Thr Thr 645 650
655 Asn Glu Ser Arg Asp Thr Trp Phe Val Gly Ile Asp Gly Lys
Asn Ile 660 665 670
Ser Thr Val Trp Leu Gly Arg Asp Asp Asn Gly Glu Thr Lys Leu Thr
675 680 685 Gly Ala Ser Gly
Ala Leu Gln Ile Tyr Lys Asp Tyr Leu Asn Arg Thr 690
695 700 Asn Ile Glu Lys Leu Ala Ile Thr
Pro Pro Thr Thr Val Lys Trp Val 705 710
715 720 Gly Ile Asn Gln Tyr Gly Asp Trp Asp Cys Glu Ser
Tyr Arg Thr Ile 725 730
735 Pro Val Trp Leu Asn Asn Gly Gln Asn Phe Cys Gly Glu Thr Ser Ser
740 745 750 Pro Ser Leu
Thr Pro Thr Thr Glu Thr Glu Thr Pro Pro Gln Glu Ser 755
760 765 Leu Trp Asp Val Leu Asp Asn Pro
Asn Pro Pro Ala Gln 770 775 780
5660PRTHelicobacter
pyloriTRANSMEMBRANE(5)..(20)DOMAIN(49)..(252)DOMAIN(290)..(590) 5Met Leu
Lys Lys Ile Phe Tyr Gly Phe Ile Val Leu Phe Leu Ile Ile 1 5
10 15 Val Gly Leu Leu Ala Val Leu
Val Ala Gln Val Trp Val Thr Thr Asp 20 25
30 Lys Asp Ile Ala Lys Ile Lys Asp Tyr Arg Pro Ser
Val Ala Ser Gln 35 40 45
Ile Leu Asp Arg Lys Gly Arg Leu Ile Ala Asn Ile Tyr Asp Lys Glu
50 55 60 Phe Arg Phe
Tyr Ala Arg Phe Glu Glu Ile Pro Pro Arg Phe Val Glu 65
70 75 80 Ser Leu Leu Ala Val Glu Asp
Thr Leu Phe Phe Glu His Gly Gly Ile 85
90 95 Asn Leu Asp Ala Val Met Arg Ala Met Ile Lys
Asn Ala Lys Ser Gly 100 105
110 Arg Tyr Thr Glu Gly Gly Ser Thr Leu Thr Gln Gln Leu Val Lys
Asn 115 120 125 Met
Val Leu Thr Arg Glu Lys Thr Leu Thr Arg Lys Leu Lys Glu Ala 130
135 140 Ile Ile Ser Ile Arg Ile
Glu Lys Val Leu Ser Lys Glu Glu Ile Leu 145 150
155 160 Glu Arg Tyr Leu Asn Gln Thr Phe Phe Gly His
Gly Tyr Tyr Gly Val 165 170
175 Lys Thr Ala Ser Leu Gly Tyr Phe Lys Lys Pro Leu Asp Lys Leu Thr
180 185 190 Leu Lys
Glu Ile Thr Met Leu Val Ala Leu Pro Arg Ala Pro Ser Phe 195
200 205 Tyr Asp Pro Thr Lys Asn Leu
Glu Phe Ser Leu Ser Arg Ala Asn Asp 210 215
220 Ile Leu Arg Arg Leu Tyr Ser Leu Gly Trp Ile Ser
Ser Asn Glu Leu 225 230 235
240 Lys Ser Ala Leu Asn Glu Val Pro Ile Val Tyr Asn Gln Thr Ser Thr
245 250 255 Gln Asn Ile
Ala Pro Tyr Val Val Asp Glu Val Leu Lys Gln Leu Asp 260
265 270 Gln Leu Asp Gly Leu Lys Thr Gln
Gly Tyr Thr Ile Lys Leu Thr Ile 275 280
285 Asp Leu Asp Tyr Gln Arg Leu Ala Leu Glu Ser Leu Arg
Phe Gly His 290 295 300
Gln Lys Ile Leu Glu Lys Ile Ala Lys Glu Lys Pro Lys Thr Asn Ala 305
310 315 320 Ser Asn Asp Lys
Asp Glu Asp Asn Leu Asn Ala Ser Met Ile Val Thr 325
330 335 Glu Thr Ser Thr Gly Lys Ile Leu Ala
Leu Val Gly Gly Ile Asp Tyr 340 345
350 Lys Lys Ser Ala Phe Asn Arg Ala Thr Gln Ala Lys Arg Gln
Phe Gly 355 360 365
Ser Ala Ile Lys Pro Phe Val Tyr Gln Ile Ala Phe Asp Asn Gly Tyr 370
375 380 Ser Thr Thr Ser Lys
Ile Pro Asp Thr Ala Arg Asn Phe Glu Asn Gly 385 390
395 400 Asn Tyr Ser Lys Asn Ser Val Gln Asn His
Ala Trp His Pro Ser Asn 405 410
415 Tyr Thr Arg Lys Phe Leu Gly Leu Val Thr Leu Gln Glu Ala Leu
Ser 420 425 430 His
Ser Leu Asn Leu Ala Thr Ile Asn Leu Ser Asp Gln Leu Gly Phe 435
440 445 Glu Lys Ile Tyr Gln Ser
Leu Ser Asp Met Gly Phe Lys Asn Leu Pro 450 455
460 Lys Asp Leu Ser Ile Val Leu Gly Ser Phe Ala
Ile Ser Pro Ile Asp 465 470 475
480 Ala Ala Glu Lys Tyr Ser Leu Phe Ser Asn Tyr Gly Thr Met Leu Lys
485 490 495 Pro Met
Leu Ile Glu Ser Ile Thr Asn Gln Gln Asn Glu Val Lys Thr 500
505 510 Phe Thr Pro Ile Glu Thr Lys
Lys Ile Thr Ser Lys Glu Gln Ala Phe 515 520
525 Leu Thr Leu Ser Ala Leu Met Asp Ala Val Glu Asn
Gly Thr Gly Ser 530 535 540
Leu Ala Arg Ile Lys Gly Leu Glu Ile Ala Gly Lys Thr Gly Thr Ser 545
550 555 560 Asn Asn Asn
Ile Asp Ala Trp Phe Ile Gly Phe Thr Pro Thr Leu Gln 565
570 575 Ser Val Ile Trp Phe Gly Arg Asp
Asp Asn Thr Pro Ile Gly Lys Gly 580 585
590 Ala Thr Gly Gly Val Val Ser Ala Pro Val Tyr Ser Tyr
Phe Met Arg 595 600 605
Asn Ile Leu Ala Ile Glu Pro Ser Leu Lys Arg Lys Phe Asp Val Pro 610
615 620 Lys Gly Leu Arg
Lys Glu Ile Val Asp Lys Ile Pro Tyr Tyr Ser Ser 625 630
635 640 Pro Asn Ser Ile Thr Pro Thr Pro Lys
Lys Thr Asp Asp Ser Glu Glu 645 650
655 Arg Leu Leu Phe 660 6850PRTKlebsiella
pneumoniaeTRANSMEMBRANE(64)..(87)DOMAIN(254)..(410)DOMAIN(444)..(733)
6Met Ala Gly Asp Asp Arg Glu Pro Ile Gly Arg Lys Gly Arg Pro Ser 1
5 10 15 Arg Pro Thr Lys
Gln Lys Val Thr Arg Arg Arg Val Arg Glu Glu Asp 20
25 30 Tyr Asp Asp Glu Tyr Asp Asp Asp Asp
Tyr Glu Asp Glu Lys Pro Val 35 40
45 Pro Arg Lys Ala Lys Gly Lys Gly Gly Lys Pro Arg Arg Lys
Arg Ser 50 55 60
Trp Leu Trp Leu Leu Val Lys Leu Gly Ile Val Phe Ala Val Leu Ile 65
70 75 80 Ala Ala Tyr Gly Val
Tyr Leu Asp Gln Lys Ile Arg Ser Arg Ile Asp 85
90 95 Gly Lys Val Trp Glu Leu Pro Ala Ala Val
Tyr Gly Arg Met Val Asn 100 105
110 Leu Glu Pro Asp Met Gln Ile Ser Lys Asn Glu Met Val Arg Leu
Leu 115 120 125 Asn
Ala Thr Gln Tyr Arg Gln Val Ser Ala Met Thr Arg Pro Gly Glu 130
135 140 Tyr Thr Val Gln Ala Asn
Ser Ile Glu Met Ile Arg Arg Pro Phe Asp 145 150
155 160 Phe Pro Asp Ser Lys Glu Gly Gln Val Arg Ala
Arg Leu Thr Phe Asp 165 170
175 Gly Asp His Leu Glu Thr Ile Glu Asn Met Asp Asn Asn Arg Gln Phe
180 185 190 Gly Phe
Phe Arg Leu Asp Pro Arg Leu Ile Thr Met Leu Gln Ser Pro 195
200 205 Asn Gly Glu Gln Arg Leu Phe
Val Lys Arg Ser Gly Phe Pro Asp Leu 210 215
220 Leu Val Asp Thr Leu Leu Ala Thr Glu Asp Arg His
Phe Tyr Glu His 225 230 235
240 Asp Gly Ile Ser Leu Tyr Ser Ile Gly Arg Ala Val Leu Ala Asn Leu
245 250 255 Thr Ala Gly
Arg Thr Val Gln Gly Ala Ser Thr Leu Thr Gln Gln Leu 260
265 270 Val Lys Asn Leu Phe Leu Ser Ser
Glu Arg Ser Tyr Trp Arg Lys Ala 275 280
285 Asn Glu Ala Tyr Met Ala Leu Ile Val Asp Ala Arg Tyr
Ser Lys Asp 290 295 300
Arg Ile Leu Glu Leu Tyr Met Asn Glu Val Tyr Leu Gly Gln Ser Gly 305
310 315 320 Asp Asn Glu Ile
Arg Gly Phe Pro Leu Ala Ser Leu Tyr Tyr Phe Gly 325
330 335 Arg Pro Val Glu Glu Leu Ser Leu Asp
Gln Gln Ala Leu Leu Val Gly 340 345
350 Met Val Lys Gly Ala Ser Val Tyr Asn Pro Trp Arg Asn Pro
Lys Leu 355 360 365
Ala Leu Glu Arg Arg Asn Leu Val Leu Arg Leu Leu Gln Gln Gln Gln 370
375 380 Val Ile Asp Gln Glu
Leu Tyr Asp Met Leu Ser Ala Arg Pro Leu Gly 385 390
395 400 Val Gln Pro Arg Gly Gly Val Ile Ser Pro
Gln Pro Ala Phe Met Gln 405 410
415 Met Val Arg Gln Glu Leu Gln Ala Lys Leu Gly Asp Lys Val Lys
Asp 420 425 430 Leu
Ser Gly Val Lys Ile Phe Thr Thr Phe Asp Ser Val Ala Gln Asp 435
440 445 Ala Ala Glu Lys Ala Ala
Ser Glu Gly Ile Pro Val Leu Lys Lys Gln 450 455
460 Arg Lys Leu Ala Asp Leu Glu Thr Ala Met Val
Val Val Asp Arg Phe 465 470 475
480 Thr Gly Glu Val Arg Ala Met Val Gly Gly Ala Glu Pro Gln Phe Ala
485 490 495 Gly Tyr
Asn Arg Ala Met Gln Ala Arg Arg Ser Ile Gly Ser Leu Ala 500
505 510 Lys Pro Ala Thr Tyr Leu Thr
Ala Leu Ser Gln Pro Asn Gln Tyr Arg 515 520
525 Leu Asn Thr Trp Ile Ala Asp Ala Pro Val Thr Ile
Arg Leu Ser Asn 530 535 540
Gly Gln Thr Trp Ser Pro Gln Asn Asp Asp Arg Arg Phe Ser Gly Gln 545
550 555 560 Val Met Leu
Val Asp Ala Leu Thr Arg Ser Met Asn Val Pro Thr Val 565
570 575 Asn Leu Gly Met Ala Leu Gly Leu
Pro Ala Val Val Asp Thr Trp Thr 580 585
590 Lys Leu Gly Ala Pro Lys Asn Gln Leu Asn Ala Val Pro
Ser Met Leu 595 600 605
Leu Gly Ala Leu Asn Leu Thr Pro Ile Glu Val Ala Gln Ala Phe Gln 610
615 620 Thr Ile Ala Ser
Gly Gly Asn Arg Ala Pro Leu Ser Ala Leu Arg Ser 625 630
635 640 Val Ile Ala Glu Asp Gly Thr Val Leu
Tyr Gln Ser Tyr Pro Gln Ala 645 650
655 Glu Arg Ala Val Pro Ala Gln Ala Ala Tyr Met Thr Leu Trp
Thr Met 660 665 670
Gln Gln Val Val Gln Arg Gly Thr Gly Arg Gln Leu Gly Ala Lys Tyr
675 680 685 Pro Gly Leu His
Leu Ala Gly Lys Thr Gly Thr Thr Asn Asn Asn Val 690
695 700 Asp Thr Trp Phe Ala Gly Ile Asp
Gly Ser Gln Val Thr Ile Thr Trp 705 710
715 720 Val Gly Arg Asp Asn Asn Gln Pro Thr Lys Leu Tyr
Gly Ala Ser Gly 725 730
735 Ala Met Ser Ile Tyr Gln Arg Tyr Leu Ala Asn Gln Thr Pro Thr Pro
740 745 750 Leu Val Leu
Thr Val Pro Glu Asp Val Val Asp Met Gly Val Asp Ser 755
760 765 Asn Gly Asn Phe Val Cys Ser Gly
Gly Met Arg Ser Leu Pro Val Trp 770 775
780 Thr Thr Gln Pro Asp Ala Leu Cys Arg Gln Gly Glu Met
Met Gln Gln 785 790 795
800 Gln Gln Leu Gln Gln Gln Glu Ala Lys Asn Pro Phe Asn Gln Ser Gly
805 810 815 Gln Gln Pro Pro
Pro Gln Gln Gln Gln Gln Gln Gln Pro Pro Lys Gln 820
825 830 Gln Glu Lys Ser Asp Gly Val Ala Gly
Trp Ile Lys Asp Met Phe Gly 835 840
845 Ser Asn 850 7798PRTNeisseria
gonorrhoeaeTRANSMEMBRANE(19)..(24)DOMAIN(52)..(263)DOMAIN(295)..(745)
7Met Ile Lys Lys Ile Leu Thr Thr Cys Phe Gly Leu Phe Phe Gly Phe 1
5 10 15 Cys Val Phe Gly
Val Gly Leu Val Ala Ile Ala Ile Leu Val Thr Tyr 20
25 30 Pro Lys Leu Pro Ser Leu Asp Ser Leu
Gln His Tyr Gln Pro Lys Met 35 40
45 Pro Leu Thr Ile Tyr Ser Ala Asp Gly Glu Val Ile Gly Met
Tyr Gly 50 55 60
Glu Gln Arg Arg Glu Phe Thr Lys Ile Gly Asp Phe Pro Glu Val Leu 65
70 75 80 Arg Asn Ala Val Ile
Ala Ala Glu Asp Lys Arg Phe Tyr Arg His Trp 85
90 95 Gly Val Asp Val Trp Gly Val Ala Arg Ala
Ala Val Gly Asn Val Val 100 105
110 Ser Gly Ser Val Gln Ser Gly Ala Ser Thr Ile Thr Gln Gln Val
Ala 115 120 125 Lys
Asn Phe Tyr Leu Ser Ser Glu Lys Thr Phe Thr Arg Lys Phe Asn 130
135 140 Glu Val Leu Leu Ala Tyr
Lys Ile Glu Gln Ser Leu Ser Lys Asp Lys 145 150
155 160 Ile Leu Glu Leu Tyr Phe Asn Gln Ile Tyr Leu
Gly Gln Arg Ala Tyr 165 170
175 Gly Phe Ala Ser Ala Ala Gln Ile Tyr Phe Asn Lys Asn Val Arg Asp
180 185 190 Leu Thr
Leu Ala Glu Ala Ala Met Leu Ala Gly Leu Pro Lys Ala Pro 195
200 205 Ser Ala Tyr Asn Pro Ile Val
Asn Pro Glu Arg Ala Lys Leu Arg Gln 210 215
220 Lys Tyr Ile Leu Asn Asn Met Leu Glu Glu Lys Met
Ile Thr Val Gln 225 230 235
240 Gln Arg Asp Gln Ala Leu Asn Glu Glu Leu His Tyr Glu Arg Phe Val
245 250 255 Arg Lys Ile
Asp Gln Ser Ala Leu Tyr Val Ala Glu Met Val Arg Arg 260
265 270 Glu Leu Tyr Glu Lys Tyr Gly Glu
Asp Ala Tyr Thr Gln Gly Phe Lys 275 280
285 Val Tyr Thr Thr Val Arg Thr Asp His Gln Lys Ala Ala
Thr Glu Ala 290 295 300
Leu Arg Lys Ala Leu Arg Asn Phe Asp Arg Gly Ser Ser Tyr Arg Gly 305
310 315 320 Ala Glu Asn Tyr
Ile Asp Leu Ser Lys Ser Glu Asp Val Glu Glu Thr 325
330 335 Val Ser Gln Tyr Leu Ser Gly Leu Tyr
Thr Val Asp Lys Met Val Pro 340 345
350 Ala Val Val Leu Asp Val Thr Lys Lys Lys Asn Val Val Ile
Gln Leu 355 360 365
Pro Gly Gly Arg Arg Val Ala Leu Asp Arg Arg Ala Leu Gly Phe Ala 370
375 380 Ala Arg Ala Val Asp
Asn Glu Lys Met Gly Glu Asp Arg Ile Arg Arg 385 390
395 400 Gly Ala Val Ile Arg Val Lys Asn Asn Gly
Gly Arg Trp Ala Val Val 405 410
415 Gln Glu Pro Leu Leu Gln Gly Ala Leu Val Ser Leu Asp Ala Lys
Thr 420 425 430 Gly
Ala Val Arg Ala Leu Val Gly Gly Tyr Asp Phe His Ser Lys Thr 435
440 445 Phe Asn Arg Ala Val Gln
Ala Met Arg Gln Pro Gly Ser Thr Phe Lys 450 455
460 Pro Phe Val Tyr Ser Ala Ala Leu Ser Lys Gly
Met Thr Ala Ser Thr 465 470 475
480 Val Val Asn Asp Ala Pro Ile Ser Leu Pro Gly Lys Gly Pro Asn Gly
485 490 495 Ser Val
Trp Thr Pro Lys Asn Ser Asp Gly Arg Tyr Ser Gly Tyr Ile 500
505 510 Thr Leu Arg Gln Ala Leu Thr
Ala Ser Lys Asn Met Val Ser Ile Arg 515 520
525 Ile Leu Met Ser Ile Gly Val Gly Tyr Ala Gln Gln
Tyr Ile Arg Arg 530 535 540
Phe Gly Phe Arg Pro Ser Glu Leu Pro Ala Ser Leu Ser Met Ala Leu 545
550 555 560 Gly Thr Gly
Glu Thr Thr Pro Leu Lys Val Ala Glu Ala Tyr Ser Val 565
570 575 Phe Ala Asn Gly Gly Tyr Arg Val
Ser Ser His Val Ile Asp Lys Ile 580 585
590 Tyr Asp Arg Asp Gly Arg Leu Arg Ala Gln Met Gln Pro
Leu Val Ala 595 600 605
Gly Gln Asn Ala Pro Gln Ala Ile Asp Pro Arg Asn Ala Tyr Ile Met 610
615 620 Tyr Lys Ile Met
Gln Asp Val Val Arg Val Gly Thr Ala Arg Gly Ala 625 630
635 640 Ala Ala Leu Gly Arg Thr Asp Ile Ala
Gly Lys Thr Gly Thr Thr Asn 645 650
655 Asp Asn Lys Asp Ala Trp Phe Val Gly Phe Asn Pro Asp Val
Val Thr 660 665 670
Ala Val Tyr Ile Gly Phe Asp Lys Pro Lys Ser Met Gly Arg Ala Gly
675 680 685 Tyr Gly Gly Thr
Ile Ala Val Pro Val Trp Val Asp Tyr Met Arg Phe 690
695 700 Ala Leu Lys Gly Lys Gln Gly Lys
Gly Met Lys Met Pro Glu Gly Val 705 710
715 720 Val Ser Ser Asn Gly Glu Tyr Tyr Met Lys Glu Arg
Met Val Thr Asp 725 730
735 Pro Gly Leu Met Leu Asp Asn Ser Gly Ile Ala Pro Gln Pro Ser Arg
740 745 750 Arg Ala Lys
Glu Asp Asp Glu Ala Ala Val Glu Asn Glu Gln Gln Gly 755
760 765 Arg Ser Asp Glu Thr Arg Gln Asp
Val Gln Glu Thr Pro Val Leu Pro 770 775
780 Ser Asn Thr Asp Ser Lys Gln Gln Gln Leu Asp Ser Leu
Phe 785 790 795
8774PRTPseudomonas
aeruginosaTRANSMEMBRANE(22)..(45)DOMAIN(151)..(367)DOMAIN(402)..(701)
8Met Thr Arg Pro Arg Ser Pro Arg Ser Arg Asn Ser Lys Ala Arg Pro 1
5 10 15 Ala Pro Gly Leu
Asn Lys Trp Leu Ser Trp Ala Leu Lys Leu Gly Leu 20
25 30 Val Gly Leu Val Leu Leu Ala Gly Phe
Ala Ile Tyr Leu Asp Ala Val 35 40
45 Val Gln Glu Lys Phe Ser Gly Arg Arg Trp Thr Ile Pro Ala
Lys Val 50 55 60
Tyr Ala Arg Pro Leu Glu Leu Phe Asn Gly Leu Lys Leu Ser Arg Glu 65
70 75 80 Asp Phe Leu Arg Glu
Leu Asp Ala Leu Gly Tyr Arg Arg Glu Pro Ser 85
90 95 Val Ser Gly Pro Gly Thr Val Ser Val Ala
Ala Ser Ala Val Glu Leu 100 105
110 Asn Thr Arg Gly Phe Gln Phe Tyr Glu Gly Ala Glu Pro Ala Gln
Arg 115 120 125 Val
Arg Val Arg Phe Asn Gly Asn Tyr Val Ser Gly Leu Ser Gln Ala 130
135 140 Asn Gly Lys Glu Leu Ala
Val Ala Arg Leu Glu Pro Leu Leu Ile Gly 145 150
155 160 Gly Leu Tyr Pro Ala His His Glu Asp Arg Ile
Leu Val Lys Leu Asp 165 170
175 Gln Val Pro Thr Tyr Leu Ile Asp Thr Leu Val Ala Val Glu Asp Arg
180 185 190 Asp Phe
Trp Asn His His Gly Val Ser Leu Lys Ser Val Ala Arg Ala 195
200 205 Val Trp Val Asn Thr Thr Ala
Gly Gln Leu Arg Gln Gly Gly Ser Thr 210 215
220 Leu Thr Gln Gln Leu Val Lys Asn Phe Phe Leu Ser
Asn Glu Arg Ser 225 230 235
240 Leu Ser Arg Lys Ile Asn Glu Ala Met Met Ala Val Leu Leu Glu Leu
245 250 255 His Tyr Asp
Lys Arg Asp Ile Leu Glu Ser Tyr Leu Asn Glu Val Phe 260
265 270 Leu Gly Gln Asp Gly Gln Arg Ala
Ile His Gly Phe Gly Leu Ala Ser 275 280
285 Gln Tyr Phe Phe Ser Gln Pro Leu Ala Glu Leu Lys Leu
Asp Gln Val 290 295 300
Ala Leu Leu Val Gly Met Val Lys Gly Pro Ser Tyr Phe Asn Pro Arg 305
310 315 320 Arg Tyr Pro Asp
Arg Ala Leu Ala Arg Arg Asn Leu Val Leu Asp Val 325
330 335 Leu Ala Glu Gln Gly Val Ala Thr Gln
Gln Glu Val Asp Ala Ala Lys 340 345
350 Leu Arg Pro Leu Gly Val Thr Arg Gln Gly Ser Met Ala Asp
Ser Ser 355 360 365
Tyr Pro Ala Phe Leu Asp Leu Val Lys Arg Gln Leu Arg Gln Asp Tyr 370
375 380 Arg Asp Glu Asp Leu
Thr Glu Glu Gly Leu Arg Ile Phe Thr Ser Phe 385 390
395 400 Asp Pro Ile Leu Gln Glu Lys Ala Glu Thr
Ser Val Asn Glu Thr Leu 405 410
415 Lys Arg Leu Ser Gly Arg Lys Gly Val Asp Gln Val Glu Ala Ala
Met 420 425 430 Val
Val Thr Asn Pro Glu Thr Gly Glu Ile Gln Ala Leu Ile Gly Ser 435
440 445 Arg Asp Pro Arg Phe Ala
Gly Phe Asn Arg Ala Leu Asp Ala Val Arg 450 455
460 Pro Ile Gly Ser Leu Ile Lys Pro Ala Val Tyr
Leu Thr Ala Leu Glu 465 470 475
480 Arg Pro Ser Lys Tyr Thr Leu Thr Thr Trp Val Gln Asp Glu Pro Phe
485 490 495 Ala Val
Lys Gly Gln Asp Gly Gln Val Trp Arg Pro Gln Asn Tyr Asp 500
505 510 Arg Arg Ser His Gly Thr Ile
Phe Leu Tyr Gln Gly Leu Ala Asn Ser 515 520
525 Tyr Asn Leu Ser Thr Ala Lys Leu Gly Leu Asp Val
Gly Val Pro Asn 530 535 540
Val Leu Gln Thr Val Ala Arg Leu Gly Ile Asn Arg Asp Trp Pro Ala 545
550 555 560 Tyr Pro Ser
Met Leu Leu Gly Ala Gly Ser Leu Ser Pro Met Glu Val 565
570 575 Ala Thr Met Tyr Gln Thr Ile Ala
Ser Gly Gly Phe Asn Thr Pro Leu 580 585
590 Arg Gly Ile Arg Ser Val Leu Thr Ala Asp Gly Gln Pro
Leu Lys Arg 595 600 605
Tyr Pro Phe Gln Val Glu Gln Arg Phe Asp Ser Gly Ala Val Tyr Leu 610
615 620 Val Gln Asn Ala
Met Gln Arg Val Met Arg Glu Gly Thr Gly Arg Ser 625 630
635 640 Val Tyr Ser Gln Leu Pro Ser Ser Leu
Thr Leu Ala Gly Lys Thr Gly 645 650
655 Thr Ser Asn Asp Ser Arg Asp Ser Trp Phe Ser Gly Phe Gly
Gly Asp 660 665 670
Leu Gln Ala Val Val Trp Leu Gly Arg Asp Asp Asn Gly Lys Thr Pro
675 680 685 Leu Thr Gly Ala
Thr Gly Ala Leu Gln Val Trp Ala Ser Phe Met Arg 690
695 700 Lys Ala His Pro Gln Ser Leu Glu
Met Pro Met Pro Glu Asn Val Val 705 710
715 720 Met Ala Trp Val Asp Ala Gln Thr Gly Gln Gly Ser
Ala Ala Asp Cys 725 730
735 Pro Asn Ala Val Gln Met Pro Tyr Ile Arg Gly Ser Glu Pro Ala Gln
740 745 750 Gly Pro Gly
Cys Gly Ser Gln Asn Pro Ala Gly Glu Val Met Asp Trp 755
760 765 Val Arg Gly Trp Leu Asn 770
9840PRTSalmonella
entericaTRANSMEMBRANE(59)..(82)DOMAIN(189)..(402)DOMAIN(439)..(730) 9Met
Ala Gly Asn Asp Arg Glu Pro Ile Gly Arg Lys Gly Lys Ser Ser 1
5 10 15 Arg Pro Val Lys Gln Lys
Val Ser Arg Arg Arg Gln His Asp Asp Asp 20
25 30 Tyr Asp Asp Asp Tyr Glu Asp Glu Glu Pro
Met Pro Arg Lys Gly Lys 35 40
45 Gly Lys Gly Arg Lys Pro Arg Gly Lys Arg Gly Trp Leu Trp
Leu Leu 50 55 60
Leu Lys Leu Phe Ile Val Phe Val Val Leu Phe Ala Ile Tyr Gly Val 65
70 75 80 Tyr Leu Asp Gln Lys
Ile Arg Ser Arg Ile Asp Gly Lys Val Trp Gln 85
90 95 Leu Pro Ala Ala Val Tyr Gly Arg Met Val
Asn Leu Glu Pro Asp Met 100 105
110 Pro Val Ser Lys Asn Glu Met Val Lys Leu Leu Glu Ala Thr Gln
Tyr 115 120 125 Arg
Leu Val Thr Lys Met Thr Arg Pro Gly Glu Phe Thr Val Gln Ala 130
135 140 Asn Ser Ile Glu Met Ile
Arg Arg Pro Phe Asp Phe Pro Asp Ser Lys 145 150
155 160 Glu Gly Gln Val Arg Ala Arg Leu Thr Phe Ser
Asp Gly Arg Leu Glu 165 170
175 Thr Ile Val Asn Leu Asp Asn Asn Arg Gln Phe Gly Phe Phe Arg Leu
180 185 190 Asp Pro
Arg Leu Ile Thr Met Leu Ser Ser Pro Asn Gly Glu Gln Arg 195
200 205 Leu Phe Val Pro Arg Ser Gly
Phe Leu Asp Leu Leu Val Asp Thr Leu 210 215
220 Leu Ala Thr Glu Asp Arg His Phe Tyr Glu His Asp
Gly Ile Ser Leu 225 230 235
240 Tyr Ser Ile Gly Arg Ala Val Leu Ala Asn Leu Thr Ala Gly Arg Thr
245 250 255 Val Gln Gly
Ala Ser Thr Leu Thr Gln Gln Leu Val Lys Asn Leu Phe 260
265 270 Leu Ser Ser Glu Arg Ser Tyr Trp
Arg Lys Ala Asn Glu Ala Tyr Met 275 280
285 Ala Leu Ile Met Asp Ala Arg Tyr Ser Lys Asp Arg Ile
Leu Glu Leu 290 295 300
Tyr Met Asn Glu Val Tyr Leu Gly Gln Ser Gly Asp Asn Glu Ile Arg 305
310 315 320 Gly Phe Pro Leu
Ala Ser Leu Tyr Tyr Phe Gly Arg Pro Val Glu Glu 325
330 335 Leu Ser Leu Asp Gln Gln Ala Leu Leu
Val Gly Met Val Lys Gly Ala 340 345
350 Ser Ile Tyr Asn Pro Trp Arg Asn Pro Lys Leu Ala Leu Glu
Arg Arg 355 360 365
Asn Leu Val Leu Arg Leu Leu Gln Gln Gln Lys Ile Ile Asp Gln Glu 370
375 380 Leu Tyr Asp Met Leu
Ser Ala Arg Pro Leu Gly Val Gln Pro Arg Gly 385 390
395 400 Gly Val Ile Ser Pro Gln Pro Ala Phe Met
Gln Met Val Arg Gln Glu 405 410
415 Leu Gln Ala Lys Leu Gly Asp Lys Ile Lys Asp Leu Ser Gly Val
Lys 420 425 430 Ile
Phe Thr Thr Phe Asp Ser Val Ala Gln Asp Ala Val Glu Lys Ala 435
440 445 Val Val Glu Gly Ile Pro
Ala Leu Lys Lys Gln Arg Lys Leu Ser Asp 450 455
460 Leu Glu Thr Ala Met Val Val Val Asp Arg Phe
Ser Gly Glu Val Arg 465 470 475
480 Ala Met Val Gly Gly Ala Glu Pro Gln Tyr Ala Gly Tyr Asn Arg Ala
485 490 495 Met Gln
Ala Arg Arg Ser Ile Gly Ser Leu Ala Lys Pro Ala Thr Tyr 500
505 510 Leu Thr Ala Leu Ser Gln Pro
Asn Leu Tyr Arg Leu Asn Thr Trp Ile 515 520
525 Ala Asp Ala Pro Ile Ser Leu Arg Gln Pro Asn Gly
Gln Val Trp Ser 530 535 540
Pro Gln Asn Asp Asp Arg Arg Tyr Ser Glu Ser Gly Lys Val Met Leu 545
550 555 560 Val Asp Ala
Leu Thr Arg Ser Met Asn Val Pro Thr Val Asn Leu Gly 565
570 575 Met Ala Leu Gly Leu Pro Ala Val
Thr Asp Thr Trp Thr Lys Leu Gly 580 585
590 Val Pro Lys Asp Gln Leu Asn Pro Val Pro Ala Met Leu
Leu Gly Ala 595 600 605
Leu Asn Leu Thr Pro Ile Glu Val Ala Gln Ala Phe Gln Thr Ile Ala 610
615 620 Ser Gly Gly Asn
Arg Ala Pro Leu Ser Ala Leu Arg Ser Val Ile Ala 625 630
635 640 Glu Asp Gly Lys Val Leu Tyr Gln Ser
Tyr Pro Gln Ala Glu Arg Ala 645 650
655 Val Pro Ala Gln Ala Ala Tyr Leu Thr Leu Trp Thr Met Gln
Gln Val 660 665 670
Val Gln Arg Gly Thr Gly Arg Gln Leu Gly Ala Lys Tyr Pro Gly Leu
675 680 685 His Leu Ala Gly
Lys Thr Gly Thr Thr Asn Asn Asn Val Asp Thr Trp 690
695 700 Phe Ala Gly Ile Asp Gly Ser Gln
Val Thr Ile Thr Trp Val Gly Arg 705 710
715 720 Asp Asn Asn Gln Pro Thr Lys Leu Tyr Gly Ala Ser
Gly Ala Met Ala 725 730
735 Ile Tyr Gln Arg Tyr Leu Ala Asn Gln Thr Pro Thr Pro Leu Val Leu
740 745 750 Thr Pro Pro
Glu Asp Val Val Asp Met Gly Val Asp Tyr Asp Gly Asn 755
760 765 Phe Val Cys Ser Gly Gly Met Arg
Thr Leu Pro Val Trp Thr Asp Asp 770 775
780 Pro Asn Thr Leu Cys Gln Gln Gly Glu Met Met Gln Gln
Gln Gln Gln 785 790 795
800 Pro Ser Gly Asn Pro Phe Asp Gln Ser Ser Gln Pro Gln Gln Pro Ala
805 810 815 Gln Gln Gln Pro
Pro Lys Glu Glu Lys Ser Asp Gly Val Ala Gly Trp 820
825 830 Ile Lys Glu Met Phe Gly Gly Asn
835 840 10914PRTBacillus
subtilisTRANSMEMBRANE(28)..(50)DOMAIN(79)..(293)DOMAIN(329)..(660) 10Met
Ser Asp Gln Phe Asn Ser Arg Glu Ala Arg Arg Lys Ala Asn Ser 1
5 10 15 Lys Ser Ser Pro Ser Pro
Lys Lys Gly Lys Lys Arg Lys Lys Gly Gly 20
25 30 Leu Phe Lys Lys Thr Leu Phe Thr Leu Leu
Ile Leu Phe Val Leu Gly 35 40
45 Val Val Gly Gly Ala Val Thr Phe Ala Val Met Val Ser Asp
Ala Pro 50 55 60
Ser Leu Asp Glu Ser Lys Leu Lys Thr Pro Tyr Ser Ser Thr Ile Tyr 65
70 75 80 Asp Lys Asn Gly Lys
Glu Ile Ala Glu Val Gly Ala Glu Lys Arg Thr 85
90 95 Tyr Val Ser Ile Asp Glu Ile Pro Asp Val
Val Lys Glu Ala Phe Ile 100 105
110 Ala Thr Glu Asp Ala Arg Phe Tyr Glu His His Gly Ile Asp Pro
Val 115 120 125 Arg
Ile Gly Gly Ala Leu Val Ala Asn Phe Lys Asp Gly Phe Gly Ala 130
135 140 Glu Gly Gly Ser Thr Ile
Thr Gln Gln Val Val Lys Asn Ser Leu Leu 145 150
155 160 Ser His Gln Lys Thr Leu Lys Arg Lys Val Gln
Glu Val Trp Leu Ser 165 170
175 Ile Gln Leu Glu Arg Asn Tyr Ser Lys Asp Glu Ile Leu Glu Met Tyr
180 185 190 Leu Asn
Arg Ile Tyr Phe Ser Pro Arg Ala Tyr Gly Ile Gly Lys Ala 195
200 205 Ala Glu Glu Phe Phe Gly Val
Thr Asp Leu Ser Lys Leu Thr Val Glu 210 215
220 Gln Ala Ala Thr Leu Ala Gly Met Pro Gln Ser Pro
Thr Ala Tyr Asn 225 230 235
240 Pro Val Lys Asn Pro Asp Lys Ala Glu Lys Arg Arg Asn Ile Val Leu
245 250 255 Ser Leu Met
Lys Lys Gln Gly Phe Ile Ser Asp Ser Gln Tyr Asn Lys 260
265 270 Ala Lys Lys Val Ala Val Lys Asp
Glu Gly Val Val Ser Gln Lys Glu 275 280
285 Tyr Glu Lys Ala Ser Thr Asn Lys Tyr Ser Ala Phe Val
Glu Glu Val 290 295 300
Met Lys Glu Ile Asp Glu Lys Ser Asp Val Asp Pro Ser Ala Asp Gly 305
310 315 320 Leu Lys Ile Tyr
Thr Thr Leu Asp Thr Lys Ala Gln Asp Lys Leu Asp 325
330 335 Glu Leu Met Asp Gly Asp Thr Val Gly
Phe Thr Glu Gly Met Gln Gly 340 345
350 Gly Val Thr Leu Leu Asp Thr Lys Asn Gly Glu Val Arg Ala
Ile Gly 355 360 365
Ala Gly Arg Asn Gln Pro Val Gly Gly Phe Asn Tyr Ala Thr Gln Thr 370
375 380 Lys Ala Gln Pro Gly
Ser Thr Ile Lys Pro Ile Leu Asp Tyr Gly Pro 385 390
395 400 Val Ile Glu Asn Lys Lys Trp Ser Thr Tyr
Glu Gln Ile Asp Asp Ser 405 410
415 Ala Tyr Thr Tyr Ser Asn Gly Lys Pro Ile Arg Asp Trp Asp Arg
Lys 420 425 430 Tyr
Leu Gly Pro Ile Ser Met Arg Tyr Ala Leu Ala Gln Ser Arg Asn 435
440 445 Ile Pro Ala Leu Lys Ala
Phe Gln Ala Val Gly Lys Asp Thr Ala Val 450 455
460 Asp Phe Ala Asn Gly Leu Gly Leu Gly Leu Thr
Lys Asp Asn Val Thr 465 470 475
480 Glu Ala Tyr Ser Ile Gly Gly Phe Gly Gly Asn Asp Gly Val Ser Pro
485 490 495 Leu Thr
Met Ala Gly Ala Tyr Ser Ala Phe Gly Asn Asn Gly Thr Tyr 500
505 510 Asn Glu Pro His Phe Val Lys
Ser Ile Glu Phe Asn Asp Gly Thr Lys 515 520
525 Leu Asp Leu Thr Pro Lys Ser Lys Ser Ala Met Ser
Asp Tyr Thr Ala 530 535 540
Phe Met Ile Thr Asp Met Leu Lys Thr Ala Val Lys Thr Gly Thr Gly 545
550 555 560 Gln Leu Ala
Gln Val Pro Gly Val Glu Val Ala Gly Lys Thr Gly Thr 565
570 575 Thr Asn Phe Asp Asp Asn Glu Val
Lys Arg Tyr Asn Ile Ala Ser Gly 580 585
590 Gly Ala Arg Asp Ser Trp Phe Val Gly Tyr Thr Pro Gln
Tyr Thr Ala 595 600 605
Ala Val Trp Thr Gly Met Gly Glu Asn Glu Ala Gly Lys Lys Ser Leu 610
615 620 Ser Ala Glu Glu
Gln Lys Val Ala Lys Arg Ile Phe Ala Gln Leu Ile 625 630
635 640 Ala Asp Val Asp Asp Gly Ser Gly Ser
Phe Glu Lys Pro Asp Ser Val 645 650
655 Val Glu Ala Thr Val Glu Lys Gly Ser Asn Pro Ala Lys Leu
Ala Gly 660 665 670
Pro Asn Thr Pro Ser Asp Lys Lys Leu Thr Glu Tyr Phe Val Lys Gly
675 680 685 Thr Ala Pro Ser
Thr Val Ser Lys Thr Tyr Glu Lys Glu Glu Lys Glu 690
695 700 Glu Thr Ala Lys Leu Ser Gly Leu
Asn Val Lys Tyr Asp Lys Asp Asn 705 710
715 720 Gln Ser Leu Thr Leu Ser Trp Asn Tyr Asp Gly Asp
Ala Thr Phe Ala 725 730
735 Val Lys Gln Ser Val Asp Gly Gly Ser Tyr Ser Glu Ile Gln Asn Ser
740 745 750 Ser Ala Lys
Glu Ala Val Ile Ser Gly Val Gln Pro Gly Ser Val Tyr 755
760 765 Lys Phe Glu Val Thr Ala Val Ser
Asp Asp Gly Lys Ser Thr Ala Ser 770 775
780 Thr Ser Tyr Glu Val Pro Lys Ala Glu Asp Asp Glu Asp
Lys Lys Asp 785 790 795
800 Gln Gln Gln Thr Asp Asp Glu Lys Gln Asp Asp Glu Lys Thr Gln Asp
805 810 815 Asp Thr Gln Thr
Asp Asp Ser Gln Lys Asp Asp Gly Gln Thr Asp Gln 820
825 830 Asp Gln Thr Asp Asp Ser Thr Asn Asp
Gln Asp Lys Lys Gln Asp Asn 835 840
845 Thr Asn Thr Asn Pro Ser Asp Asn Asn Asn Gln Asp Gln Ser
Asn Asp 850 855 860
Asn Asp Asn Asp Asn Ser Asn Asn Gln Asp Thr Ser Asp Gly Asp Ser 865
870 875 880 Asn Ser Gly Lys Asn
Asp Ser Thr Gly Ser Asp Thr Asn Lys Asn Lys 885
890 895 Thr Asp Thr Ser Asn Lys Thr Gln Thr Asn
Ser Ser Ser Ile Glu Lys 900 905
910 Thr Asn 11897PRTClostridium
difficileTRANSMEMBRANE(47)..(71)DOMAIN(100)..(373)DOMAIN(418)..(731)
11Met Asn Asn Asp Asn Asp Lys Asn Gly Asn Lys Ile Arg Arg Lys Lys 1
5 10 15 Val Ser Ser Ser
Gly Asn Thr Asn Lys Pro Ile Asn Arg Thr Ser Ala 20
25 30 Asn Lys Thr Arg Ser Thr Lys Asn Lys
Lys Lys Ser Lys Lys Ser Asp 35 40
45 Lys Phe Lys Lys Leu Arg Val Phe Gly Ile Val Phe Leu Val
Leu Leu 50 55 60
Val Val Gly Thr Ala Gly Thr Ala Gly Leu Val Phe Ala Ser Leu Arg 65
70 75 80 Asp Val Thr Pro Val
Thr Glu Ala Val Leu Asp Lys Gln Thr Asn Gln 85
90 95 Thr Thr Thr Ile Lys Tyr Ala Asn Gly Lys
Thr Leu Ser Thr Ala Pro 100 105
110 Ser Val Asn Lys Lys Thr Pro Val Pro Leu Asp Lys Ile Ser Pro
Tyr 115 120 125 Leu
Gln His Ala Val Ile Ala Ile Glu Asp Glu Arg Phe Tyr Glu His 130
135 140 Lys Gly Val Asp Ile Lys
Gly Leu Phe Arg Ser Val Leu Lys Thr Leu 145 150
155 160 Thr Gly Thr Lys Gln Gly Gly Ser Thr Ile Pro
Met Gln Val Ser Lys 165 170
175 Met Leu Leu Thr Thr Glu Gln Gln Thr Ile Pro Arg Lys Ile Lys Asp
180 185 190 Ile Tyr
Tyr Ala His Glu Met Ser Lys Thr Val Ser Lys Asp Lys Ile 195
200 205 Leu Glu Thr Tyr Leu Asn Asn
Phe Phe Val Gly Arg Gly Leu Ala Gly 210 215
220 Ala Glu Ala Gly Ala Arg Gly Tyr Phe Asp Lys Ser
Ala Ala Asp Leu 225 230 235
240 Thr Leu Ala Glu Ser Ala Leu Leu Ala Gly Ser Thr Lys Asn Pro Ser
245 250 255 Arg Phe Ser
Ala Tyr Lys Thr Ser Lys Leu Glu Gly Asn Glu Thr Lys 260
265 270 Glu Asp Leu Glu Asn Lys Leu Leu
Phe Phe Val Asn Thr Thr Asp Asp 275 280
285 Asp Leu Asp Asp Pro Thr Gln Val Asp Phe Asp Met Ile
Glu Lys Ile 290 295 300
Lys Ser Trp Glu Leu Ile Ser Asn Asp Thr Tyr Arg Gln Leu Lys Ala 305
310 315 320 Gly Thr Leu Val
Val Arg Lys Ala Val Ser Asn Pro Glu Ala Lys Lys 325
330 335 Arg Gln Glu Ile Val Leu Lys Lys Met
Leu Glu Leu Lys Tyr Ile Lys 340 345
350 Gln Ser Glu Tyr Asp Glu Ala Ile Lys Ala Lys Ile Glu Ile
Lys Leu 355 360 365
Pro Gln Ser Ser Asp Lys Val Ser Ser Ser Val Glu Asp Leu Ile Glu 370
375 380 Ser Glu Val Ile Asn
Ala Leu Met Glu Gln Gly His Thr Asn Asp Glu 385 390
395 400 Ala Gln Asn Leu Phe Tyr Asn Gly Gly Leu
Ile Val Asn Thr Thr Ile 405 410
415 Asp Pro Lys Met Gln Asp Ala Leu Glu Glu Glu Phe Asp Arg Asn
Ser 420 425 430 Asn
Phe Pro Gly His Met Val Gly Pro Asp Gly Val Ser Gln Pro Gln 435
440 445 Ala Ala Met Val Ile Leu
Asp Tyr Lys Asn Gly Glu Ile Arg Ala Leu 450 455
460 Ala Gly Gly Arg Asn Ile Ser Gly Arg Lys Thr
Leu Asn Arg Ala Thr 465 470 475
480 Asn Pro His Gln Pro Gly Ser Ser Ile Lys Pro Leu Ala Ile Tyr Thr
485 490 495 Pro Ala
Ile Asp Thr Leu Lys Ile Thr Gln Ala Thr Ala Leu Ser Asp 500
505 510 Ser Arg Gly Gly Tyr Lys Phe
Glu Glu Asn Asn Lys Trp Asn Pro Arg 515 520
525 Thr Thr Thr Ala Gly His Gly Ser Met Ser Leu Arg
Lys Ala Leu Ala 530 535 540
Lys Ser Ser Asn Thr Ile Ala Val Lys Thr Ala Glu Met Leu Gly Asp 545
550 555 560 Ser Tyr Asp
Glu Cys Val Asp Ile Met Met Asp Tyr Leu Lys Asn Phe 565
570 575 Gly Ile Thr Thr Leu Lys Asn Asn
His Ser Gly Ser Ser Glu Ala Ser 580 585
590 Asp Arg Lys Phe Pro Ser Leu Thr Leu Gly Gly Met Ala
Asn Gly Ile 595 600 605
Thr Pro Leu Gln Met Ala Ala Ala Tyr Gly Thr Leu Ala Asn His Gly 610
615 620 Ile Tyr Val Glu
Pro Ser Ile Phe Thr Thr Ile Thr Thr Phe Asp Gly 625 630
635 640 Gln Leu Leu Val Lys Asn Ala Pro Glu
Glu His Lys Val Val Asp Pro 645 650
655 Glu Val Ala Tyr Val Val Thr Asp Met Leu Glu Ser Val Ile
Thr Glu 660 665 670
Gly Ile Gly Gly Val Ala Thr Leu Pro Lys Gly Met Pro Val Ala Gly
675 680 685 Lys Thr Gly Thr
Thr Asn Ser Ala Tyr Asp Ala Trp Phe Val Gly Tyr 690
695 700 Thr Pro Tyr Tyr Val Gly Ala Thr
Tyr Ile Gly Asp Asp Ala Gly Arg 705 710
715 720 Lys Asp Asp Ser Gly Asn Thr Ile Lys Arg Arg Glu
Val Pro His Gly 725 730
735 Ser Thr Ser Thr Ala Lys Leu Trp Glu Lys Ile Met Glu Lys Ile His
740 745 750 Ala Asn Leu
Thr Val Thr Glu Phe Glu Val Pro Lys Asn Val Tyr Phe 755
760 765 Thr Lys Ile Asn Leu Glu Asp Gly
Gly Lys Gln Ser Ser Gly Ser Lys 770 775
780 Ala Ala Phe Ile Glu Gly Thr Ala Pro Thr Lys Val Ser
Ser Gln Pro 785 790 795
800 Ser Ser Glu Asp Thr Lys Lys Pro Asp Asn Asn Gln Pro Glu Glu Asn
805 810 815 Asn Asn Asn Asn
Asn Asn Thr Gly Asp Asn Gly Gly Gly Ser Thr Pro 820
825 830 Pro Asp Asn Gly Gly Asn Asn Gly Gly
Gly Ser Thr Thr Pro Pro Asp 835 840
845 Asn Gly Gly Asn Asn Gly Gly Gly Ser Thr Thr Pro Pro Asp
Asn Gly 850 855 860
Gly Asn Asn Gly Gly Gly Ser Thr Thr Pro Pro Asp Gly Gly Gly Asn 865
870 875 880 Asn Gly Gly Gly Ser
Thr Thr Pro Pro Asp Gly Gly Gly Thr Pro Ala 885
890 895 Thr 12781PRTEnterococcus
faecalisTRANSMEMBRANE(15)..(45)DOMAIN(82)..(307)DOMAIN(364)..(666) 12Leu
Pro Arg Lys Asn Thr Arg Lys Lys Gln Asn Arg Arg Lys Lys Glu 1
5 10 15 Lys Trp Phe Val Pro Lys
Ile Ile Phe Arg Val Phe Gln Ser Leu Thr 20
25 30 Val Phe Ile Thr Val Leu Leu Leu Met Phe
Ala Ala Leu Gly Ile Gly 35 40
45 Ile Gly Ala Gly Tyr Phe Ala Tyr Leu Val Glu Asp Thr Gln
Leu Pro 50 55 60
Thr Lys Lys Ala Leu Gln Thr Glu Leu Gly Asn Ile Thr Glu Thr Ser 65
70 75 80 Lys Ile Val Tyr Ala
Asp Asn Thr Glu Ile Ser Lys Ile Gln Thr Asp 85
90 95 Leu Met Arg Thr Thr Ile Ser Ser Asp Lys
Ile Ser Pro Leu Leu Lys 100 105
110 Thr Ala Ile Ile Ser Thr Glu Asp Glu Tyr Phe Asp Lys His Gln
Gly 115 120 125 Tyr
Val Pro Lys Ala Val Leu Arg Ala Leu Val Ser Glu Ala Thr Gly 130
135 140 Ile Gly Ser Ser Gly Gly
Ser Thr Leu Thr Gln Gln Leu Val Lys Gln 145 150
155 160 Gln Ile Leu Thr Asp Glu Thr Thr Phe Lys Arg
Lys Ala Asn Glu Ile 165 170
175 Ile Leu Ser Ala Gln Val Glu Lys Tyr Phe Ser Lys Asp Glu Ile Ile
180 185 190 Ala Thr
Tyr Leu Asn Val Ser Pro Phe Gly Arg Asn Asn Lys Gly Gln 195
200 205 Asn Ile Ala Gly Val Gln Glu
Ala Ala Arg Gly Ile Phe Gly Val Asp 210 215
220 Ala Lys Asp Val Thr Leu Pro Gln Ala Ala Tyr Ile
Ala Gly Leu Pro 225 230 235
240 Gln Ser Pro Ile Thr Tyr Ser Pro Tyr Thr Asn Thr Gly Ala Leu Lys
245 250 255 Glu Asp Leu
Ser Ala Gly Leu Ala Arg Lys Asp Phe Val Leu Phe Ser 260
265 270 Met Tyr Arg Glu Gly Gln Ile Thr
Lys Glu Gln Tyr Glu Glu Ala Lys 275 280
285 Ala Tyr Asp Leu Thr Lys Asp Phe Leu Pro Gln Gln Ile
Ala Glu Gln 290 295 300
Thr Asp Arg Glu Phe Leu Tyr Tyr Thr Val Met Asn Glu Ala Thr Arg 305
310 315 320 Ile Ile Ala Gln
Gln Leu Ala Glu Lys Asp Asn Ala Asp Met Ser Asp 325
330 335 Ser Ser Val Ser Asp Ala Tyr Tyr Gln
Lys Ala Gln Gln Thr Ile Gln 340 345
350 Asn Lys Gly Tyr Thr Ile His Ser Thr Ile Asp Lys Asp Val
Tyr Ala 355 360 365
Ala Leu Gln Asn Gly Val Glu Asn Tyr Gly Tyr Met Leu Asp Asp Gly 370
375 380 Ala Gly Ser Gln Val
Glu Thr Gly Asn Val Leu Met Asp Asn Arg Thr 385 390
395 400 Gly Arg Ile Tyr Gly Phe Val Gly Gly Arg
Asn Tyr Ser Gln Asn Gln 405 410
415 Asn Asn His Ala Phe Asp Thr Glu Arg Gln Ala Gly Ser Ser Ile
Lys 420 425 430 Pro
Val Leu Val Tyr Gly Pro Ala Ile Asp Met Gly Leu Ile Gly Ser 435
440 445 Glu Ser Arg Val Ser Asp
Tyr Ala Thr Thr Trp Gln Glu Gly Glu Asn 450 455
460 Ala Gly Glu Lys Ile Val Asn Ala Thr Asn Glu
Gly Ser Asn Thr Phe 465 470 475
480 Gln Thr Val Arg Glu Ser Leu Glu Trp Ser Asn Asn Ile Pro Ala Tyr
485 490 495 His Leu
Tyr Gln Asp Val Leu Asn Ser Gly Gly Ser Lys Gln Tyr Ala 500
505 510 Tyr Glu His Tyr Leu Ala Lys
Met Asn Tyr Pro Ala Asn Asp Asn Trp 515 520
525 Gly Val Glu Ser Ala Pro Leu Gly Thr Val Asp Val
Thr Thr Leu Gln 530 535 540
Gln Thr Asn Gly Phe Gln Ala Leu Ala Asn Gly Gly Val Tyr Glu Glu 545
550 555 560 Gly Tyr Ile
Ile Asp Ser Ile Thr Asp Asn Ala Gly Asn Val Ile Tyr 565
570 575 Lys His Glu Ser Asn Ser Val Arg
Ile Tyr Ser Glu Ala Thr Ala Ser 580 585
590 Ile Met Asn Asp Met Met Arg Ser Val Ile Asn Ala Lys
Ile Thr Thr 595 600 605
Pro Phe Lys Asp Ala Ile Ser Ser Leu Asn Gly Asn Leu Gly Lys Ala 610
615 620 Asp Trp Val Gly
Lys Thr Gly Ser Thr Asn Glu Tyr Arg Asp Ser Trp 625 630
635 640 Leu Ile Val Ser Thr Pro Ser Ile Thr
Ile Ser Ser Trp Ala Gly His 645 650
655 Asp Asp Asn Thr Gly Met Asp Ser Lys Ala Arg Ile Arg Ser
Ala Asn 660 665 670
Tyr Leu Ala Asn Leu Ile Asn Gln Ala Tyr Gln Ala Lys Pro Asp Ile
675 680 685 Phe Gly Thr Asp
Glu Lys Phe Glu Leu Ser Ser Asp Val Met Lys Lys 690
695 700 Lys Val Ser Gly Phe Thr Gly Gln
Thr Pro Gly Lys Val Thr Val Asn 705 710
715 720 Lys Lys Ser Ile Gln Thr Pro Gly Lys Thr Val Glu
Ser Leu Trp Ala 725 730
735 Lys Asn Gly Pro Lys Lys Ser Ala Phe Lys Phe Gly Val Gly Gly Thr
740 745 750 Asp Glu Asn
Tyr Thr Asp Tyr Trp Asn Thr Ala Ser Ala Tyr Ala Arg 755
760 765 Ala Asn Gln Ala Thr Lys Glu Ser
Asp Glu Lys Asp Asp 770 775 780
13803PRTEnterococcus
faeciumTRANSMEMBRANE(28)..(58)DOMAIN(95)..(321)DOMAIN(380)..(743) 13Met
Tyr His Phe Ile Glu Val Lys Leu Leu Lys Asn Asn Ser Ser Asn 1
5 10 15 Asn Lys Gln Lys Pro Thr
Thr Ser Ser Gly Gly Asn Val Phe Leu Leu 20
25 30 Ile Leu Asn Val Ile Ile Arg Val Phe Gln
Ser Leu Val Val Phe Gly 35 40
45 Val Ile Leu Ile Val Leu Gly Gly Ser Leu Gly Leu Gly Ile
Gly Met 50 55 60
Gly Tyr Phe Ala Phe Leu Val Glu Asp Thr Gln Pro Pro Thr Lys Glu 65
70 75 80 Glu Leu Gln Lys Glu
Ile Ser Asp Ile Thr Glu Val Ser Lys Met Thr 85
90 95 Tyr Ala Asp Gly Thr Pro Ile Ala Asn Ile
Lys Ser Asp Leu Ile Arg 100 105
110 Thr Arg Ile Asn Gly Asp Gln Met Ser Pro Leu Leu Lys Lys Ala
Ile 115 120 125 Ile
Ser Thr Glu Asp Glu Tyr Phe Glu Glu His His Gly Val Val Pro 130
135 140 Lys Ala Leu Val Arg Ala
Leu Ile Ser Asp Ala Thr Gly Ile Gly Gly 145 150
155 160 Ser Ser Gly Gly Ser Thr Leu Thr Gln Gln Leu
Val Lys Gln Gln Ile 165 170
175 Leu Thr Asp Glu Thr Thr Phe Lys Arg Lys Ala Asn Glu Ile Leu Leu
180 185 190 Ala Leu
Arg Ile Glu Lys Tyr Phe Ser Lys Asp Glu Ile Val Thr Thr 195
200 205 Tyr Leu Asn Val Ser Pro Phe
Gly Arg Asn Asn Lys Gly Glu Asn Ile 210 215
220 Ala Gly Val Glu Glu Ala Ala Lys Gly Leu Phe Gly
Lys Ser Ala Lys 225 230 235
240 Asp Leu Asn Leu Pro Gln Ala Ala Phe Ile Ala Gly Leu Pro Gln Ser
245 250 255 Pro Ile Val
Tyr Thr Pro Tyr Thr Asn Thr Gly Ala Leu Lys Asp Asp 260
265 270 Leu Ser Leu Gly Met Lys Arg Lys
Asp Phe Val Leu Phe Ser Met Tyr 275 280
285 Arg Glu Lys Ala Ile Ser Gln Lys Glu Tyr Glu Glu Ala
Lys Ala Tyr 290 295 300
Asp Leu Lys Lys Asp Phe Leu Pro Thr Glu Gln Ala Asn Val Asn Thr 305
310 315 320 Glu Gly Tyr Leu
Tyr Tyr Thr Val Leu Asp Lys Ala Val Glu Ile Val 325
330 335 Met Asp Leu Asp Met Lys Lys Ala Lys
Val Asn Arg Asp Asp Leu Asp 340 345
350 Gln Val Gly Leu Asp Gln Tyr Glu Glu Gln Ala Arg Arg Glu
Ile Gln 355 360 365
Ser Gln Gly Tyr Thr Ile Gln Ser Thr Ile Asp Gln Asn Ile Tyr Asn 370
375 380 Thr Met Gln Thr Ala
Val Ala Asn Tyr Gly Tyr Leu Leu Asp Asp Gly 385 390
395 400 Thr Ala Asp Val Asn Gly Asn Thr Met Ile
Glu Thr Gly Asn Ile Leu 405 410
415 Met Asp Asn Ala Thr Gly Arg Ile Leu Gly Phe Ile Gly Gly Arg
Asn 420 425 430 Phe
Asp Ile Asn Gln Asn Asn His Ala Phe Asn Ala Asp Arg Gln Val 435
440 445 Gly Ser Thr Ile Lys Pro
Ile Ser Val Tyr Gly Pro Ala Ile Asp Gln 450 455
460 Gly Ile Ile Gly Ser Glu Ser Arg Leu Ala Asn
Tyr Pro Thr Thr Tyr 465 470 475
480 Ala Asp Gly Arg Glu Phe Val Asn Ser Thr Asn Val Asp Leu Asn Gln
485 490 495 Phe Val
Thr Val Arg Asn Ala Leu Asn Trp Ser Phe Asn Ile Pro Val 500
505 510 Val His Val Asn Asn Glu Leu
Arg Lys Lys Met Gly Asp Asp Asn Phe 515 520
525 Ser Tyr Asn His Tyr Leu Ser Lys Met Asn Tyr Pro
Ala Ser Asp Ala 530 535 540
Trp Ala Tyr Glu Ser Ala Pro Leu Gly Ser Val Glu Thr Asn Val Val 545
550 555 560 Thr Gln Thr
Asn Gly Phe Gln Ala Leu Ala Asn Lys Gly Lys Tyr Gln 565
570 575 Lys Ala Tyr Met Ile Glu Lys Ile
Thr Asp Asn Ser Gly His Val Val 580 585
590 Tyr Glu His Lys Asp Glu Gly Thr Gln Val Tyr Ser Pro
Ala Thr Ala 595 600 605
Ser Ile Met Asn Asp Leu Leu Arg Ser Val Val Asp Ser Ala Asn Thr 610
615 620 Thr Lys Phe Lys
Pro Thr Leu Ala Gly Leu Asn Pro His Leu Ala Ser 625 630
635 640 Ala Asp Trp Val Gly Lys Thr Gly Thr
Thr Asp Glu Phe Lys Asp Ser 645 650
655 Trp Leu Ile Val Ser Thr Pro Thr Val Thr Leu Ser Ser Trp
Ala Gly 660 665 670
His Asp Leu Pro Ala Pro Met Thr Met Thr Ser Gly Asp Asn Asn Gly
675 680 685 Asn Tyr Met Ala
Asn Leu Ala Asn Ala Leu Tyr Tyr Ala Asn Pro Glu 690
695 700 Leu Phe Gly Ile Gly Gln Lys Phe
Glu Leu Asp Pro Ser Val Ile Lys 705 710
715 720 Ser Lys Val Ser Glu Phe Thr Gly Glu Lys Pro Gly
Ser Ile Thr Tyr 725 730
735 Asn Gly Ala Lys Phe Asn Thr Pro Gly Lys Thr Thr Thr Ser Tyr Tyr
740 745 750 Ala Lys Asp
Gly Ala Pro Gln Ser Thr Tyr Lys Phe Gly Ile Gly Gly 755
760 765 Thr Asp Ser Asn Tyr Ala Ser Tyr
Trp Gly Asn Leu Ala Pro Arg Ala 770 775
780 Thr Thr Asn Asn Asn Asn Asn Asn Asn Lys Asn Asn Asp
Asn Lys Lys 785 790 795
800 Asn Asn Asn 14727PRTStaphylococcus
aureusTRANSMEMBRANE(27)..(49)DOMAIN(77)..(295)DOMAIN(337)..(631) 14Met
Thr Glu Asn Lys Gly Ser Ser Gln Pro Lys Lys Asn Gly Asn Asn 1
5 10 15 Gly Gly Lys Ser Asn Ser
Lys Lys Asn Arg Asn Val Lys Arg Thr Ile 20
25 30 Ile Lys Ile Ile Gly Phe Met Ile Ile Ala
Phe Phe Val Val Leu Leu 35 40
45 Leu Gly Ile Leu Leu Phe Ala Tyr Tyr Ala Trp Lys Ala Pro
Ala Phe 50 55 60
Thr Glu Ala Lys Leu Gln Asp Pro Ile Pro Ala Lys Ile Tyr Asp Lys 65
70 75 80 Asn Gly Glu Leu Val
Lys Thr Leu Asp Asn Gly Gln Arg His Glu His 85
90 95 Val Asn Leu Lys Asp Val Pro Lys Ser Met
Lys Asp Ala Val Leu Ala 100 105
110 Thr Glu Asp Asn Arg Phe Tyr Glu His Gly Ala Leu Asp Tyr Lys
Arg 115 120 125 Leu
Phe Gly Ala Ile Gly Lys Asn Leu Thr Gly Gly Phe Gly Ser Glu 130
135 140 Gly Ala Ser Thr Leu Thr
Gln Gln Val Val Lys Asp Ala Phe Leu Ser 145 150
155 160 Gln His Lys Ser Ile Gly Arg Lys Ala Gln Glu
Ala Tyr Leu Ser Tyr 165 170
175 Arg Leu Glu Gln Glu Tyr Ser Lys Asp Asp Ile Phe Gln Val Tyr Leu
180 185 190 Asn Lys
Ile Tyr Cys Ser Asp Gly Val Thr Gly Ile Lys Ala Ala Ala 195
200 205 Lys Tyr Tyr Phe Asn Lys Asp
Leu Lys Asp Leu Asn Leu Ala Glu Glu 210 215
220 Ala Tyr Leu Ala Gly Leu Pro Gln Val Pro Asn Asn
Tyr Asn Ile Tyr 225 230 235
240 Asp His Pro Lys Ala Ala Glu Asp Arg Lys Asn Thr Val Leu Tyr Leu
245 250 255 Met His Tyr
His Lys Arg Ile Thr Asp Lys Gln Trp Glu Asp Ala Lys 260
265 270 Lys Ile Asp Leu Lys Ala Asn Leu
Val Asn Arg Thr Pro Glu Glu Arg 275 280
285 Gln Asn Ile Asp Thr Asn Gln Asp Ser Glu Tyr Asn Ser
Tyr Val Asn 290 295 300
Phe Val Lys Ser Glu Leu Met Asn Asn Lys Ala Phe Lys Asp Glu Asn 305
310 315 320 Leu Gly Asn Val
Leu Gln Ser Gly Ile Lys Ile Tyr Thr Asn Met Asp 325
330 335 Lys Asp Val Gln Lys Thr Leu Gln Asn
Asp Val Asp Asn Gly Ser Phe 340 345
350 Tyr Lys Asn Lys Asp Gln Gln Val Gly Ala Thr Ile Leu Asp
Ser Lys 355 360 365
Thr Gly Gly Leu Val Ala Ile Ser Gly Gly Arg Asp Phe Lys Asp Val 370
375 380 Val Asn Arg Asn Gln
Ala Thr Asp Pro His Pro Thr Gly Ser Ser Leu 385 390
395 400 Lys Pro Phe Leu Ala Tyr Gly Pro Ala Ile
Glu Asn Met Lys Trp Ala 405 410
415 Thr Asn His Ala Ile Gln Asp Glu Ser Ser Tyr Gln Val Asp Gly
Ser 420 425 430 Thr
Phe Arg Asn Tyr Asp Thr Lys Ser His Gly Thr Val Ser Ile Tyr 435
440 445 Asp Ala Leu Arg Gln Ser
Phe Asn Ile Pro Ala Leu Lys Ala Trp Gln 450 455
460 Ser Val Lys Gln Asn Ala Gly Asn Asp Ala Pro
Lys Lys Phe Ala Ala 465 470 475
480 Lys Leu Gly Leu Asn Tyr Glu Gly Asp Ile Gly Pro Ser Glu Val Leu
485 490 495 Gly Gly
Ser Ala Ser Glu Phe Ser Pro Thr Gln Leu Ala Ser Ala Phe 500
505 510 Ala Ala Ile Ala Asn Gly Gly
Thr Tyr Asn Asn Ala His Ser Ile Gln 515 520
525 Lys Val Val Thr Arg Asp Gly Glu Thr Ile Glu Tyr
Asp His Thr Ser 530 535 540
His Lys Ala Met Ser Asp Tyr Thr Ala Tyr Met Leu Ala Glu Met Leu 545
550 555 560 Lys Gly Thr
Phe Lys Pro Tyr Gly Ser Ala Tyr Gly His Gly Val Ser 565
570 575 Gly Val Asn Met Gly Ala Lys Thr
Gly Thr Gly Thr Tyr Gly Ala Glu 580 585
590 Thr Tyr Ser Gln Tyr Asn Leu Pro Asp Asn Ala Ala Lys
Asp Val Trp 595 600 605
Ile Asn Gly Phe Thr Pro Gln Tyr Thr Met Ser Val Trp Met Gly Phe 610
615 620 Ser Lys Val Lys
Gln Tyr Gly Glu Asn Ser Phe Val Gly His Ser Gln 625 630
635 640 Gln Glu Tyr Pro Gln Phe Leu Tyr Glu
Asn Val Met Ser Lys Ile Ser 645 650
655 Ser Arg Asp Gly Glu Asp Phe Lys Arg Pro Ser Ser Val Ser
Gly Ser 660 665 670
Ile Pro Ser Ile Asn Val Ser Gly Ser Gln Asp Asn Asn Thr Thr Asn
675 680 685 Arg Ser Thr His
Gly Gly Ser Asp Thr Ser Ala Asn Ser Ser Gly Thr 690
695 700 Ala Gln Ser Asn Asn Asn Thr Arg
Ser Gln Gln Ser Arg Asn Ser Gly 705 710
715 720 Gly Leu Thr Gly Ile Phe Asn 725
15821PRTStreptococcus
pneumoniaeTRANSMEMBRANE(41)..(71)DOMAIN(271)..(336)DOMAIN(396)..(688)
15Met Gln Asn Gln Leu Asn Glu Leu Lys Arg Lys Met Leu Glu Phe Phe 1
5 10 15 Gln Gln Lys Gln
Lys Asn Lys Lys Ser Ala Arg Pro Gly Lys Lys Gly 20
25 30 Ser Ser Thr Lys Lys Ser Lys Thr Leu
Asp Lys Ser Ala Ile Phe Pro 35 40
45 Ala Ile Leu Leu Ser Ile Lys Ala Leu Phe Asn Leu Leu Phe
Val Leu 50 55 60
Gly Phe Leu Gly Gly Met Leu Gly Ala Gly Ile Ala Leu Gly Tyr Gly 65
70 75 80 Val Ala Leu Phe Asp
Lys Val Arg Val Pro Gln Thr Glu Glu Leu Val 85
90 95 Asn Gln Val Lys Asp Ile Ser Ser Ile Ser
Glu Ile Thr Tyr Ser Asp 100 105
110 Gly Thr Val Ile Ala Ser Ile Glu Ser Asp Leu Leu Arg Thr Ser
Ile 115 120 125 Ser
Ser Glu Gln Ile Ser Glu Asn Leu Lys Lys Ala Ile Ile Ala Thr 130
135 140 Glu Asp Glu His Phe Lys
Glu His Lys Gly Val Val Pro Lys Ala Val 145 150
155 160 Ile Arg Ala Thr Leu Gly Lys Phe Val Gly Leu
Gly Ser Ser Ser Gly 165 170
175 Gly Ser Thr Leu Thr Gln Gln Leu Ile Lys Gln Gln Val Val Gly Asp
180 185 190 Ala Pro
Thr Leu Ala Arg Lys Ala Ala Glu Ile Val Asp Ala Leu Ala 195
200 205 Leu Glu Arg Ala Met Asn Lys
Asp Glu Ile Leu Thr Thr Tyr Leu Asn 210 215
220 Val Ala Pro Phe Gly Arg Asn Asn Lys Gly Gln Asn
Ile Ala Gly Ala 225 230 235
240 Arg Gln Ala Ala Glu Gly Ile Phe Gly Val Asp Ala Ser Gln Leu Thr
245 250 255 Val Pro Gln
Ala Ala Phe Leu Ala Gly Leu Pro Gln Ser Pro Ile Thr 260
265 270 Tyr Ser Pro Tyr Glu Asn Thr Gly
Glu Leu Lys Ser Asp Glu Asp Leu 275 280
285 Glu Ile Gly Leu Arg Arg Ala Lys Ala Val Leu Tyr Ser
Met Tyr Arg 290 295 300
Thr Gly Ala Leu Ser Lys Asp Glu Tyr Ser Gln Tyr Lys Asp Tyr Asp 305
310 315 320 Leu Lys Gln Asp
Phe Leu Pro Ser Gly Thr Val Thr Gly Ile Ser Arg 325
330 335 Asp Tyr Leu Tyr Phe Thr Thr Leu Ala
Glu Ala Gln Glu Arg Met Tyr 340 345
350 Asp Tyr Leu Ala Gln Arg Asp Asn Val Ser Ala Lys Glu Leu
Lys Asn 355 360 365
Glu Ala Thr Gln Lys Phe Tyr Arg Asp Leu Ala Ala Lys Glu Ile Glu 370
375 380 Asn Gly Gly Tyr Lys
Ile Thr Thr Thr Ile Asp Gln Lys Ile His Ser 385 390
395 400 Ala Met Gln Ser Ala Val Ala Asp Tyr Gly
Tyr Leu Leu Asp Asp Gly 405 410
415 Thr Gly Arg Val Glu Val Gly Asn Val Leu Met Asp Asn Gln Thr
Gly 420 425 430 Ala
Ile Leu Gly Phe Val Gly Gly Arg Asn Tyr Gln Glu Asn Gln Asn 435
440 445 Asn His Ala Phe Asp Thr
Lys Arg Ser Pro Ala Ser Thr Thr Lys Pro 450 455
460 Leu Leu Ala Tyr Gly Ile Ala Ile Asp Gln Gly
Leu Met Gly Ser Glu 465 470 475
480 Thr Ile Leu Ser Asn Tyr Pro Thr Asn Phe Ala Asn Gly Asn Pro Ile
485 490 495 Met Tyr
Ala Asn Ser Lys Gly Thr Gly Met Met Thr Leu Gly Glu Ala 500
505 510 Leu Asn Tyr Ser Trp Asn Ile
Pro Ala Tyr Trp Thr Tyr Arg Met Leu 515 520
525 Arg Glu Asn Gly Val Asp Val Lys Gly Tyr Met Glu
Lys Met Gly Tyr 530 535 540
Glu Ile Pro Glu Tyr Gly Ile Glu Ser Leu Pro Met Gly Gly Gly Ile 545
550 555 560 Glu Val Thr
Val Ala Gln His Thr Asn Gly Tyr Gln Thr Leu Ala Asn 565
570 575 Asn Gly Val Tyr His Gln Lys His
Val Ile Ser Lys Ile Glu Ala Ala 580 585
590 Asp Gly Arg Val Val Tyr Glu Tyr Gln Asp Lys Pro Val
Gln Val Tyr 595 600 605
Ser Lys Ala Thr Ala Thr Ile Met Gln Gly Leu Leu Arg Glu Val Leu 610
615 620 Ser Ser Arg Val
Thr Thr Thr Phe Lys Ser Asn Leu Thr Ser Leu Asn 625 630
635 640 Pro Thr Leu Ala Asn Ala Asp Trp Ile
Gly Lys Thr Gly Thr Thr Asn 645 650
655 Gln Asp Glu Asn Met Trp Leu Met Leu Ser Thr Pro Arg Leu
Thr Leu 660 665 670
Gly Gly Trp Ile Gly His Asp Asp Asn His Ser Leu Ser Arg Arg Ala
675 680 685 Gly Tyr Ser Asn
Asn Ser Asn Tyr Met Ala His Leu Val Asn Ala Ile 690
695 700 Gln Gln Ala Ser Pro Ser Ile Trp
Gly Asn Glu Arg Phe Ala Leu Asp 705 710
715 720 Pro Ser Val Val Lys Ser Glu Val Leu Lys Ser Thr
Gly Gln Lys Pro 725 730
735 Gly Lys Val Ser Val Glu Gly Lys Glu Val Glu Val Thr Gly Ser Thr
740 745 750 Val Thr Ser
Tyr Trp Ala Asn Lys Ser Gly Ala Pro Ala Thr Ser Tyr 755
760 765 Arg Phe Ala Ile Gly Gly Ser Asp
Ala Asp Tyr Gln Asn Ala Trp Ser 770 775
780 Ser Ile Val Gly Ser Leu Pro Thr Pro Ser Ser Ser Ser
Ser Ser Ser 785 790 795
800 Ser Ser Ser Ser Asp Ser Ser Asn Ser Ser Thr Thr Arg Pro Ser Ser
805 810 815 Ser Arg Ala Arg
Arg 820
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