U.S. patent application number 13/472114 was filed with the patent office on 2013-06-06 for crystal structure of a marr family polypeptide.
This patent application is currently assigned to Trustees of Boston University. The applicant listed for this patent is Michael N. Alekshun, James F. Head, Stuart B. Levy, Barbara A. Seaton. Invention is credited to Michael N. Alekshun, James F. Head, Stuart B. Levy, Barbara A. Seaton.
Application Number | 20130144038 13/472114 |
Document ID | / |
Family ID | 26974573 |
Filed Date | 2013-06-06 |
United States Patent
Application |
20130144038 |
Kind Code |
A1 |
Alekshun; Michael N. ; et
al. |
June 6, 2013 |
CRYSTAL STRUCTURE OF A MarR FAMILY POLYPEPTIDE
Abstract
The crystal structure of the product, crystals of the MarR
protein, a regulator of multiple antibiotic resistance in
Escherichia coli, and methods of crystallization of the MarR
protein are described.
Inventors: |
Alekshun; Michael N.;
(Marlboro, NJ) ; Levy; Stuart B.; (Boston, MA)
; Head; James F.; (Newton, MA) ; Seaton; Barbara
A.; (Newton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Alekshun; Michael N.
Levy; Stuart B.
Head; James F.
Seaton; Barbara A. |
Marlboro
Boston
Newton
Newton |
NJ
MA
MA
MA |
US
US
US
US |
|
|
Assignee: |
Trustees of Boston
University
Boston
MA
Trustees of Tufts College
Medford
MA
|
Family ID: |
26974573 |
Appl. No.: |
13/472114 |
Filed: |
May 15, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12488275 |
Jun 19, 2009 |
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13472114 |
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11707404 |
Feb 16, 2007 |
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12488275 |
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10196655 |
Jul 15, 2002 |
7202339 |
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11707404 |
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60305404 |
Jul 13, 2001 |
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60388622 |
Jun 13, 2002 |
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Current U.S.
Class: |
530/350 ;
378/70 |
Current CPC
Class: |
C07K 14/245 20130101;
G01N 23/20 20130101; C07K 2299/00 20130101 |
Class at
Publication: |
530/350 ;
378/70 |
International
Class: |
C07K 14/245 20060101
C07K014/245; G01N 23/20 20060101 G01N023/20 |
Goverment Interests
GOVERNMENT FUNDING
[0002] This invention was made with government support under
GM51661 awarded by The National Institutes of Health. The
government may, therefore, have certain rights in the invention.
Claims
1. A crystallized MarR family polypeptide, wherein said
crystallized MarR family polypeptide is crystallized under
appropriate conditions such that the three dimensional structure of
said MarR family polypeptide can be determined.
2. The crystallized MarR family polypeptide of claim 1, wherein the
crystallized MarR family polypeptide is a structural homolog of
crystallized MarR.
3. The crystallized MarR family polypeptide of claim 1, wherein
said MarR family polypeptide is selected from the group consisting
of MarR, SlyA, EmrR (MprA), PapX, PrsX, HpcR, Ec17kD of Escherichia
coli; MarR, SlyA, EmrR of Salmonella typhimurium; MexR of
Pseudomonas aeruginosa; PecS of Erwinia chrysanthemi; BadR of
Rhodopseudomonas palustris; OrfE of Burkholderia pseudomallei;
YdcH, YhbI, YkmA, YkoM, Orf7, YfiV, YetL, YdgJ, YwoH, YwaE, YwhA,
Hpr, YybA, YxaD, YsmB, YusO, YpoP, YkvE of Bacillus subtilus;: Orf7
of Bacillus firmus;: Orf145, Orf141 of Staphylococcus sciuri;: CinR
of Butyrivibrio fibrisolvens;: Orf158 of Sphingomonas
aromaticivorans;: NhhD of Rhodococcus rhodochrous; Orf1 of
Streptomyces peucetius;: 14.7 kD, Rv1404, Rv0737, Rv0042c, Yz08
(15.6 kD) of Mycobacterium tuberculosis; Yz08 (15.6 kD) of
Mycobacterium leprae;: MTH313 of Methanobacterium
thermoautotrophicum; Lrs14 of Sulfolobus solfataricus; CinR of
Archaeoglobus fulgidus; PetP of Rhodobacter capsulatus; and SlyA
(E293909) of Sinorhizobium meliloti.
4. The crystallized MarR family polypeptide of claim 1, wherein
said MarR family polypeptide is MarR.
5. The crystallized MarR family polypeptide of claim 4, wherein the
peptide sequence of MarR comprises the amino acid sequence shown in
SEQ ID NO:2.
6. The crystallized MarR family polypeptide of claim 4, wherein
said three dimensional structure has the space group of
I4.sub.122.
7. The crystallized MarR family polypeptide of claim 1, wherein
said appropriate conditions include the presence of a MarR family
polypeptide modulator.
8. The crystallized MarR family polypeptide of claim 7, wherein
said MarR family polypeptide inhibitor is salicylate.
9. The crystallized MarR family polypeptide of claim 1, wherein
said three dimensional structure is determined to a resolution of 5
.ANG. or better.
10.-12. (canceled)
13. The crystallized MarR family polypeptide of claim 4, wherein
said crystallized MarR family polypeptide has a structure defined
by the atomic coordinates given in FIG. 7 or FIG. 8.
14. (canceled)
15. The crystallized MarR family polypeptide of claim 1, wherein
said MarR family polypeptide has a winged-helix structure.
16. A crystallized MarR family polypeptide defined by the atomic
coordinates given in FIG. 7 or FIG. 8.
17. (canceled)
18. A method for determining the three dimensional structure of
MarR family polypeptide, comprising crystallizing the MarR family
polypeptide under appropriate conditions such that crystals are
formed; and analyzing the crystallized MarR family polypeptide,
such that the three dimensional structure of the MarR family
polypeptide is determined.
19. The method of claim 18, wherein said crystallized MarR family
polypeptide is analyzed by x-ray diffraction techniques.
20. The method of claim 18, wherein said MarR family polypeptide is
MarR.
21. The method of claim 20, wherein MarR comprises the amino acid
sequence set forth in SEQ ID NO:2.
22. The method of claim 18, wherein said appropriate conditions
comprise the presence of a MarR family polypeptide modulator.
23. The method of claim 22, wherein said MarR family polypeptide
modulator is salicylate.
24. The method of claim 18, wherein said three dimensional
structure is determined to a resolution of 5 .ANG. or better.
25.-27. (canceled)
28. The method of claim 18, wherein said MarR family polypeptide is
selected from the group consisting of: MarR, SlyA, EmrR (MprA),
PapX, PrsX, HpcR, Ec17kD of Escherichia coli; MarR, SlyA, EmrR of
Salmonella typhimurium; MexR of Pseudomonas aeruginosa; PecS of
Erwinia chrysanthemi; BadR of Rhodopseudomonas palustris; OrfE of
Burkholderia pseudomallei; YdcH, YhbI, YkmA, YkoM, Orf7, YfiV,
YetL, YdgJ, YwoH, YwaE, YwhA, Hpr, YybA, YxaD, YsmB, YusO, YpoP,
YkvE of Bacillus subtilus;: Orf7 of Bacillus firmus;: Orf145,
Orf141 of Staphylococcus sciuri;: CinR of Butyrivibrio
fibrisolvens;: Orf158 of Sphingomonas aromaticivorans;: NhhD of
Rhodococcus rhodochrous; Orf1 of Streptomyces peucetius;: 14.7 kD,
Ry1404, Rv0737, Rv0042c, Yz08 (15.6 kD) of Mycobacterium
tuberculosis; Yz08 (15.6 kD) of Mycobacterium leprae;: MTH313 of
Methanobacterium thermoautotrophicum; Lrs14 of Sulfolobus
solfataricus; CinR of Archaeoglobus fulgidus; PetP of Rhodobacter
capsulatus; and SlyA (E293909) of Sinorhizobium meliloti.
29. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 12/488,275, filed Jun. 19, 2009; which is a
continuation application of U.S. application Ser. No. 11/707,404,
filed Feb. 16, 2007; which is a continuation application of U.S.
application Ser. No. 10/196,655, filed Jul. 15, 2002, now U.S. Pat.
No. 7,202,339, issued Apr. 10, 2007; which claims priority to U.S.
Provisional Patent Application Ser. No. 60/388,622, filed on Jun.
13, 2002; and U.S. Provisional Patent Application Ser. No.
60/305,404, filed on Jul. 13, 2001. The entire contents of all of
the above applications and patents are hereby incorporated herein
by reference.
BACKGROUND OF THE INVENTION
[0003] The Mar phenotype in E. coli is attributed largely to the
action of MarA, the expression of which is regulated by MarR
(Alekshun, M. N. supra (1997)). MarA is a transcription factor that
autoactivates expression of the marRAB operon and regulates the
expression of a global network of more than 60 chromosomal genes
(Martin, R. G. et al. J. Bact. 178, 2216-2223 (1996); Barbosa, T.
M. & Levy, S. B. J. Bact. 182, 3467-3474 (2000)). Mar mutants
in isolates of clinical origin have now been identified
(Maneewannakul, K. & Levy, S. B. Antimicrob. Agents Chemother.
40, 1695-1698 (1996); Oethinger, M. et al. Antimicrob. Agents
Chemother. 42, 2089-2094 (1998); Linde, H. J. et al. Antimicrob.
Agents Chemother. 44, 1865-1868 (2000); Ziha-Zarifi, I., et al.
Antimicrob. Agents Chemother. 43, 287-291 (1999); Koutsolioutsou, A
et al. Antimicrob. Agents Chemother. 45, 38-43 (2001)).
Constitutive overexpression of MarA or a MarA homolog in many of
these strains is a key contributor to the maintenance of the
resistance phenotype, particularly with respect to the
fluoroquinolones, and recent studies have documented the selection
of Mar mutants, bearing mutations in MarR, MexR, or other
homologous loci, in E. coli, Pseudomonas aeruginosa, and other
organisms during antimicrobial chemotherapy (Oethinger, M supra;
Linde, H. J. et al.; supra; Ziha-Zarifi, I. et al. supra; Kern, W.
V., et al. Antimicrob. Agents Chemother. 44, 814-820 (2000)).
[0004] MarR is a regulator of multiple antibiotic resistance in
Escherichia coli. It is the prototypic member of a family of
regulatory proteins found in the Bacteria and the Archaea that play
important roles in the development of antibiotic resistance, a
global health problem. In the absence of an appropriate stimulus,
MarR negatively regulates expression of the marRAB operon (Cohen,
S. P., et al. 1993. J. Bacteriol. 175: 1484-1492; Martin, R. G. and
Rosner, J. L. 1995. Proc. Natl. Acad. Sci. 92: 5456-5460; Seoane,
A. S. and Levy, S. B. 1995. J. Bacteriol. 177: 414-3419, 1995). DNA
footprinting experiments suggest that MarR dimerizes at two
locations, sites I and II, health problem. In the absence of an
appropriate stimulus, MarR negatively regulates expression of the
marRAB operon (Cohen, S. P., et al. 1993. J. Bacteria 175:
1484-1492.; Martin, R. G. and Rosner, J. L. 1995. Proc. Natl. Acad.
Sci. 92: 5456-5460; Seoane, A. S. and Levy, S. B. 1995. J. Bacteria
177: 3414-3419., 1995). DNA footprinting experiments suggest that
MarR dimerizes at two locations, sites I and II, within the mar
operator (marO) (Martin and Rosner, 1995, supra). Site I is
positioned among the -35 and -10 hexamers and site II spans the
putative MarR ribosome binding site (reviewed in Alekshun, M. N.
and Levy, S. B. 1997. Antimicrob. Agents Chemother. 10:
2067-2075).
[0005] MarR is a member of a newly recognized family of regulatory
proteins (Alekshun, M. N. and Levy, S. B. 1997. Antimicrob. Agents
Chemother. 10:. 2067-2075. Sulavik, M. C., et al. 1995. Mol. Med.
1: 436-446) and many functional homologues have been identified in
a variety of important human pathogens and have been found to
regulate a variety of different processes. For example, some MarR
homologues have been found to control expression of multiple
antibiotic resistance operons, some regulate tissue-specific
adhesive properties, some control expression of a cryptic
hemolysin, some regulate protease production, and some regulate
sporulation. Proteins of the MarR family control an assortment of
biological functions including resistance to multiple antibiotics,
organic solvents, household disinfectants, and oxidative stress
agents, collectively termed the multiple antibiotic resistance
(Mar) phenotype (Alekshun, M. N. & Levy, S. B. Trends
Microbiol. 7, 410-413 (1999)). These proteins also regulate the
synthesis of pathogenic factors in microbes that infect humans and
plants (Miller, P. F. & Sulavik, M. C. Mol. Microbiol. 21,
441-448 (1996)). Insight into the three dimensional structure of
MarR family proteins would be of great value in designing drugs
that interact with this family of proteins and modulate MarR
function, for example, antibiotic resistance and virulence.
SUMMARY OF THE INVENTION
[0006] The instant invention advances the prior art by providing
the crystal structure of a MarR family polypeptide, MarR. The
crystal structure was solved for both the MarR polypeptide and the
MarR polypeptide bound to salicylate.
[0007] The invention pertains at least in part to a crystallized
MarR family polypeptide. The MarR family polypeptide is
crystallized under appropriate conditions such that its three
dimensional structure can be determined. In another embodiments,
the crystallized MarR family polypeptide is given by the atomic
coordinates given in FIG. 7 or 8.
[0008] In another embodiment, the invention pertains, at least in
part, to a method for determining the three dimensional structure
of MarR family polypeptide. The method includes crystallizing the
MarR family polypeptide under appropriate conditions such that
crystals are formed, and analyzing it, such that its three
dimensional structure is determined.
[0009] In yet another embodiment, the invention pertains to a
method of making a crystal of a MarR family polypeptide. The method
includes contacting MarR with a modulator to form a complex and
allowing crystals of the complex to grow, e.g., by using hanging
droplet vapor diffusion.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 shows the sequence alignment of MarR with
representative members of the MarR family (SEQ ID NOS 2-8,
respectively, in order of appearance).
[0011] FIG. 2 is a ribbon representation of the cocrystal structure
of the MarR dimer with salicylate viewed with the subunit two-fold
axis near vertical.
[0012] FIG. 3 is an electrostatic surface representation of the
MarR dimer.
[0013] FIG. 4 is a C.alpha. trace of a MarR subunit in stereo
representation.
[0014] FIG. 5 is a representation of the N-/C-terminal domain
represented by a surface around the van der Waals radii of the side
chain atoms only of the hydrophobic core residues. Helices leading
to and from the domain are shown in ribbon representation.
[0015] FIG. 6 is a diagram which shows interactions between the
DNA-binding domains of the dimer in the region of the Arg 73-Asp
67' salt bridges. The stereo view is coincident with the 2-fold
rotation axis of the dimer. Electron density shown is a
2F.sub.O-F.sub.C map contoured at 1.sigma..
[0016] FIG. 7 shows the atomic coordinates of the MarR--salicylate
co-crystal (residues 7-144 of SEQ ID NO:2).
[0017] FIG. 8 shows the atomic coordinates of the MarR crystal
without salicylate (residues 12-144 and 9-144 of SEQ ID NO:2).
[0018] FIG. 9 is a ribbon representation of the MarR dimer with the
two-fold axis near vertical.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The invention pertains, at least in part, to crystallized
MarR family polypeptides. The crystallized MarR family polypeptides
are crystallized under appropriate conditions such that the three
dimensional structure can be determined using methods described
herein and/or art recognized techniques.
[0020] The term "MarR family polypeptide" includes molecules
related to MarR, e.g., having certain shared structural and
functional features. MarR family polypeptides also include those
which are structural homologs of MarR. The structural homologs
include those having a crystallized form which are structurally
similar to that of crystallized MarR. MarR family members, in
addition to having similarity to MarR, may bind to DNA and regulate
transcription. While some MarR family members negatively control
transcription (e.g., MarR), others have positive/activator
functions (e.g., SlyA, BadR, NhhD, and MexR). MarR family
polypeptides comprise DNA and protein binding domains. In addition,
MarR family polypeptides can interact with a variety of
structurally unrelated compounds that regulate their activity.
[0021] Exemplary MarR family members are taught in the art and can
be found, e.g., in Sulavik et al. (1995. Molecular Medicine.
1:436), Miller and Sulavik (1996. Molecular Microbiology. 21:441)
in which alignments of MarR and related proteins are shown, or
through the use of BLAST searches and other techniques known in the
art. Exemplary MarR family polypeptides are also illustrated in the
following chart:
TABLE-US-00001 MarR Family Polypeptides Gram-negative Escherichia
coli MarR SlyA EmrR (MprA) PapX PrsX HpcR Ec17 kD Salmonella
typhimurium MarR SlyA EmrR Pseudomonas aeruginosa MexR Erwinia
chrysanthemi PecS Rhodopseudomonas palustris BadR Burkholderia
pseudomallei OrfE Gram-positive Bacillus subtilus YdcH YhbI YkmA
YkoM Orf7 YfiV YetL YdgJ YwoH YwaE YwhA Hpr YybA YxaD YsmB YusO
YpoP YkvE Bacillus firmus Orf7 Staphylococcus sciuri Orf145 Orf141
Butyrivibrio fibrisolvens CinR Sphingomonas aromaticivorans Orf158
Rhodococcus rhodochrous NhhD Streptomyces peucetius Orf1 Acid-fast
Mycobacterium tuberculosis 14.7 kD Rv1404 Rv0737 Rv0042c Yz08 (15.6
kD) Mycobacterium leprae Yz08 (15.6 kD) Archaea Methanobacterium
thermoautotrophicum MTH313 Sulfolobus solfataricus Lrs14
Archaeoglobus fulgidus CinR Purple non-sulfur Rhodobacter
capsulatus PetP Sinorhizobium meliloti SlyA (E293909)
[0022] Preferably, the MarR family polypeptide is MarR. Other
preferred MarR family polypeptides include: EmrR, Ec17kD, and
MexR.
[0023] In a further embodiment, the MarR family polypeptide has a
winged-helix structure, such as the three dimensional structure of
MarR.
[0024] FIG. 1 shows a sequence alignment of MarR with
representative MarR family polypeptides. The MarR secondary
structure elements were identified in its crystal structure and are
illustrated in FIG. 1 (e.g., as tubes for .alpha.-helices (.alpha.)
and arrows for .beta.-sheets (.beta.) and the single wing region
(W1)). The numbering in FIG. 1 is according to the MarR primary
sequence. The MarR family polypeptides used for the alignment were
from the following organisms: MarR, E. coli; MprA (EmrR), E. coli;
MexR, Pseudomonas aeruginosa; YS87, Mycobacterium tuberculosis;
SlyA, Salmonella typhimurium; PecS, Erwinia chrysanthemi; CinR,
Butyrivibrio fibrisolvens.
[0025] In a further embodiment, MarR comprises, consists
essentially of, or consists of the polypeptide sequence shown in
Sequence Listing SEQ ID NO:1. Other MarR family polypeptides of
interest include EmrR, YS87, PecS, CinR, SlyA, Ec17kD, MexR,
etc.
[0026] In another embodiment, the MarR family polypeptide is found,
for example, in one of the following organisms Escherichia coli,
Salmonella typhimurium, Salmonella enterica, Enterobacter cloacae,
Enterobacter aerogenes, Erwinia chrysanthemi, Yrsinia pestis,
Yersinia enterocolitica, Kluyvera cryocrescens, Edwardsiella tarda,
Pseudomonas aeruginosa, Vibrio cholera, Xanthomonas axonopodis,
Xanthomonas campestris, Ralstonia solanacearum, Burkholderia
pseudomallei, Burkholderia cepacia, Vogesella indigofera,
Mesorhizobium loti, Agrobacterium tumefaciens, Sinorhizobium
meliloti, Brucella melitensis, Caulobacter crescentus, Bacillus
anthracis, Bacillus subtilis, Bacillus halodurans, Listeria
monocytogenes, Listeria innocua, Listeria welshimeri,
Staphylococcus sciuri, Streptococcus criceti, Streptococcus
pneumoniae, Clostridium perfringens, Clostridium difficile,
Streptomyces coelicolor, Streptomyces avermitilis, Mycobacterium
tuberculosis, Mycobacterium leprae, Corynebacterium glutamicum,
Thermotoga maritima, Methanosarcina acetivorans, Methanosarcina
mazei, and Sulfolobus solfataricus.
[0027] In another embodiment, the MarR family polypeptide is from
an organism belonging to one of the following biological
classifications: Enterobacteriaceae, Enterobacter, Yersinia,
Kluyvera, Edwardsiella, Xanthomonas group, Xanthomonadales,
Pseudomonaceae/Moraxellaceae group, Pseudomonadaceae, Vibrionaceae
group, Burkholderia/Oxalobacter/Ralstonia group, Ralstonia group,
Burkholderia group, Neisseriaceae, Vogesella, Rhizobiaceae group,
Phyllobacteriaceae, Mesorhizobium, Rhizobiaceae, Sinorhizobium,
Brucellaceae, Brucella, Caulobacter group, Firmicutes,
Bacillus/Clostridium group, Bacilli, Bacillales, Bacillus,
Bacillaceae, Bacillus cereus group, Listeria, Listeriaceae,
Staphylococcaceae, Staphylococcus, Streptococcus, Lactobacillales,
Streptococcaceae, Clostridium, Clostridiaceae, Clostridiales,
Clostridia, Actinomycetales, Actinobacteria, Actinobacteridae,
Streptomyces, Streptomycineae, Streptomycetaceae,
Corynebacterineae, Mycobacterium, Mycobacteriaceae,
Corynebacteriaceae, Corynebacterium, Nostocales, Nostocaceae,
Nostoc, Thermotogae, Thermotogales, Thermotogaceae; Thermotoga,
Methanosarcina, Euryarchaeota, Methanococci; Methanosarcinales,
Methanosarcinaceae, Crenarchaeota, Thermoprotei; Sulfolobales,
Sulfolobaceae, Sulfolobus, Proteobacteria, Pectobacterium,
Cyanobacteria, or Archaea.
[0028] In one embodiment, the MarR family polypeptides of the
invention are naturally occurring. In another embodiment, the
subject crystal structures can be generated using non-naturally
occurring forms of MarR family polypeptides, e.g. mutants or
synthetic forms of MarR family polypeptides not found in
nature.
[0029] In one embodiment, the MarR family polypeptide comprises one
or more conservative mutations as compared to the wild type protein
for the particular MarR family polypeptide. The term "MarR family
polypeptide" also includes fragments of MarR family polypeptides
which minimally retain at least a portion of the tertiary structure
of the MarR family protein.
[0030] MarR family member polypeptide sequences are "structurally
related" to one or more known MarR family members, preferably to
MarR. This structural relatedness is shown by sequence similarity
between two MarR family polypeptide sequences or between two MarR
family nucleotide sequences. Sequence similarity can be shown,
e.g., by optimally aligning MarR family member sequences using an
alignment program for purposes of comparison and comparing
corresponding positions. To determine the degree of similarity
between sequences, they will be aligned for optimal comparison
purposes (e.g., gaps may be introduced in the sequence of one
protein for nucleic acid molecule for optimal alignment with the
other protein or nucleic acid molecules). The amino acid residues
or bases and corresponding amino acid positions or bases are then
compared. When a position in one sequence is occupied by the same
amino acid residue or by the same base as the corresponding
position in the other sequence, then the molecules are identical at
that position. If amino acid residues are not identical, they may
be similar. As used herein, an amino acid residue is "similar" to
another amino acid residue if the two amino acid residues are
members of the same family of residues having similar side chains.
Families of amino acid residues having similar side chains have
been defined in the art (see, for example, Altschul et al. 1990. J.
Mol. Biol. 215:403) including basic side chains (e.g., lysine,
arginine, histidine), acidic side chains (e.g., aspartic acid,
glutamic acid), uncharged polar side chains (e.g., glycine,
asparagine, glutamine, serine, threonine, tyrosine, cysteine),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine, tryptophan), beta-branched side
chains (e.g., threonine, valine, isoleucine) and aromatic side
chains (e.g., tyrosine, phenylalanine, tryptophan). The degree
(percentage) of identity or similarity between sequences,
therefore, can be calculated as a function of the number of
identical or similar positions shared by two sequences (i.e., %
homology=# of identical or similar positions/total # of
positions.times.100). Alignment strategies are well known in the
art; see, for example, Altschul et al. supra for optimal sequence
alignment.
[0031] MarR family polypeptides share some amino acid sequence
similarity with MarR. The nucleic acid and amino acid sequences of
MarR as well as other MarR family polypeptides are available in the
art. For example, the nucleic acid and amino acid sequence of MarR
can be found, e.g., on GeneBank (accession number M96235 or in
Cohen et al. 1993. J. Bacteriol. 175:1484, or in SEQ ID NO:1).
[0032] The nucleic acid and protein sequences of MarR can be used
as "query sequences" to perform a search against databases (e.g.,
either public or private) to, for example, identify other MarR
family members having related sequences. Such searches can be
performed, e.g., using the NBLAST and XBLAST programs (version 2.0)
of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST
nucleotide searches can be performed with the NBLAST program,
score=100, wordlength=12 to obtain nucleotide sequences homologous
to MarR family nucleic acid molecules. BLAST protein searches can
be performed with the XBLAST program, score=50, wordlength=3 to
obtain amino acid sequences homologous to MarR protein molecules of
the invention. To obtain gapped alignments for comparison purposes,
Gapped BLAST can be utilized as described in Altschul et al.,
(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST
and Gapped BLAST programs, the default parameters of the respective
programs (e.g., BLAST and NBLAST) can be used. See
http://www.ncbi.nlm.nih.gov.
[0033] MarR family members can also be identified as being similar
based on their ability to specifically hybridize to the complement
of nucleic acid sequences specifying MarR. Such stringent
conditions are known to those skilled in the art and can be found
e.g., in Current Protocols in Molecular Biology, John Wiley &
Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example
of stringent hybridization conditions are hybridization in 6.times.
sodium chloride/sodium citrate (SSC) at about 45.degree. C.,
followed by one or more washes in 0.2X SSC, 0.1% SDS at
50-65.degree. C. Conditions for hybridizations are largely
dependent on the melting temperature Tm that is observed for half
of the molecules of a substantially pure population of a
double-stranded nucleic acid. Tm is the temperature in .degree. C.
at which half the molecules of a given sequence are melted or
single-stranded. For nucleic acids of sequence 11 to 23 bases, the
Tm can be estimated in degrees C. as 2(number of A+T
residues)+4(number of C+G residues).
[0034] Hybridization or annealing of nucleic acid molecules should
be conducted at a temperature lower than the Tm, e.g., 15.degree.
C., 20.degree. C., 25.degree. C. or 30.degree. C. lower than the
Tm. The effect of salt concentration (in M of NaCl) can also be
calculated, see for example, Brown, A., "Hybridization" pp.
503-506, in The Encyclopedia of Molec. Biol., J. Kendrew, Ed.,
Blackwell, Oxford (1994).
[0035] Preferably, the nucleic acid sequence of a MarR family
member identified in this way is at least about 10%, 20%, more
preferably at least about 30%, more preferably at least about 40%
identical and most preferably at least about 50%, or 60% identical
or more with a MarR nucleotide sequence. Preferably, MarR family
members have an amino acid sequence at least about 20%, more
preferably at least about 30%, more preferably at least about 40%
identical and most preferably at least about 50%, or 60% or more
identical with a MarR amino acid sequence. However, it will be
understood that the level of sequence similarity among microbial
regulators of gene transcription, even though members of the same
family, is not necessarily high. This is particularly true in the
case of divergent genomes where the level of sequence identity may
be low, e.g., less than 20% (e.g., B. burgdorferi as compared e.g.,
to B. subtilis). For example, the level of amino acid sequence
homology between MarR and PecS is about 31% and the level of amino
acid sequence homology between MarR and PapX is about 28% when
determined as described above. Accordingly, structural similarity
among MarR family members can also be determined based on
"three-dimensional correspondence" of amino acid residues. As used
herein, the language "three-dimensional correspondence" is meant to
includes residues which spatially correspond, e.g., are in the same
functional position of a MarR family protein member as determined,
e.g., by x-ray crystallography, but which may not correspond when
aligned using a linear alignment program. The language
"three-dimensional correspondence" also includes residues which
perform the same function, e.g., bind to DNA or bind the same
cofactor, as deteiinined, e.g., by mutational analysis.
[0036] Preferred MarR family polypeptides include: MarR, EmrR,
Ec17kD, MexR, PapX, SlyA, Hpr, PecS, Hpr, MprA, (EmrR), as well as
the other peptides listed in the chart above or known in the art.
In a more preferred embodiment, a MarR family polypeptide is
selected from the group consisting of MarR, EmrR, Ec171kD, and
MexR. In a particularly preferred embodiment, a MarR family
polypeptide is MarR.
[0037] In addition to sharing structural similarity, MarR family
members have a MarR family polypeptide activity, i.e., they bind to
DNA and regulate transcription. Some MarR family members positively
regulate transcription (e.g., SlyA, BadR, NhhD, or MexR), while
others negatively regulate transcription (e.g., MarR). While all
MarR family members bind to DNA and regulate transcription, the
different loci controlled by each family member regulate different
processes in microbes. For example, MarR family polypeptides can
control the expression of microbial loci involved in: regulation of
antibiotic resistance [e.g., MarR (Cohen et al. 1993. J. Bacteriol.
175:1484), EmrR (Lomovskaya and Lewis. 1992. Proc. Natl. Acad. Sci.
89:8938), and Ec17kD (Sulavik et al. 1995. Mol. Med. 1:436), and
MexR (Poole et al. 1996. Antimicrob. Agents. Chemother. 40:2021)],
regulation of tissue-specific adhesive properties [e.g., PapX
(Marklund et al., 1992. Mol. Microbiol. 6:2225)], regulation of
expression of a cryptic hemolysin [e.g., SlyA (Ludwig et al. 1995
249:4740)], regulation of protease production [e.g., Hpr from B.
subtilis (Perago and Hoch. 1988. J. Bacteriol. 170:2560) and PecS
from Erwinia chrysanthemi (Reverchon et al., 1994. Mol. Microbiol.
11:1127)] and regulation of sporulation [e.g., Hpr (Perego and
Hoch. 1988. J. Bacteriol. 170:2560)], regulation of the breakdown
of plant materials [e.g., CinR (Dalymple and Swadling 1997
Microbiology)] sensing of phenolic compounds [(e.g., Sulvik et al.
1995. Mol. Med. 1:436], and repress marRAB expression when
introduced into E. coli [e.g., Ec17kd (Markhmd et al. 1992. Mol.
Microbiol. 6:2225) and MprA (EmrR) (del Castillo et al., 1991. J.
Bacteriol. 173:3924)]. The activity of MarR family polypeptides is
antagonized by salicylate (Lomovskaya et al., 1995. J. Bacteriol.
177:2328; Sulavik et al. 1995. Mol. Med. 1:436).
[0038] Preferred MarR family polypeptide activities include
regulation of multiple drug resistance and/or regulation of
virulence.
[0039] In addition to full length MarR family polypeptide fragments
MarR family polypeptide which are useful in making crystals are
also within the scope of the invention. Accordingly, MarR family
polypeptides for use in the instant invention can be full length
MarR family member proteins or fragments thereof. Thus, a MarR
family polypeptide can comprise, consist essentially of, or consist
of an amino acid sequence derived from the full length amino acid
sequence of a MarR family member. For example, in one embodiment, a
polypeptide comprising a MarR family polypeptide DNA interacting
domain or a polypeptide comprising a MarR family member protein
interacting domain can be used.
[0040] In addition, naturally or non-naturally occurring variants
of these polypeptides and nucleic acid molecules which retain the
same functional activity, e.g., the ability to bind to DNA and
regulate transcription. Such variants can be made, e.g., by
mutation using techniques which are known in the art.
Alternatively, variants can be chemically synthesized.
[0041] For example, it will be understood that the MarR family
polypeptides described herein, are also meant to include
equivalents thereof. For instance, mutant forms of MarR family
polypeptides which are functionally equivalent, (e.g., have the
ability to bind to DNA and to regulate transcription from an
operon) can be made using techniques which are well known in the
art. Mutations can include, e.g., at least one of a discrete point
mutation which can give rise to a substitution, or by at least one
deletion or insertion. For example, random mutagenesis can be used.
Mutations can be made by random mutagenesis or using cassette
mutagenesis. For the former, the entire coding region of a molecule
is mutagenized by one of several methods (chemical, PCR, doped
oligonucleotide synthesis) and that collection of randomly mutated
molecules is subjected to selection or screening procedures. In the
latter, discrete regions of a protein, corresponding either to
defined structural or functional determinants (e.g., the first or
second helix of a helix-turn-helix domain) are subjected to
saturating or semi-random mutagenesis and these mutagenized
cassettes are re-introduced into the context of the otherwise wild
type allele. In one embodiment, PCR mutagenesis can be used. For
example, Megaprimer PCR can be used (O. H. Landt, Gene
96:125-128).
[0042] In addition, other portions of the above described
polypeptides suitable for use in the claimed assays, such as those
which retain their function (e.g., the ability to bind to DNA, to
regulate transcription from an operon) or those which are critical
for binding to regulatory molecules (such as compounds) can be
easily determined by one of ordinary skill in the art, e.g., using
standard truncation or mutagenesis techniques and used in the
instant assays. Exemplary techniques are described by Gallegos et
al. (1996. J. Bacteriol. 178:6427).
[0043] It shall be understood that the instant invention also
pertains to isolated MarR family member polypeptides, portions
thereof, and the nucleic acid molecules encoding them, including
naturally occurring and mutant forms.
Preparation of MarR Family Polypeptides
[0044] Preferred MarR family polypeptides for use in the instant
invention are synthesized, isolated or recombinant polypeptides. In
one embodiment, MarR family polypeptides can be made from nucleic
acid molecules. Nucleic acid molecules encoding MarR family
polypeptides can be used to produce MarR family polypeptides for
use in the instant assays. For example, nucleic acid molecules
encoding a MarR family polypeptide can be isolated (e.g., isolated
from the sequences which naturally flank it in the genome and from
cellular components) and can be used to produce a MarR family
polypeptide. In one embodiment, a nucleic acid molecule which has
been (1) amplified in vitro by, for example, polymerase chain
reaction (PCR); (2) recombinantly produced by cloning, or (3)
purified, as by cleavage and gel separation; or (4) synthesized by,
for example, chemical synthesis can be used to produce MarR family
polypeptides. As used herein, the term "nucleic acid" refers to
polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic
acid (RNA).
[0045] Nucleic acid molecules specifying MarR family polypeptides
can be placed in a vector. The term "vector" refers to a nucleic
acid molecule capable of transporting another nucleic acid molecule
to which it has been linked. The term "expression vector" includes
any vector, (e.g., a plasmid, cosmid or phage chromosome)
containing a gene construct in a form suitable for expression by a
cell (e.g., linked to a promoter). In the present specification,
"plasmid" and "vector" are used interchangeably, as a plasmid is a
commonly used form of vector. Moreover, the invention is intended
to include other vectors which serve equivalent functions.
[0046] Exemplary expression vectors for expression of a gene
encoding a MarR family polypeptide and capable of replication in a
bacterium, such a bacterium from a genus selected from the group
consisting of Escherichia, Bacillus, Streptomyces, Streptococcus,
or in a cell of a simple eukaryotic fungus such as a Saccharomyces
or, Pichia, or in a cell of a eukaryotic organism such as an
insect, a bird, a mammal, or a plant, are known in the art. Such
vectors may carry functional replication-specifying sequences
(replicons) both for a host for expression, for example a
Streptomyces, and for a host, for example, E. coli, for genetic
manipulations and vector construction. See e.g. U.S. Pat. No.
4,745,056. Suitable vectors for a variety of organisms are
described in Ausubel, F. et al., Short Protocols in Molecular
Biology, Wiley, New York (1995), and for example, for Pichia, can
be obtained from Invitrogen (Carlsbad, Calif.).
[0047] Useful expression control sequences, include, for example,
the early and late promoters of SV40, adenovirus or cytomegalovirus
immediate early promoter, the lac system, the trp system, the TAC
or TRC system, T7 promoter whose expression is directed by T7 RNA
polymerase, the major operator and promoter regions of phage
lambda, the control regions for fd coat protein, the promoter for
3-phosphoglycerate kinase or other glycolytic enzymes, the
promoters of acid phosphatase, e.g., Pho5, the promoters of the
yeast .alpha.-mating factors, the polyhedron promoter of the
baculovirus system and other sequences known to control the
expression of genes of prokaryotic or eukaryotic cells or their
viruses, and various combinations thereof. A useful translational
enhancer sequence is described in U.S. Pat. No. 4,820,639.
[0048] It should be understood that the design of the expression
vector may depend on such factors as the choice of the host cell to
be transformed and/or the type of protein desired to be
expressed.
[0049] "Transcriptional regulatory sequence" is a generic term to
refer to DNA sequences, such as initiation signals, enhancers,
operators, and promoters, which induce or control transcription of
nucleic acid sequences with which they are operably linked. It will
also be understood that a recombinant gene encoding a MarR family
polypeptide can be under the control of transcriptional regulatory
sequences which are the same or which are different from those
sequences which control transcription of the naturally-occurring
MarR family gene. Exemplary regulatory sequences are described in
Goeddel; Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, Calif. (1990). For instance, any of a
wide variety of expression control sequences, that control the
expression of a DNA sequence when operatively linked to it, may be
used in these vectors to express DNA sequences encoding the MarR
family proteins of this invention.
[0050] Appropriate vectors are widely available commercially and it
is within the knowledge and discretion of one of ordinary skill in
the art to choose a vector which is appropriate for use with a
given microbial cell. The sequences encoding MarR family
polypeptides can be introduced into a cell on a self-replicating
vector or may be introduced into the chromosome of a microbe using
homologous recombination or by an insertion element such as a
transposon.
[0051] Such vectors can be introduced into cells using standard
techniques, e.g., transformation or transfection. The terms
"transformation" and "transfection" mean the introduction of a
nucleic acid, e.g., an expression vector, into a recipient or
"host" cell. The term "transduction" means transfer of a nucleic
acid sequence, preferably DNA, from a donor to a recipient cell, by
means of infection with a virus previously grown in the donor,
preferably a bacteriophage. Nucleic acids can also be introduced
into microbial cells by transformation using calcium chloride or
electroporation.
[0052] "Cells," "host cells," "recipient cells, are terms used
interchangeably herein. It is understood that such terms refer not
only to the particular subject cell but to the progeny or potential
progeny of such a cell. In preferred embodiments, cells used to
express MarR family polypeptides for purification, e.g., host
cells, comprise a mutation which renders any endogenous MarR family
polypeptide nonfunctional or causes the endogenous polypeptide to
not be expressed. In other embodiments, mutations may also be made
in other related genes of the host cell, such that there will be no
interference from the endogenous host loci.
[0053] Purification of a MarR family polypeptides, e.g.,
recombinantly expressed polypeptides, can be accomplished using
techniques known in the art. For example, if the MarR family
polypeptide is expressed in a form that is secreted from cells, the
medium can be collected. Alternatively, if the MarR family
polypeptide is expressed in a form that is retained by cells, the
host cells can be lysed to release the MarR family polypeptide.
Such spent medium or cell lysate can be used to concentrate and
purify the MarR family polypeptide. For example, the medium or
lysate can be passed over a column, e.g., a column to which
antibodies specific for the MarR family member polypeptide have
been bound. Alternatively, such antibodies can be specific for a
non-MarR family member polypeptide which has been fused to the MarR
family polypeptide (e.g., as a tag) to facilitate purification of
the MarR family member polypeptide. Other means of purifying MarR
family member polypeptides are known in the art.
Architecture of the MarR-Salicylate Co-Crystal Structure
[0054] The term "three dimensional structure" includes both
pictorial representations of MarR family polypeptides (e.g., such
as those shown for MarR with salicylate and MarR without salicylate
in the Figures) as well as atomic coordinates (e.g., such as those
given in FIG. 7 for MarR-salicylate cocrystal, or in FIG. 8 for
MarR without salicylate) and other renditions of the shape, size,
or symmetry of a MarR family polypeptide of interest. In a further
embodiment, the three dimensional structure of the crystallized
MarR family polypeptide is determined to a resolution of 5 .ANG. or
better, 3 .ANG. or better, 2.5 .ANG. or better, or, advantageously,
2.3 .ANG. or better. The three dimensional structure of MarR, a
MarR family polypeptide, is described in greater detail below.
[0055] MarR consists of a dimer with approximate overall dimensions
of 50.times.55.times.45 .ANG. (corresponding to the width, height
and depth of the molecule in the orientation shown in FIG. 2).
There is one monomer in the asymmetric unit of the crystal with the
dimer composed of subunits related by a crystallographic two-fold
rotation. The dimeric structure is consistent with the results of
earlier in vitro experiments suggesting that MarR binds the mar
operator (marO) as a dimer (Martin, R. G. et al. supra (1996);
Martin, R. G. & Rosner, J. L. Proc. Natl. Acad. Sci. U.S.A. 92,
5456-5460 (1995)). Another family member, MprA (EmrR) (FIG. 1) is
also believed to function as a dimer (Brooun, A., et al. J. Bact.
181, 5131-5133 (1999)).
[0056] Each MarR subunit is an .alpha./.beta. protein with
approximate dimensions of 35.times.25.times.60 .ANG. and can be
divided into two domains as shown in FIG. 2. FIG. 2 is a ribbon
representation of the co-crystal structure of the MarR dimer viewed
with the subunit 2-fold axis near vertical. The secondary structure
elements of one subunit are colored according to the scheme used in
FIG. 1. The N- and C-terminal regions are closely juxtaposed and
intertwine with the equivalent regions of the second subunit to
form a domain that holds the subunits together (FIG. 5). This
N-/C-terminal domain is linked to the remainder of the protein by
two long antiparallel helices in each subunit. These helices lead
to a globular domain that is likely to be responsible for DNA
binding (see below). Although the globular DNA-binding domains of
the dimer are adjacent to one another, they make minimal contact
with each other and are situated to function independently. The
overall organization of the N-/C-terminal domain and the two
DNA-binding domains results in the formation of an approximately 6
.ANG. wide channel through the center of the dimer (FIGS. 3 and 4).
The electrostatic surface potential (FIG. 3) is consistent with the
putative DNA-binding regions being strongly electropositive, as
observed in other such winged-helix DNA-binding proteins (Gajiwala,
K. S. & Burley, S. K. Curr. Opin. Str. Biol. 10, 110-116
(2000)).
[0057] Genetic and biochemical data have previously identified the
N-terminus of MarR to be important for mediating protein-protein
contacts between repressor subunits and have demonstrated that the
C-terminus is important for protein function (Alekshun, M. N., et
al. Mol. Microbiol. 35, 1394-404 (2000); Linde, H. J. et al.
supra). The present structure shows that a-helices in the N- and
C-terminal regions of each monomer fold around and interdigitate
with those of the other subunit to form a well-packed hydrophobic
core (FIG. 5) burying a surface area of 3,570 .ANG..sup.2 (the
total buried surface area for the whole dimer is 3,700
.ANG..sup.2). The dimer is further stabilized in this region by
several intermolecular hydrogen bonds, notably that between the
c-amino group of Lys 24 and the main chain carbonyl oxygen of Pro
144' in the C-terminus of the second subunit and that between the
main chain carbonyl oxygen of Glu 10 and the side chain amino group
of Lys 140'.
[0058] While the DNA-binding lobe of each subunit also forms a
well-packed hydrophobic core, the only interactions between these
lobes of the two subunits are salt bridges formed between Asp 67
and Arg 73' and the reciprocal pair (FIG. 6). These salt bridges
stabilize the relationship between the two lobes of the dimer in
the crystal form of the protein but if disrupted by other
interactions, such as might occur during the binding of MarR to
marO, the two lobes would be able to act independently. Relative
movement of the lobes would require distortion of the helices that
link them to the N-/C-terminal domain. The long linker helix region
encompassing residues 103-126 (.alpha.5/.alpha.5') (FIG. 2) appears
poorly ordered in the region of Gly 116, as is the loop (residues
128-131) that connects this helix to the C-terminal helix
(.alpha.6/.alpha.6') (FIGS. 1 and 2). It is possible that
flexibility at these sites in MarR helps to accommodate relative
shifts of the two lobes of the dimer that might occur on binding to
DNA.
Architecture of the MarR Crystal Structure
[0059] The MarR without salicylate structure is a dimer (FIG. 9)
and both subunits of the dimer are in the asymmetric unit. These
individual subunits are joined by extensive protein-protein
interactions mediated by amino acids within both the N- and
C-termini of the monomers (FIG. 9). Like the MarR-salicylate
structure, MarR without salicylate is an .alpha./.beta. protein.
The MarR without salicylate structure is, however, conformationally
different from the salicylate bound protein in that the caliper
created by the dimer is more closed in the form of the protein
without salicylate. Thus, the channel through the center of the
dimer has been lost.
[0060] The overall architecture of the MarR without salicylate
structure is comparable to that of the salicylate bound protein.
The presumed DNA binding lobes or domains are linked to the
remainder of the protein by two long .alpha.-helices. The
positioning of the two DNA binding lobes in the MarR without
salicylate structure is fixed by hydrogen bonds between the two
lobes. This arrangement is believed to be mediated by interactions
between Asp 67 and Arg 77'. In addition, Asp 26 is involved in
hydrogen bonds with the side chains of Lys 44 and Lys 25. Together,
the presumed recognition helices within the DNA binds lobes overlap
by approximately one helical turn.
The DNA Binding Domain
[0061] Previous studies have shown the region spanning amino acids
61-121 in MarR to be required for its DNA binding activity
(Alekshun, M. N et al., supra, (2000)). In the crystal structure,
amino acids 55-100 [.beta.1-.alpha.3-.alpha.4-.beta.2-W1
(wing)-.beta.3] adopt the winged-helix fold (Clark, K. L. et al.
Nature 364, 412-420 (1993)). The overall topology [H1 (.alpha.2)-S1
(.beta.1)-H2 (.alpha.3)-H3 (.alpha.4, recognition helix)-S2
(.beta.2)-W1-S3 (.beta.3)] of this region is similar to other
winged-helix DNA binding proteins (the terminology applied for
these and subsequent structural elements is according to Gajiwala
and Burley, supra (2000)) except that a third strand of sheet
present in most members of the group appears to be represented in
this MarR structure by an interaction with Ile 55 (.beta.1) (FIG.
1). The presence of this single residue as the third component in
the sheet interaction is very similar to that observed in OmpR
(Martinez-Hackert, E. & Stock, A. M. Structure 5, 109-124
(1997)), a winged helix protein, where Leu 180 interacts with the
two strands of the antiparallel sheet that forms part of the "wing"
in this transcription factor.
[0062] Within the winged-helix family of DNA-binding proteins,
there are multiple modes of DNA binding. Members such as
HNF-3.gamma. use the recognition helix (H3) of the motif as the
primary determinant for DNA-protein interactions in the major
groove, and a wing region(s) (W1) to form minor groove or
phosphodiester backbone nucleoprotein contacts (Clark, K. L. et al.
supra (1993)). Others, such as hRFX1, use W1 to interact with the
major groove and the H3 helix makes only a single minor groove
contact (Gajiwala, K. S. et al. Nature 403, 916-921 (2000)). The
juxtaposition of the DNA-binding lobes in the present structure
does not allow for modeling of the whole dimer onto a B-DNA
representation of the operator. However, since mutations in both
.alpha.4 (H3) and W1 affect the DNA binding activity of MarR it is
expected that amino acids from each of these regions would
contribute to the DNA binding activity of the protein. For example,
mutations in .alpha.4, including an R73C change, abolish MarR DNA
binding activity in whole cells and in vitro (Alekshun, M. N et
al., supra, (2000)). In the present crystal structure, it is the
side chain of Arg 73 that is hydrogen bonded to Asp 67' of the
other subunit, an interaction that stabilizes the relative
orientation of the two DNA-binding lobes (FIG. 6). Also, an R94C
mutation at the tip of W1 is inactive in a whole cell assay while a
G95S "superrepressor" mutation increases the DNA binding activity
of MarR 30-fold in vitro (Alekshun, M. N et al., supra, (2000);
Alekshun, M. N. & Levy, S. B. J. Bact. 181, 3303-3306 (1999)).
In the absence of protein-DNA co-crystal structures, the precise
mechanism by which these mutations affect the DNA binding activity
of the protein is uncertain.
[0063] Footprinting experiments have suggested that MarR binds as a
dimer at two separate but very similar sites in marO, the protein
protects .about.21-bp of DNA on both strands at a single site, and
does not bend its target (Martin, R. G. et al., supra (1996);
Martin, R. G. et al. Proc. Natl. Acad. Sci. U.S.A. 92, 5456-5460
(1995)). Each MarR binding site is composed of two half-sites whose
organization is such that they are on different faces of the DNA
double helix (Alekshun, M. N. et al. Mol. Microbiol. 35, 1394-404
(2000)), an arrangement that is very similar to the hRFX1 binding
site (Gajiwala, K. S. et al. Nature 403, 916-921 (2000)). For MarR
to bind as a dimer, with each winged-helix DNA binding domain
contacting one half-site on B-DNA, geometric constraints suggest
only a few possible modes of binding. One scenario, involving the
binding of a single dimer to one MarR binding site, would require
reorientation of the DNA binding lobes so that each could reach one
half-site. This would be analogous to the binding of an E2F-DP
heterodimer (a eukaryotic transcription factor in which each
subunit also has a winged-helix DNA binding domain) to its cognate
binding site (Zheng, N. et al. Genes Dev. 13, 666-74. (1999)). A
second scenario would involve the binding of two dimers, on
opposite faces of the double helix, to a single MarR binding site.
This model would be analogous to the binding of DtxR (a bacterial
protein with a winged-helix DNA binding domain) to its target,
although in DtxR the half-sites are on the same face of the DNA
helix (Pohl, E. et al. J. Biol. Chem. 273, 22420-22427 (1998);
White, A. et al. Nature 394, 502-506 (1998)).
[0064] The term "appropriate conditions" include those conditions
which result in the formation of a crystal which can by analyzed to
a resolution of 5.0 .ANG. or less. The crystals may be formed using
suitable art recognized techniques, such as hanging droplet vapor
diffusion. In one embodiment, the temperature of crystallization of
the MarR family polypeptide is from about 1.degree. C. to about
30.degree. C., from about 10.degree. C. to about 25.degree. C.,
from about 15.degree. C. to about 20.degree. C., or abut 17.degree.
C. In a further embodiment, the conditions are selected such that
crystals of said MarR family polypeptide grow within an acceptable
time and reach dimensions which are suitable for structural
determination, e.g., by using X-ray diffraction. In one embodiment,
the acceptable time is 8 weeks or less, 6 weeks or less, 4 weeks or
less, or 3 weeks or less. In an embodiment, the dimensions of the
crystal are approximately 0.1 mm or greater per side, 0.2 mm or
greater per said, or approximately. 0.3 mm per side or greater.
[0065] In a further embodiment, the appropriate conditions include
a cocrystallization agent which interacts with the protein such
that the three dimensional structure of the protein can be
determined.
[0066] The term "cocrystallization agent" includes substances which
can be crystallized with the MarR family polypeptide such that the
three dimensional structure can be determined. In an embodiment,
the coocrystallization agent is a MarR family polypeptide
modulator. The term "MarR family polypeptide modulator" includes
compounds which interact with MarR, either to inhibit or enhance
the activity of MarR, such that they alter its activity in its
non-crystallized form. In one embodiment, the MarR family
polypeptide modulator is a MarR inhibitor (e.g., salicylate,
plombagin, or DNP). In an embodiment, the concentration of the
salicylate is about 100 mM or less, 150 mM or less, 200 mM or less,
or 250 mM or less.
[0067] The crystal structure or MarR has been solved using crystals
grown in the presence and in the absence of high concentrations
(250 mM) of sodium salicylate. This agent, at millimolar
concentrations, is known to inhibit MarR activity both in vitro and
in whole cells (Alekshun, M. N. supra (1999)). It is routinely used
as a model inhibitor of MarR to induce MarA expression in E. coli
and S. typhimurium (Cohen, S. P. et al. J. Bact. 175, 7856-7862
(1993); Sulavik, M. C. et al. J. Bact. 179, 1857-1866 (1997)) and
thus, to confer a Mar phenotype (Alekshun, M. N. supra (1999)). In
one example, salicylate was included in the current crystal growth
conditions to provide stable crystals. In another example, the
crystal structure of MarR was determined using MarR without
salicylate.
[0068] Electron density that is consistent with bound salicylate is
apparent at two sites on each subunit in the present structure
(FIG. 6). These sites are on the surface of the molecule on either
side of the proposed DNA-binding helix .alpha.4 (H3). In one site
(SAL-A), the salicylate hydroxyl is hydrogen bonded to the hydroxyl
side chain of Thr 72 in the .alpha.4 (H3) helix and the salicylate
carboxylate hydrogen bonds to the guanidinium group of Arg 86. In
the other site (SAL-B), the salicylate hydroxyl hydrogen bonds to
the backbone carbonyl of Ala 70 and its carboxyl hydrogen bonds to
Arg 77. In each of these sites, the salicylate ring sits over a
hydrophobic side chain in the pocket; Pro 57 in SAL-A and Met 74 in
SAL-B and other surface hydrophobes are also located laterally
within 3.5 .ANG. of the unsubstituted side of the ring. Although
SAL-B is solvent exposed, SAL-A packs in the crystal with Val 96 of
a symmetry mate situated 3.6 .ANG. above the salicylate ring and
adjacent to the SAL-A site of this symmetry mate. Since both SAL-A
and SAL-B are close to the DNA binding helix, they may be
positioned to influence DNA binding.
[0069] The crystal structure of MarR was solved by multiwavelength
anomalous dispersion methods using protein containing
selenomethionine. Diffraction data were collected to 2.3 .ANG. from
crystals of both seleno and native protein.
[0070] The invention is further illustrated by the following
examples, which should not be construed as further limiting. The
contents of all references, pending patent applications and
published patents, cited throughout this application are hereby
expressly incorporated by reference.
EXEMPLIFICATION. OF THE. INVENTION
EXAMPLE 1
Crystallization of MarR with Salicylate
[0071] Protein Production and Purification
[0072] Native and selenomethionine (Se-Met) containing MarR was
prepared from E. coli BL21(DE3) (Novagen) bearing pMarR-WT, a wild
type MarR expression vector that has been previously described
(Alekshun, M. N. & Levy, S. B. J. Bact. 181, 4669-4672 (1999)).
Native MarR was produced in whole cells according to previous
methods (Alekshun, M. N. & Levy, S. B. J. Bact. 181, 4669-4672
(1999)). Se-Met MarR was produced by diluting an overnight culture
of E. coli BL21(DE3)+pMarR-WT 1:1000 in M9 medium supplemented with
2 mM MgSO.sub.4, 0.2% glucose, 0.1 mM CaCl.sub.2, 0.00005%
thiamine, 0.04 mg ml.sup.-1 each of the following amino acids
phenylalanine, leucine, isoleucine, valine, serine, threonine,
tyrosine, histidine, lysine, aspartic acid, glutamic acid,
tryptophan, and tryptophan, and kanamycin (Miller, J. H. In
Experiments in Molecular Genetics. (Cold Spring Harbor
Laboratories, Cold Spring Harbor, N.Y.; 1972). This culture was
grown at 37.degree. C. to an OD600.apprxeq.0.6 and 100 mg each of
amino acids threonine, lysine-hydrochloride, phenylalanine, 50 mg
each of amino acids leucine, isoleucine, and valine (single letter
abbreviations), and 60 mg L-(+)-selenomethionine (Sigma) were then
added. The culture was grown for 15 min at 37.degree. C.; IPTG was
subsequently added to a final concentration of 1 mM and protein
production was allowed to proceed for 14.5 hr at 37.degree. C. Cell
pellets were collected and processed as previously described
(Alekshun, M. N. & Levy, S. B. J. Bact. 181, 4669-4672
(1999)).
[0073] Frozen cell pellets containing native or Se-Met MarR were
resuspended in 100 mM sodium phosphate buffer (pH 7.4) containing a
bacterial protease inhibitor cocktail (Sigma) and sonicated on ice.
All buffers contained 2 mM DTT when Se-Met MarR was prepared.
Insoluble matter was removed by centrifugation at 4.degree. C. at
30,000.times.g for 40 min. The supernatant was passed over
prepacked 5 ml SP-sepharose HiTrap columns (Amersham Pharmacia
Biotech) previously equilibrated with 10 mM sodium phosphate buffer
(pH 7.4). The column was washed with 50 ml of 10 mM sodium
phosphate buffer (pH 7.4) and the pure proteins were eluted with a
linear gradient (0-0.5 M) of NaCl in 10 mM sodium phosphate buffer
(pH 7.4). Protein containing fractions were dialyzed vs. 10 mM
HEPES (pH 7.4), 200 mM NaCl, and 1 mM DTT, or 2 mM DTT in the case
of Se-Met MarR, and the protein in these samples was judged to be
greater than 99% pure via SDS-PAGE and electrospray ionization mass
spectrophotometry. The latter also demonstrated that more than 95%
of the three methionine residues in Se-Met MarR were substituted
with selenomethionine.
[0074] Crystallization:
[0075] MarR crystals were originally grown in 18% PEG MME 5000, 200
mM ammonium sulfate, 100 mM citrate buffer (pH 5.6) but showed
anisotropic disorder in the diffraction data that made them
unsuitable for structure determination. To stabilize the protein,
the citrate was substituted by the known inhibitor salicylate.
Crystals of the MarR-salicylate complex were grown at 17.degree. C.
by hanging droplet vapor diffusion. 6 .mu.l of a 11.4 mg ml.sup.-1
protein solution in 200 mM NaCl, 20 mM HEPES (pH 7.4), and 10 mM
DTT were added to 2 .mu.l of reservoir buffer (18% PEG MME 5000, 50
mM ammonium sulfate, 250 mM sodium salicylate, 10 mM DTT, and 15%
glycerol, pH 5.5), and 0.8 .mu.l 15% heptanetriol. The droplets
were equilibrated with 1 ml of reservoir buffer. Crystals grew
within 1 week reaching dimensions of approximately 0.3 mm per
side.
[0076] X-Ray Data Collection, Structure Determination, and
Refinement:
[0077] Diffraction data were collected at the Brookhaven National
Synchrotron Light Source, beamline X8C. Crystals were flash frozen
in mother liquor at the beam line before data collection. All data
were processed and reduced using DENZO and SCALEPACK (Otwinowski,
Z. In CCP4 Proceedings. 56-62 (Daresbury Laboratory, Warrington,
UK, 1993). The space group of the MarR-salicylate co-crystals was
determined to be I4.sub.122 with one molecule in the asymmetric
unit and with unit cell dimensions of a=b=62.0 .ANG., c=132.9
.ANG., .alpha.=.beta.=.gamma.=90.degree. for both the native and
the selenoprotein. Data were collected on the selenoprotein
crystals at three wavelengths to enable MAD phasing. Phases were
determined from the MAD data using the program SOLVE (Terwilliger,
T. C. & Berendzen, J. Acta. Crystallogr. D. 55, 849-861
(1999)). This showed two selenium sites per asymmetric unit, with
the third selenomethionine, at the N-terminus, apparently
disordered. Maps were solvent-flattened using the program DM and
the model was built into density using the program O (Collaborative
Computational Project, Number 4. Acta Crystallogr. D. 50, 760-763
(1994); Jones, T. A. et al. Acta Crystallogr. A 47, 110-119
(1991)). Model and refinement parameters for salicylate were
obtained from the Hetero Compound Information Center (Kleywegt, G.
J. & Jones, T. A. Acta Crystallogr. D. 54, 1119-1131 (1998)).
Model refinement was performed using CNS and cycles of rebuilding
and refinement continued to give the final model (Brunger, A. T. et
al. Acta. Crystallogr. D. 54, 905-921 (1998)). Model quality was
assessed by sa-omit, Fo-Fc, maps generated over the whole molecule
omitting no more than 7% of the structure at a time. The model
extends from residue 6 to the C-terminus at residue 144. In common
with several other transcription factors (e.g. TetR, (1A6I), ArgR
(1B4B) and TreR (1BYK)), MarR shows relatively high thermal
mobility throughout the structure, as reflected by the B-factors.
Certain regions appear to be particularly mobile, including the
extended structure at the N-terminus, the tip of the "wing"
(residues 91-94), parts of the .alpha.5 helix, especially around
Gly 116 and the connecting loop (128-131) between the .alpha.5 and
the C-terminal .alpha.6 helix. Consistent with the high B-factors,
the molecule shows few well-ordered solvent molecules. PROCHECK
reports overall g-factors of 0.25 (dihedrals) and 0.55 (main chain
covalent forces) and shows that 91% of the residues fall within the
most favored region of the Ramachandran plot, with only residue Ala
53 in a disallowed region. This residue is located at the start of
the loop connecting the .alpha.2 and .alpha.3 helices.
[0078] The coordinates of the MarR-salicylate cocrystal are shown
in FIG. 7. Table 1 shows data collection, phasing, and refinement
statistics for the MarR-salicylate co-crystal.
TABLE-US-00002 TABLE 1 Data set Native Se-met edge Se-met peak
Se-met remote Wavelength (.ANG.) 1.072 0.9795 0.9793 0.9500
Resolution range (.ANG.) 50-2.3 50-2.3 50-2.3 50-2.3 Measured
reflections 56,495 84,173 96,582 87,365 Unique reflections 6,069
5,534 5,564 5,472 Completeness (%) 99.5 (100) 91.3 (99.8) 91.7
(99.8) 90.4 (99.7) overall (final <I/.sigma.I> (final shell)
21.1 (12.0) 12.2 (7.2) 12.0 (7.0) 12.9 (7.9) R.sub.merge(%) (final
shell) 6.0 (20.0) 6.4 (29.7) 5.7 (30.3) 4.9 (25.5) Rano (%) 4.9 5.0
3.5 Overall FOM 0.59/0.71 (centric/acentric) Resolution 50-2.3
Rfree 28.7% Rcryst 24.7% Atoms/AU Protein 1078 Salicylate 20 Water
18 Average B (.ANG..sup.2) main chain 49.7 side chain 59.2
salicylate 42.7 water 50.0 R.m.s. deviation Bonds (.ANG.) 0.009
Angles (.degree.) 1.3 indicates data missing or illegible when
filed
EXAMPLE 2
Crystallization of MarR
[0079] MarR was produced and purified as described in Example
1.
[0080] Crystallization:
[0081] Crystals of MarR were grown by hanging droplet vapor
diffusion. 3 .mu.l of a 10 mg ml.sup.-1 2:1 (mol:mol) DNA-protein
solution in 200 mM NaCl, 20 mM HEPES, pH 7.4, 20 mM TRIS-HCl, pH
8.0, and 2 mM MgCl.sub.2 was added to 1 .mu.l of reservoir buffer
(23% PEG MME 5000, 100 mM sodium citrate, 200 mM ammonium sulfate,
10 mM DTT, 10% glycerol, 5% Isopropanol, pH 5.6), and 0.4 .mu.l 15%
heptanetriol. The droplets were equilibrated with 0.5 ml of
reservoir buffer.
[0082] X-Ray Data Collection, Structure Determination, and
Refinement:
[0083] Diffraction data were collected at the Brookhaven National
Synchrotron Light Source, beamline X8C. Crystals were flash frozen
in mother liquor at the beam line before data collection. All data
were processed and reduced using DENZO and
[0084] SCALEPACK (Otwinowski, Z. In CCP4 Proceedings. 56-62
(Daresbury Laboratory, Warrington, UK, 1993).
[0085] The coordinates of the MarR crystal without salicylate are
shown in FIG. 8. Table 2 shows data collection, phasing, and
refinement statistics for the MarR crystal.
TABLE-US-00003 TABLE 2 Space group C222 Unit cell (.ANG.) a = 65.8,
b = 137.7, c = 96.4 Resolution 50-2.7 Rfree 26.7% Rcryst 23.2%
Atoms/AU Protein 2093 Water 14 Average B (.ANG..sup.2) main chain
40.0 side chain 48.0 Water 32.6 R.m.s. deviation Bonds (.ANG.)
0.009 Angles (.degree.) 1.3
EQUIVALENTS
[0086] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments and methods described
herein. Such equivalents are intended to be encompassed by the
scope of the following claims.
[0087] All patents, patent applications, and literature references
cited herein are hereby expressly incorporated by reference. The
entire contents of Alekshun et al. "The Crystal Structure of MarR a
Regulator of Multiple Antibiotic Resistance at 2.3 .ANG.
resolution," Nature Structural Biology 8(8) is hereby incorporated
herein by reference.
Sequence CWU 1
1
81125PRTEscherichia coli 1Met Val Asn Gln Lys Lys Asp Arg Leu Leu
Asn Glu Tyr Leu Ser Pro1 5 10 15Leu Asp Ile Thr Ala Ala Gln Phe Lys
Val Leu Cys Ser Ile Arg Cys 20 25 30Ala Ala Cys Ile Thr Pro Val Glu
Leu Lys Lys Val Leu Ser Val Asp 35 40 45Leu Gly Ala Leu Thr Arg Met
Leu Asp Arg Leu Val Cys Lys Gly Trp 50 55 60Val Glu Arg Leu Pro Asn
Pro Asn Asp Lys Arg Gly Val Leu Val Lys65 70 75 80Leu Thr Thr Gly
Gly Ala Ala Ile Cys Glu Gln Cys His Gln Leu Val 85 90 95Gly Gln Asp
Leu His Gln Glu Leu Thr Lys Asn Leu Thr Ala Asp Glu 100 105 110Val
Ala Thr Leu Glu Tyr Leu Leu Lys Lys Val Leu Pro 115 120
1252144PRTEscherichia coli 2Met Lys Ser Thr Ser Asp Leu Phe Asn Glu
Ile Ile Pro Leu Gly Arg1 5 10 15Leu Ile His Met Val Asn Gln Lys Lys
Asp Arg Leu Leu Asn Glu Tyr 20 25 30Leu Ser Pro Leu Asp Ile Thr Ala
Ala Gln Phe Lys Val Leu Cys Ser 35 40 45Ile Arg Cys Ala Ala Cys Ile
Thr Pro Val Glu Leu Lys Lys Val Leu 50 55 60Ser Val Asp Leu Gly Ala
Leu Thr Arg Met Leu Asp Arg Leu Val Cys65 70 75 80Lys Gly Trp Val
Glu Arg Leu Pro Asn Pro Asn Asp Lys Arg Gly Val 85 90 95Leu Val Lys
Leu Thr Thr Gly Gly Ala Ala Ile Cys Glu Gln Cys His 100 105 110Gln
Leu Val Gly Gln Asp Leu His Gln Glu Leu Thr Lys Asn Leu Thr 115 120
125Ala Asp Glu Val Ala Thr Leu Glu Tyr Leu Leu Lys Lys Val Leu Pro
130 135 1403176PRTEscherichia coli 3Met Asp Ser Ser Phe Thr Pro Ile
Glu Gln Met Leu Lys Phe Arg Ala1 5 10 15Ser Arg His Glu Asp Phe Pro
Tyr Gln Glu Ile Leu Leu Thr Arg Leu 20 25 30Cys Met His Met Gln Ser
Lys Leu Leu Glu Asn Arg Asn Lys Met Leu 35 40 45Lys Ala Gln Gly Ile
Asn Glu Thr Leu Phe Met Ala Leu Ile Thr Leu 50 55 60Glu Ser Gln Glu
Asn His Ser Ile Gln Pro Ser Glu Leu Ser Cys Ala65 70 75 80Leu Gly
Ser Ser Arg Thr Asn Ala Thr Arg Ile Ala Asp Glu Leu Glu 85 90 95Lys
Arg Gly Trp Ile Glu Arg Arg Glu Ser Asp Asn Asp Arg Arg Cys 100 105
110Leu His Leu Gln Leu Thr Glu Lys Gly His Glu Phe Leu Arg Glu Val
115 120 125Leu Pro Pro Gln His Asn Cys Leu His Gln Leu Trp Ser Ala
Leu Ser 130 135 140Thr Thr Glu Lys Asp Gln Leu Glu Gln Ile Thr Arg
Lys Leu Leu Ser145 150 155 160Arg Leu Asp Gln Met Glu Gln Asp Gly
Val Val Leu Glu Ala Met Ser 165 170 1754147PRTPseudomonas
aeruginosa 4Met Asn Tyr Pro Val Asn Pro Asp Leu Met Pro Ala Leu Met
Ala Val1 5 10 15Phe Gln His Val Arg Thr Arg Ile Gln Ser Glu Leu Asp
Cys Gln Arg 20 25 30Leu Asp Leu Thr Pro Pro Asp Val His Val Leu Lys
Leu Ile Asp Glu 35 40 45Gln Arg Gly Leu Asn Leu Gln Asp Leu Gly Arg
Gln Met Cys Arg Asp 50 55 60Lys Ala Leu Ile Thr Arg Lys Ile Arg Glu
Leu Glu Gly Arg Asn Leu65 70 75 80Val Arg Arg Glu Arg Asn Pro Ser
Asp Gln Arg Ser Phe Gln Leu Phe 85 90 95Leu Thr Asp Glu Gly Leu Ala
Ile His Gln His Ala Glu Ala Ile Met 100 105 110Ser Arg Val His Asp
Glu Leu Phe Ala Pro Leu Thr Pro Val Glu Gln 115 120 125Ala Thr Leu
Val His Leu Leu Asp Gln Cys Leu Ala Ala Gln Pro Leu 130 135 140Glu
Asp Ile1455139PRTMycobacterium tuberculosis 5Met Gly Leu Ala Asp
Asp Ala Pro Leu Gly Tyr Leu Leu Tyr Arg Val1 5 10 15Gly Ala Val Leu
Arg Pro Glu Val Ser Ala Ala Leu Ser Pro Leu Gly 20 25 30Leu Thr Leu
Pro Glu Phe Val Cys Leu Arg Met Leu Ser Gln Ser Pro 35 40 45Gly Leu
Ser Ser Ala Glu Leu Ala Arg His Ala Ser Val Thr Pro Gln 50 55 60Ala
Met Asn Thr Val Leu Arg Lys Leu Glu Asp Ala Gly Ala Val Ala65 70 75
80Arg Pro Ala Ser Val Ser Ser Gly Arg Ser Leu Pro Ala Thr Leu Thr
85 90 95Ala Arg Gly Arg Ala Leu Ala Lys Arg Ala Glu Ala Val Val Arg
Ala 100 105 110Ala Asp Ala Arg Val Leu Ala Arg Leu Thr Ala Pro Gln
Gln Arg Glu 115 120 125Phe Lys Arg Met Leu Glu Lys Leu Gly Ser Asp
130 1356146PRTSalmonella typhimurium 6Met Lys Leu Glu Ser Pro Leu
Gly Ser Asp Leu Ala Arg Leu Val Arg1 5 10 15Ile Trp Arg Ala Leu Ile
Asp His Arg Leu Lys Pro Leu Glu Leu Thr 20 25 30Gln Thr His Trp Val
Thr Leu His Asn Ile His Gln Leu Pro Pro Asp 35 40 45Gln Ser Gln Ile
Gln Leu Ala Lys Ala Ile Gly Ile Glu Gln Pro Ser 50 55 60Leu Val Arg
Thr Leu Asp Gln Leu Glu Asp Lys Gly Leu Ile Ser Arg65 70 75 80Gln
Thr Cys Ala Ser Asp Arg Arg Ala Lys Arg Ile Lys Leu Thr Glu 85 90
95Lys Ala Glu Pro Leu Ile Ala Glu Met Glu Glu Val Ile His Lys Thr
100 105 110Arg Gly Glu Ile Leu Ala Gly Ile Ser Ser Glu Glu Ile Glu
Leu Leu 115 120 125Ile Lys Leu Ile Ala Lys Leu Glu His Asn Ile Met
Glu Leu His Ser 130 135 140His Asp1457166PRTErwinia chrysanthemi
7Met Ala Arg Tyr Leu Glu Val Ser Asp Ile Val Gln Gln Trp Arg Asn1 5
10 15Glu Arg Pro Asp Leu Asp Val Glu Pro Met Leu Val Ile Gly Thr
Leu 20 25 30Ser Arg Val Ser Leu Leu Ile Asp Arg Ala Leu Asp Lys Val
Phe Ser 35 40 45Lys Tyr Lys Leu Ser Ala Arg Glu Phe Asp Ile Leu Ala
Thr Leu Arg 50 55 60Arg Arg Gly Ala Pro Tyr Ala Tyr Ser Pro Ser Gln
Ile Val Asn Ala65 70 75 80Leu Met Ile Asn Asn Ser Thr Leu Thr Ser
Arg Leu Asp Arg Leu Glu 85 90 95Gln Ala Gly Trp Leu Arg Arg Met Pro
Ile Glu Gly Asp Arg Arg Ser 100 105 110Val Asn Ile Gln Leu Thr Asp
Glu Gly Phe Ala Leu Ile Asn Arg Val 115 120 125Val Glu Glu His Val
Glu Asn Glu Arg Asp Ile Leu Ser Pro Phe Ser 130 135 140Glu Glu Glu
Lys Thr His Leu Arg Ala Leu Leu Gly Arg Val Glu Lys145 150 155
160His Leu Val Asn Asn Arg 1658142PRTButyrivibrio fibrisolvens 8Met
Lys Tyr Asp Lys Phe Phe Met Ala Leu Leu Gly Ala Tyr Ala Ala1 5 10
15His Gly Asp Ala Ser Arg Gln Ile Phe Asn Asp Tyr Gly Leu Thr Glu
20 25 30Ala Gln Pro Lys Ile Leu Tyr Ile Leu Gly Phe Asn Glu Gly Ile
Val 35 40 45Gln Lys Asp Phe Ala Lys Leu Cys Ala Ile Lys Pro Ser Thr
Met Thr 50 55 60Val Gln Leu Ala Arg Leu Glu Lys Asp Gly Leu Ile Arg
Arg Glu Ser65 70 75 80Cys Tyr Ile Ser Gly Gly Lys Lys Ala Tyr Arg
Val Tyr Leu Thr Lys 85 90 95Arg Gly Lys Glu Ile Ala Asp Ser Leu Ile
Glu Arg Ile Asp Asn Leu 100 105 110Glu Asp Ile Ser Phe Lys Gly Phe
Thr Ala Lys Glu Gln Ala Thr Leu 115 120 125Leu Ser Leu Leu Glu Arg
Val Glu Asp Asn Leu Arg Gly Arg 130 135 140
* * * * *
References