staphylococcus aureus (sa) plays a pathogenic role in nosocomial

1

Three-dimensional structure of MecI : molecular basis for transcriptional regulation of staphylococcal methicillin resistance *.

Raquel García-Castellanos 1, Aniebrys Marrero 1, Goretti Mallorquí-Fernández 1, Jan Potempa 2, Miquel Coll 1 & F.Xavier Gomis-Rüth 1,3

1 Institut de Biologia Molecular de Barcelona, C.I.D. – C.S.I.C. C/ Jordi Girona, 18 – 26; 08034 Barcelona (Spain).

2 Department of Microbiology, Faculty of Biotechnology, Jagiellonian University, Krakow (Poland) and Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602 (USA).

3 To whom correspondence should be addressed. Tel. +3493 400 61 44; Fax. +3493 204 59 04; e-mail. [email protected]

Running Title: Crystal structure of methicillin repressor

Keywords: Bacterial antibiotic resistance; MecI protein; transcriptional regulatory element; DNA-binding protein; winged helix protein.

* This work was supported in part by grants BIO2000-1659 and BIO2003-00132 (to F.X.G.-R.), and BIO2002-00379 (to M.C.), both from the Spanish Ministry for Science and Technology; and SGR2001-00346 from the Generalitat de Catalunya to M.C.. R.G.C. is recipient of

an FPI Ph.D.-fellowship from the Spanish Ministry for Science and Technology, G.M.F. from the University of Girona, and A.M.N. acknowledges a postgraduate fellowship from “Fundación Carolina”, Spanish Ministry of Foreign Affairs.

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on July 24, 2003 as Manuscript M307199200 by guest on M

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Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is the main cause of nosocomial and community-onset infections that affect

millions of people worldwide. Some MRSA infections have become essentially untreatable by �-lactams due to acquired molecular

machineries enabling antibiotic resistance. Evasion from methicillin challenge is mainly achieved by the synthesis of a penicillin-binding

protein (PBP) of low affinity for antibiotics, MecA, which replaces regular PBPs in cell-wall turnover when these have been inactivated by

antibiotics. MecA synthesis is regulated by a signal-transduction system consisting of the sensor/transducer MecR1 and the 14-kD

transcriptional repressor MecI alias (methicillin repressor), which constitutively blocks mecA transcription. The three-dimensional structure

of MecI reveals a dimer of two independent winged-helix domains, each of which binds a palindromic DNA-operator half site, and two

intimately intertwining dimerisation domains, of novel spiral-staircase architecture and hold together by a hydrophobic core. Limited

proteolytic cleavage by cognate MecR1 within the dimerisation domains results in loss of dimer interaction surface, dissociation and

repressor release, which triggers MecA synthesis. Structural information on components of the MecA regulatory pathway, in particular on

methicillin repressor, the ultimate transcriptional trigger of mecA-encoded methicillin resistance, is expected to lead to the development of

new antimicrobial drugs.

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Introduction

In industrialised countries, as many as 60% of all hospital-acquired infections are caused by drug-resistant microbes, with

derived costs of $10 billion and about 14,000 casualties per year in the US alone. Virulent Staphylococcus aureus strains cause most

nosocomial and community-onset infections by producing toxins and virulence factors, especially in immuno-compromised patients. These

strains may affect skin, urinary and respiratory tracts, soft tissue, burns, ulcers, bone, joints, and the cardiovascular system. In this way,

they cause the potentially lethal toxic-shock syndrome, pneumonia, meningitis, endocarditis, osteomyelitis, and severe invasive and

metastatic infections. They are also responsible for 40-50% of all septicaemias (1). When penicillin was introduced in 1941, the pathogen

could be combated efficiently. Since then, however, bacterial strains refractory to �-lactam antibiotics (BLAs) 1 began to appear (2). These

bacterial strains spread worldwide causing high morbidity and mortality when untreated, which required the development of new drugs, like

penicillinase-resistent semi-synthetic �-lactams (e.g. methicillin), in the 1960s. However, the appearance of new insensible bacterial

strains has continued and even worsened, leading to the appearance of multiple-drug resistance. The glycopeptides vancomycin and

teicoplanin are the last effective drugs for the moment (3).

Methicillin-resistant S. aureus (MRSA) accounts for more than 30% of all pathogenic S. aureus isolates (3). It has caused several

outbreaks in acute-care and nursing wards since its appearance in 1961, less than one year after the introduction of methicillin (4). MRSA

remains endemic in many hospitals due to the selective pressure created by the therapeutic and prophylactic use of antibiotics, and its

incidence is increasing in many countries (5). Furthermore, outbreaks of non-nosocomial MRSA infections are becoming more common,

posing a serious public-health threat. Since 1996, strains have appeared that are essentially untreatable by BLAs, vancomycin or latest-

generation antibiotics (6). Recently, an international consortium has set up a database of epidemic MRSA strains to enable early

identification (7). Such efforts must be complemented by discoveries of the molecular mechanisms of methicillin resistance and by novel

therapeutic alternatives to control and defeat the pathogenic agent responsible.

Constitutive penicillin-binding proteins (PBPs) are membrane-bound penicillinoil serine D,D-transpeptidases, structurally related

to �-lactamases, which cross-link the peptidoglycan in the cell wall of Gram-positive bacteria (8,9). BLAs target PBPs on the outer surface

of the cytoplasmic membrane due to structural analogy with their natural substrate, D-alanyl-D-alanine-terminated peptides. PBPs are

acylated by BLAs at their active-site serine residues, subsequently deacylating very slowly. This prevents PBPs from performing their

regular functions efficiently, leading to cell wall loosening. This is followed by a non-lytic killing event, and, finally, bacteriolysis (10,11).

1 The abbreviations used are: BLA, �-lactam antibiotic; bp, base pair; CCF, correlation coefficient in structure-factor amplitudes; DBD, DNA-binding domain; DD, dimerisation domain; ds, double-stranded; ESRF, European Synchrotron Radiation Facility; GA, glutaraldehyde; HTH, helix-turn-helix motif; LB, Luria-Bertani; MRSA, methicillin-resistant Staphylococcus aureus; PBP, penicillin-binding protein; PDB, Protein Data Bank; SCCmec, staphylococcal chromosome cassettes mec element; SeMet, selenomethionine; TEV, tobacco etch virus; VM, Matthews-parameter, crystal volume per unit of protein molecular mass.


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Bacterial resistance occurs mainly through excreted �-lactamases (penicillinases), which inactivate penicillins by hydrolysis of their �-

lactam ring (12). MRSA, however, bases its resistance on the production of MecA (alias PBP2a and PBP2’). This enzyme is a facultative

PBP with reduced �-lactam affinity at the clinically achievable drug concentrations and is capable of essential cell-wall construction when

the housekeeping �-lactam-sensitive PBPs are switched off (13). In this way, MecA confers imperviousness to all BLAs. It is encoded by

mecA, localised within 32-to-60-kb DNA regions called mec complexes or staphylococcal chromosome cassettes mec (SCCmec elements;

(14,15)), mobile elements that can also encode resistance to non-�-lactam antibiotics. SCCmec elements have been acquired through

horizontal transfer from an unknown heterologous source (16). MecA-synthesis is regulated by a signal-transduction system consisting of

an integral-membrane zinc-dependent metalloprotease sensor/signal transducer, MecR1, and a constitutive transcriptional repressor,

methicillin repressor MecI, both located immediately upstream from the mecA promoter on mec elements and counter-transcribed

(4,17,18). The MecR1-MecI-MecA system has a close functional and structural relative encoded by the �-lactamase divergon (bla), the

BlaR1-BlaI-BlaZ (alias BlaR-BlaI-BlaP or PenJ-PenI-PenP) protein triad (17,19). In this case, the encoded effector is a �-lactamase, BlaP

alias BlaZ or PenP (20).

MecR1 is present in S. aureus and Staphylococcus sciuri, whereas BlaR1 (alias BlaR, PenR1 or PenJ) has been found in

Bacillus licheniformis, Staphylococcus epidermis, Staphylococcus haemolyticus and several S. aureus strains (21). These proteins are

either plasmid-encoded, chromosomal or transposon-mediated. MecR1/BlaR1 proteins are made up by homologous N-terminal ~330-

residue transmembrane metalloprotease domains linked to extracellular ~260-residue homologous PBP-like penicillin-sensor moieties (22).

MecI/BlaI repressors are encoded by the same operon as MecR1/BlaR1. They are ~125-amino-acid proteins found in a number

of bacterial genomes (Fig. 1) and are highly homologous to each other, with S. aureus MecI and BlaI sharing 61% and S. aureus MecI and

B. licheniformis BlaI/PenI 43% sequence homology (23). These proteins are dimeric and may interact with cognate operators as dimers.

These repressors have been proposed to consist of an N-terminal DNA-binding domain (DBD), featuring a helix-turn-helix (HTH) motif, and

a C-terminal dimerisation domain (DD) (24,25). BlaI binds to two separate palindromic operators in S. aureus (R1-dyad and Z-dyad)

encompassing the –10 region of blaZ and the –35 of blaR1-blaI, though not co-operatively, independently and not inducing any significant

DNA bending (19,26). For B. licheniformis, binding to three bla/pen regions has been reported, two including the blaZ/P and one the blaR1-

blaI promoters (25,27). Finally, MecI binds to an extended region containing two consecutive palindromes and covering the mecA -10 and

the mecR1-mecI –35 promoter sequences (18). The regulatory regions of the bla and mec elements are 57% identical (17). Indeed, both

repressor systems seem to be interchangeable to block transcription of both structural and regulatory genes in S. aureus (28,29). The

sensor/transducers BlaR1 and MecR1, however, are not interchangeable and they have distinct kinetics: while S. aureus BlaR1 induces

BlaZ synthesis within minutes, MecR1 takes hours to induce synthesis of MecA (9).


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Based on biochemical data mainly from the bla divergon proteins, the MecR1/MecI system is thought to work as follows. Dimeric

MecI constitutively blocks mecA transcription via inhibition of mRNA synthesis initiation or elongation. Through recognition of regulatory

regions of the counter-transcribed mecR1-mecI operon, the repressor also regulates its own transcription and that of the signal

sensor/transducer. MecR1 detects BLAs in the extracellular space via its PBP-like penicillin-sensor. Upon protein acylation, a

conformational change within MecR1 leads to autocatalytic activation of the integral-membrane metalloprotease domain. The active

protease facing the cytosol specifically cleaves MecI, directly or indirectly. In the case of the BlaR1-BlaI-BlaZ system, some authors have

postulated that a further, chromosome-encoded regulatory element, BlaR2, is required for BlaI inactivation (25,30). This system highlights

the key role of methicillin repressor as the eventual transcriptional regulator of MRSA response (14,18,31-34).


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Experimental procedures

Cloning and overexpression of MecI

S. aureus strain N315 (MRSA) bacterial cells were grown in Luria-Bertani (LB) broth and lysed with lysostaphin (25 �g/ml). The

DNA was extracted as described (35) and used as a template for PCR amplification of the MecI coding sequence. Taq polymerase

(Fermentas) was used with oligonucleotides 5’-GTTAATACCATGGATAATAAAACGTATG-3’ as forward primer and 5’-

AAACACAACTAGTTTATTTTTTATTCAAT-3’ as reverse primer (purchased from Roche). The reaction yielded a 393-base-pair (bp)

fragment containing the correct coding sequence within restriction sites for NcoI (5’) and BcuI (3’). After digestion with these enzymes, the

fragment was inserted into expression vector pPROEXTM Hta (Life Technologies). DNA sequencing confirmed that the MecI-coding

sequence was properly placed after the sequence encoding the 6His-tag and the tobacco-etch virus (TEV) protease recognition site.

Reactions with restriction endonucleases (Fermentas) and ligation with T4 DNA ligase (Roche) were performed as recommended by the

suppliers. The recombinant plasmid obtained was introduced into E. coli DH5� cells by heat-shock treatment. Freshly transformed cells

were cultured overnight in LB medium containing 100 �g/ml ampicillin. For overexpression, 4 ml of these cultures was added to 500 ml of

LB medium, also 100 �g/ml in ampicillin. Cells were grown at 37ºC and induced with 1mM isopropyl �-D-1-thiogalactopyranoside at an

OD600 of 0.5. After 3 h incubation at the same temperature, cells were harvested by centrifugation and frozen. The selenomethionine

(SeMet) variant was obtained in the same way, except that the cells were added to 500 ml of minimal medium lacking methionine and

implemented with 25 mg of SeMet (Sigma) 30 min before induction.

Purification and crystallisation of MecI

Pelleted cells were gently melted at 4ºC, resuspended in 30 ml of buffer A (20 mM Tris-HCl pH 8.0, 0.5 M NaCl, 20 mM

imidazole) and disrupted by sonication. The soluble fraction was separated by ultracentrifugation and the supernatant was passed through

0.22 �m filters and loaded onto a Hi-Trap chelating column (Pharmacia), charged with Ni2SO4 and equilibrated with buffer A. The 6His-

TEVsite-MecI fusion protein (17.8 kD) was eluted with a linear gradient of buffer B (20 mM Tris-HCl pH 8.0, 0.5 M NaCl, 400 mM

imidazole). The total protein concentration (9 mg/ml) was measured according to Bradford (Bio-Rad) using lysozyme as a standard. EDTA

and 1,4-dithio-D,L-threitol were added to the fractions containing the fusion protein to a final concentration of 0.5 and 1 mM, respectively.

TEV protease was added at a 1:500 (protease:fusion protein) molar ratio to remove the 6His-tag. After overnight digestion at 22ºC, the

sample was subjected to gel filtration on a Superdex 75 HR 10/65 column (Pharmacia) equilibrated with buffer C (20 mM Tris-HCl pH 7.4,

0.2 M NaCl). 3 ml of MecI (14.9 kD) in buffer C was obtained at a final concentration of 1.25 mg/ml (as calculated with the experimentally

determined molar extinction coefficient, �=35413 M-1 cm-1) and stored at 4ºC. The purity of the protein was assessed by SDS-PAGE and


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mass spectrometry and its N-terminus was found to be GAMDNK (amino-acid one-letter code) by chemical sequencing, including a

glycine-alanine dipeptide from the TEV-protease recognition sequence prior to the first methionine of native MecI. The protein crystallised

spontaneously in the chromatography collection tubes overnight at 4ºC in the form of well-shaped, ordered crystals. The same crystals

were obtained with SeMet-MecI.

DNA-binding assay

To test the DNA-binding capability of MecI, two complementary oligonucleotides (5’-CAAAATTACAACTGTAATATCGGAG-3’ and

5’-GCTCCGATATTACAGTTGTAATTTT-3’; purchased from MWG Biotech AG) were annealed in buffer D (20 mM Tris-HCl pH 7.4, 0.1 M

NaCl) to render 180 nmol of a 25-bp double-stranded (ds) DNA comprising the sequence protected from DNase I attack of the Z-dyad of

the bla divergon (19), with an additional one-bp overhang on either side (C/G). Purified MecI (85 �M) in buffer C was mixed with DNA in

buffer D at a 2:1.1 protein:dsDNA molar ratio. The mixture was diluted 1:4 in 20 mM Tris-HCl pH 7.4 and incubated 15 minutes at 4ºC

before being loaded onto a gel filtration Superdex 75 HR 10/65 column equilibrated with buffer E (20 mM Tris-HCl pH 7.4, 50 mM NaCl). A

single peak was obtained and analysed with a band-shift assay in a 5% acrylamide gel with 5% glycerol and 50 mM Hepes pH 8.3, using

annealed oligonucleotides as a negative control.

Analytical gel filtration and cross-linking experiments

To assess the concentration-dependent aggregation states of MecI, a peak fraction of the last purification step, which rendered

crystals, was diluted with buffer C and subjected to analytical gel filtration on a Superdex 75 HR 10/65 column previously calibrated with

protein markers of known molecular mass and equilibrated with buffer F (50 mM Hepes pH 7.4, 0.2 M NaCl). Resulting peaks revealed by

SDS-PAGE a single band corresponding to a protein monomer.

140 pmol of purified MecI was cross-linked at room temperature with glutaraldehyde (GA) at different concentrations in a total

volume of 50 �L of buffer for 2 minutes, followed by quenching with 12 �l of 50 mM Tris-HCl pH 6.8. The products were resolved on 15%

SDS-PAGE and visualised by silver staining.

Structure solution and refinement

Native MecI crystals belong to the orthorhombic space group P212121, harbour one MecI dimer per asymmetric unit (Matthews-

parameter (VM)= 3.1 Å3/Da; 59.5% solvent content; (36)), and diffract beyond 2.4 Å resolution. SeMet-derivatised protein crystals are fairly

isomorphous to the native ones and diffract to 2.8 Å resolution (Table 1). A cryo-protecting protocol was established consisting of soaking

crystals in a mixture containing crystal supernatant protein solution and increasing (5%-steps) glycerol concentrations (up to 25% v/v) at 4


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°C, allowing the crystals to equilibrate for 15 min after each step. Complete diffraction datasets were collected from a single N2 flash-cryo-

cooled (Oxford Cryosystems) crystal each on an ADSC Quantum4 charged-coupled device detector at beamline ID29 of the European

Synchrotron Radiation Facility (ESRF; Grenoble, France). For the SeMet-derivative, a multiple-wavelength anomalous diffraction

experiment was carried out at three wavelengths, corresponding to the absorption maximum (12662.53 eV)), the inflection point (12660.95

eV) and a hard-remote wavelength (12900 eV). These values were determined from the crystal to be measured by means of a

fluorescence spectrum around the theoretical selenium K-edge value. All diffraction data were processed with program MOSFLM v. 6.2.2

and scaled, merged and reduced with SCALA, within the CCP4-suite of programs ((37); see Table 1).

The heavy-atom model of the SeMet derivative could not be determined in P212121 by any procedure. All tests for hemihedral

twinning were negative. Consequently, the diffraction data were reprocessed as P21, choosing the short axis as unique axis b. With this

setting, 10 out of 16 theoretical selenium-sites were found with SOLVE v. 1.18 (38), giving a figure of merit of 0.33 for the resolution range

of 50 to 2.9 Å (see Table 1). Subsequent density modification by means of DM within CCP4 increased this value to 0.66, rendering a map

where some helical segments could be identified. The four molecules in the P21 asymmetric unit were delimited and non-crystallographic-

symmetry operators were calculated. A second DM step, including averaging, delivered a �A-weighted electron-density map (Fig. 3a),

which enabled straightforward tracing of the whole polypeptide chain employing a Silicon-Graphics graphic workstation and program

TURBO-FRODO (Biographics, Marseille). A model comprising a dimer was submitted to a molecular replacement calculation, employing

program AMORE (39), against the high-resolution native dataset processed as P212121 (15-to-4 Å resolution range). A unique solution was

obtained at 81.3, 67.9, 105.2, 0.2248, 0.4739, 0.4103 (�, �, �� in Eulerian angles; x, y, z, as fractional unit-cell co-ordinates) with a

correlation coefficient in structure-factor amplitudes (CCF) of 72.4% and a crystallographic Rfactor of 34.5% (for definitions, see Table 1 and

(39); second highest peak, CCF 49.6, Rfactor 47.0%). This calculation confirmed P212121 and ruled out those other primitive orthorhombic

space groups. Manual model building alternated with crystallographic refinement utilising CNS v. 1.1 (40) and REFMAC5 (including TLS

refinement) within CCP4, until the final model was obtained. It features protein residues Lys4A-123A and Met1B-Lys122B, corresponding

to monomers A and B, two glycerol molecules (Gol201W and Gol202W), one chloride anion (Cl1203W), and 69 solvent molecules

(Hoh204W-Hoh272W). All residues are placed in allowed regions of the Ramachandran plot, except for Lys23 of each polypeptide chain

(��=64º, ��=-61º; ��=72º, ��=-57º). These residues and their side chains are, however, unambiguously defined by electron

density at the end of helices �1.

Miscellaneous

Figures were prepared with TURBO-FRODO, SETOR (41), and GRASP (42). Superimpositions were performed with TURBO-

FRODO, cavities were ascertained with GRASP, close contacts and interaction surfaces were calculated with CNS. Three-dimensional


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protein structure comparisons were done with the DALI-server at www.ebi.ac.uk/dali. The final co-ordinates have been deposited with the

Protein Data Bank (PDB) at EBI, Hinxton (UK), at www.ebi.ac.uk/msd (access code XXXX).


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Results and discussion

Oligomerisation behaviour and DNA binding of methicillin repressor in vitro.

It has been shown that BlaI from both S. aureus and B. licheniformis forms dimers in solution (24-26). For MecI, oligomerising

behaviour was observed and mixed BlaI/MecI heterodimers have been described (9,18). Analytical size-exclusion chromatography on a

calibrated column showed a concentration-dependent monomer/dimer equilibrium in the conditions assayed (pH 7.4, 0.2 M NaCl). At

concentrations above 1 mg/ml (67 �M), which eventually lead to crystallisation, the protein is almost completely dimeric (Fig. 2a). Under

progressive dilution, a peak corresponding to a monomer appears, which becomes single at 4 �M. These values are in accordance with B.

licheniformis BlaI, for which a dissociation constant of 25 �M was estimated by ultracentrifugation (25), and are corroborated by cross-

linking experiments. Fig. 2b shows that increasing concentrations of glutaraldehyde resulted in the formation of dimers with an apparent

molecular mass of ~30 kD.

A 43-bp region within the mec regulatory region fragment encompassing two consecutive 15-bp inverted repeats and the

promoter-operator sequences for mecR1-mecI and mecA is protected by methicillin repressor and by BlaI from DNase I attack (9,18).

Though MecI represses mecA transcription more strongly than BlaI, no differences are found with shorter synthetic mec and bla operator

sequences (43). The protected region corresponds to two successive palindromes and suggests binding of several MecI molecules to this

elongated stretch of dsDNA. Other studies have revealed that both S. aureus BlaI and MecI bind identically and protect two regions of 25

bp (R1-dyad; affects blaI-blaR1 promoter) and 24 bp (Z-dyad; around blaZ promoter) of the bla divergon (19). In this latter case, a 5-bp

spacer is found between the two regions. With the future aim of obtaining crystals of MecI in complex with dsDNA, the better delimitated

and shorter sequence around the Z-dyad of the bla divergon was chosen to perform a band-shift assay. To this end, protein and annealed

25-bp oligonucleotide were mixed in an approx. 2:1 (protein:DNA) molar ratio and subjected to size-exclusion chromatography. A single

peak was obtained, with no indication of excess of either protein or DNA. The purified complex analysed by native PAGE showed different

migratory properties from control DNA (see Fig. 2c). Taken together, these results indicate that MecI binds to the 25-bp dsDNA as a dimer,

confirming previous studies.

Overall structure and oligomerisation state

The methicillin repressor protomer is elongated and asymmetric, with approx. overall dimensions of 60 x 45 x 30 Å (see Fig. 3b)

and, as predicted, is subdivided into an N-terminal compact globular domain, MecI-DBD (residues Met1-Val73; see Fig. 1), and a C-

terminal domain, MecI-DD, with the shape of a spiral staircase (residues Glu74-Lys123).


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MecI-DBD starts with a segment in extended conformation and is stabilised by intramolecular interactions from Tyr6 onwards. At

Ser10, the chain enters helix �1. This regular secondary structural element is followed by a short segment in extended conformation, loop

�1�2 (Tyr24-Ser26). At Ala25, a double main-chain interaction anchors this stretch with Tyr69 of a �-ribbon (see below). Helix �2 starts at

Ser26, with its axis rotated by 60 ° clockwise with respect to �1. At Lys38, helix �2 ends and the main chain starts at Ser41 with helix �3

after a short three-residue spacer. This helix, running until Lys55, runs with its axis rotated 135 ° counter-clockwise rotated away from �2.

Both helices �1 and �2 contribute to the presentation of the following helix to the molecular surface. A glycine-mediated change in the

direction of the main-chain after �3 leads to the first residue of a �-ribbon structure. This is made up by strand �1 (Phe57 to Asp63),

followed by a tight 1,4 turn of type I, and a second strand, �2 (Ile66-Ser71). A total of seven inter-main chain hydrogen bonds stabilise this

ribbon, conferring it a rigid structure. MecI-DBD finishes at Val73 after �2.

The overall fold topology of MecI-DBD is �1-�2-�3-�1-wing-�2, in accordance with a “winged-helix” architecture, first identified

in the complex of hepatocyte nuclear factor-3 DBD with DNA (44). It is common to many prokaryotic and eukaryotic transcription factors,

and had been partially anticipated for BlaI (25). This fold is characterised by a �1-�1-�2-�3-�2-wing1-�3-wing2 topology (45). In our

structure, �1 is reduced to just one residue (Ala25, see above), while the position of the second wing is occupied by the first helix of the

MecI-DD (see below). The winged-helix topology encompasses a central HTH motif, identified for the first time in bacteriophage � cro

repressor (46), modified by variations in the length of the connecting turn and the angle between the helices. It is engaged in DNA major-

groove recognition and made up in MecI by helices �2, loop �2�3, and �3 (“recognition helix”). The fold further harbours a “wing”, in most

cases engaged in minor- and/or major-groove interactions (45,47), and constituted in MecI by the tip of the �-ribbon. The MecI-DBD

globular structure is compact and maintained by an extended hydrophobic core traversing the domain (Fig. 3b). The side chains of Ile8,

Val15, Met16, Ile18, Ile19, Ala25, Leu30, Ile31, Ile34, Trp40, Ile45, Ile49, Leu52, Phe57, Leu58, and Val73 participate in this core, which is

delimited on its left by Tyr69, close to the molecular surface, and on its right by both Trp13 and Trp40. The latter two indole rings create a

hydrophobic interface with the DD.

MecI-DD starts at Glu74 and is a right-handed superhelical tail consisting of three consecutive helices, �4 to �6 (Figs. 3b-d).

These are not totally equivalent within each monomer and somewhat flexible, in particular around the C-terminal helix. Helix �4 protrudes

from the DBD moiety and continues to Tyr91. At this residue, a tight 1,4-turn of type II features loop �4�5, prior to �5 (Phe95-Glu106),

with its axis rotated clockwise 90 ° with respect to �4. At its end, loop �5�6 consists of two residues and links it with the C-terminal helix

�6. This helix runs roughly antiparallel to �5 and extends to Asn121. The side chains of the three helices facing the central superhelical

axis are mainly hydrophobic (Tyr80, Phe 86, Ile87, Val90, and Tyr91 from �4; Leu98, Val99, Phe102, Val103 from �5; and Leu108,


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Ile113, Leu116, Ile119, and Leu120 from or before �6). This structure, together with the lack of stabilising interactions between these

helices, explains why dimers predominate at higher protein concentrations whereas only monomers are found in diluted samples.

The functional and stable structural unit is a dimer, as present in the asymmetric unit, with the overall shape of a triangle, 75 Å in

width, 45 Å in height, and 35 Å in depth (Fig. 3c). A total of 63 inter-monomeric close contacts (< 4 Å) are observed, among them 18

hydrogen bonds, 20 van-der-Waals contacts, and 3 salt bridges. Dimerisation occludes 3,677 Å2 or 40% of the total surface area of a

monomer, and is almost perfectly self complementary (see Fig. 4a). Both protomers display close structural similarity, as indicated by an

r.m.s.d. of 0.47 Å for the 90 C� atoms deviating less than 1 Å (Fig. 3b). Among the DBDs, this equivalence is almost total, while the DDs

display a certain degree of variability with greater deviations, in particular around the C-terminal helices �6. Within the dimer, the globular

DBDs are adjacent to each other but not in contact, apparently well situated to function independently (Figs. 3c,d; see below). The dimeric

structure is mainly maintained by the DDs, which display a previously unobserved arrangement. They are closely juxtaposed and

intertwine like two surface- and side chain-complementary protein superhelices in such a manner that three layers are formed, each made

up by the equivalent helices of each monomer running in an antiparallel manner. Within each layer, the interior side chains interdigitate in a

zipper-like fashion (Figs. 3a,c,d and 4a). The first layer, consisting of helices �4, is rotated 90 ° counter-clockwise with respect to layers

two and three, which are constituted by helices �5 and helices �6, respectively, which are parallel. This three-layered structure is held

together by an elongated hydrophobic core running from the interface between helices �4 on one end to the interface between helices �6

at the other end. In particular, residues Phe86, Ile87, Tyr91, Phe95, Leu98, Val99, Phe102, Val103, Leu108, Ile113, Leu116, Ile119, and

Leu120 participate in van-der-Waals contacts. This inner core is, however, not completely solid but shows two cavities, of 117 and 22 Å3

(Fig. 4e), which may account for the certain flexibility observed within the DDs. Besides residues from the DDs, two side chains at the

beginning of helix �1 of each monomer, Trp13 and Asn17, also participate in the dimerisation interface, mainly with the last turn of helix

�4. In particular, Asn17 C-caps it through its side chain.

Similarities with other proteins and working hypothesis for the recognition of operator DNA

MecI shares structural similarity with DNA-binding proteins harbouring the winged-helix motif (45,47). The closest similarity is

encountered with MarR and SmtB, despite negligible sequence similarity (12% and 6%, respectively, in the aligned stretches). MarR is a

regulator of multiple antibiotic resistance from E. coli (PDB 1jgs; (48)) and SmtB a metal-tuneable trans-acting dimeric transcriptional

regulator from Synechococcus, which represses its own synthesis and that of metallothionein (PDB 1smt; (49)). This topological

equivalence is restricted, however, to the winged-helix DBDs and ranges from helix �1 to the beginning of the dimerisation helix �4 of

MecI (Fig. 4c). The dimeric arrangement is different in all three proteins. In both MarR and SmtB, the recognition helices are not properly

positioned for operator recognition in the structures described. On the other hand, the DD of MecI displays a novel fold, which merely


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bears a topological similarity with a three-helix segment of a functionally-unrelated archaeal endonuclease, I-DmoI. This three-helical

segment does not, however, form a distinct folding unit in I-DmoI but belongs to two separate domains (PDB 1b24).

Biochemical studies indicate that dimeric MecI binds to its operator dyads in such a way that each monomer recognises one half

of the operator, while the central sequence is unprotected. This goes along with a lack of DNA bending upon protein binding (19). Within

the dimer, the putative recognition helices �3 of the HTH motif are solvent-presented in parallel with a spacing of about 30 Å, geometrically

appropriate to interact with the B-DNA major groove, and the wings are positioned so as to interact with the adjacent minor groove (Fig.

3c). The surface potential indicates that the exposed surfaces of helices �3 and those of the lateral wings are positively charged, thus

electrostatically suited to recognise the B-DNA backbone (Figs. 4a,b), as observed in other winged-helix DNA-binding proteins (45).

Despite limited overall topological similarity, the positioning, orientation and distance of the recognition helices of the winged HTH-motifs

are equivalent to those observed in the structure of Corynebacterium diphtheriae toxin in its complex with its cognate tox operator. This

protein shares with MecI the general winged-helix DBD architecture (Fig. 4d) but shows a completely unrelated nickel-dependent DD (PDB

1ddn; (50)). In the latter complex, one diphtheria toxin dimer binds on one side to the major groove of a 19-bp dsDNA at two successive

turns of the double helix and a second dimer binds independently on the opposite side of the DNA helix. The wing, shorter than in MecI,

only establishes one interaction with the phosphodiester backbone in the minor groove, mediated by an arginine. Diphtheria toxin binds its

cognate operator without causing significant global bending, with equivalent positions of the recognition helices 28 Å away. A similar value

(30 Å) has been reported for HTH replication terminator protein in a DNA-complex (PDB 1f4k).

Based on these similarities, a working model for the interaction of MecI with its cognate dsDNA can be constructed using the

dimer in our unliganded structure and canonic B-DNA encompassing the target sequence of the bla Z-dyad (Fig. 4f; (19)). According to this

model and on looking on just one half of the operator, Lys65 and Ile66 from the tip of the wing could be engaged in interactions with the

phosphate backbone or with the O2 atom of one of the three consecutive thymine bases found in the 3’-end of the complementary strand

of the operator (Fig. 4f; �). This would imply only a minor rearrangement of the wing to fit snugly into the dsDNA minor groove, as

observed in the DNA-bound structure of the DBD of signal-transduction transcriptional activator PhoB (51). At the N-terminus of helix �1,

the phosphate backbone around positions 11-12 of the coding strand could complement the side chain of Ser9 in N-capping the regular

secondary structure element, the same could occur with the N-terminus of helix �2 with the complementary DNA backbone around

position 6 (Fig. 4f; � and �). Alternatively, these serine residues could undergo a 180 °-rotation about their �1 angles and be engaged in

phosphate recognition themselves. Also, the N-terminal Lys4 could play a role in backbone recognition, as anticipated by BlaI mutant

studies (26).

The main interaction with DNA would be established by the recognition helix �3, at the centre of one dyad half. In particular,

solvent-exposed side chains in the unbound structure would be engaged in nucleoprotein-complex stabilisation as follows (Fig. 4f; �).


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Lys43 could target the phosphodiester backbone or a guanine or thymine base at complementary strand positions 7-9; Arg46 could contact

the backbone or coding-strand thymine at position 6; Thr47 and Thr50, placed at the centre of the recognition helix, could recognise

thymine O4 or guanine O6 atoms of complementary-strand positions 8-10; Arg51, could interact with the backbone or with complementary-

strand bases; finally, Lys54 and Lys55 could bind backbone phosphate oxygen atoms.

Interactions of the left-hand side of the operator would be symmetric. This model would not entail any significant bending and it

would leave the central guanine at position 13 freely accessible, in accordance with biochemical experiments (19). The DBDs would bind

independently, with no interactions between them. Oligomerisation on longer dsDNA is not ruled out, as in the case of the segment

encompassing the two consecutive operators within the mec region (see above). However, co-operativity seems unlikely, as it would have

to account for intensive interactions between adjacent dimers via the wings, which are more probably engaged in DNA recognition. Some

interactions between adjacent wings can, however, not be ruled out.

Limited proteolysis inactivates methicillin repressor

BlaI and MecI are specifically cleaved at a single bond between Asn101 and Phe102 by activated BlaR1 or MecR1 (34). This

limited proteolysis explains why both MecR1/BlaR1 (which are also autoproteolytically activated) and MecI/BlaI both require synthesis

induction, as they are turned over during the signal-transduction process (26). Cleavage disables dimerisation and the repressing

capability of MecI, thus allowing transcription of mecA. Once the extracellular BLA concentration diminishes, proteolytic cleavage of MecI

ceases, and the intracellular concentration of the full-length inhibitor molecule increases, suppressing unnecessary MecA synthesis.

(9,25,26,30,31,33,34). The scissile bond is strongly conserved among members of the MecI/BlaI family (Fig. 1) and fully affects MecI-DD,

as it is located in the middle of helix �5 (Figs. 3c,d). Interestingly, the scissile bond is not directly accessible from the exterior. This may

explain the requirements for another component, such as BlaR2, which may melt �5 locally and thus facilitate access by MecR1/BlaR1

metalloproteinase (18,30,52). After proteolytic cleavage, the interaction surface would be reduced to 43% of the intact dimerisation surface,

with only 28 close contacts. This drastic reduction in interactions, together with the two cavities observed (Fig. 4e) in MecI-DD, contributing

to a certain sponginess (see above), would ultimately result in the dimer coming apart, as the DBDs recognising each half of the dyad do

not interact. A further conceivable mechanism foresees that the repressor is not stoichiometrically cleaved, in accordance with previous

suggestions (26,33). In this case, a heterodimer containing a wild-type monomer and a free DD could be present in the cytoplasm, which is

unable to bind DNA. In either case, failure to recognise its target sequences would result in transcription of mecA, but also of mecR1 and

mecI.


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Conclusion

Because a protein HTH motif recognises the major groove of dsDNA only through one face by means of its recognition helix, only

five or six base pairs may be directly contacted. Accordingly, DNA-binding proteins harbouring one copy of this motif need to oligomerise to

recognise larger operators. A further restriction is introduced by the nature of the cognate sequence. Palindromes or pseudosymmetric

operators mostly require the binding of protein molecules to homo-oligomerise also employing a dyad, which requires self-complementary

protein interaction surfaces. Transcription factors binding DNA as dimers may either dimerise first and then associate with DNA or two

monomers can bind sequentially and assemble their dimerisation interface while binding DNA. Based on equilibrium ultracentrifugation and

chemical cross-linking experiments, it has been suggested that B. licheniformis BlaI/PenI binds as a preformed dimer and co-operatively

when recognising the different operators within the bla divergon (24,25). The dimeric arrangement of methicillin repressor described here

confirms our hypothesis and others findings (19,24-26) that S. aureus MecI -and therefore probably also BlaI and other members of the

MecI/BlaI family of (putative) transcriptional regulators, see Fig. 1- may also bind as a preformed dimer. An additional argument reads that

it is actually dimer disruption through limited proteolysis, which disables DNA binding. For BlaI it has been shown, that it fails to retard a

synthetic dsDNA encompassing just one half site of the palindrome (26).Therefore, it is unlikely that a monomer can bind to an operator

half site and then recruit the second monomer. More likely, MecI interacts as a preformed dimer with target DNA with each DBD

recognising one half of the pseudo-palindromic sequence and without any interaction between them. In the case of mec, the two dyads

recognised by MecI are consecutive, with a distance of 10 bp between their centres (18). This positions the operators on the same face of

the DNA helix. If each operator is recognised by a repressor dimer, interaction should be possible between the wings in the minor groove.

In the case of bla, there is a complete turn between the recognised R1- and Z1-dyads. Interaction between the dimers seems thus unlikely,

in particular if the dsDNA is not bent (19). This is in agreement with the separation of the probable recognition helices �3.

The current structure of methicillin repressor, the ultimate transcriptional regulator of mecA-mediated methicillin resistance in

MRSA, provides the first step in the understanding of the molecular basis of its function and underlying regulatory process, as well as

those of related proteins of the BlaI/MecI family. This information may work the switches to develop novel therapies as drugs disrupting this

regulatory pathway responsible for resistance rescue the effectiveness of BLAs against MRSA.


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Acknowledgements

S. aureus strain N315 (MRSA) was kindly provided by K. Hiramatsu and co-workers at Juntendo University, Japan. M. Espinosa

and G. del Solar from the Centro de Investigaciones Biológicas, C.S.I.C., Madrid, supervised MRSA bacterial growth and preparation of

DNA. The help provided by EMBL and ESRF synchrotron local contacts during data collection, in particular by W. Shepard, is further

acknowledged (ESRF, Grenoble). Robin Rycroft, Pablo Fuentes-Prior, and Maria Solà are thanked for critical review of the manuscript.


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Legends to the Figures

Fig. 1. MecI/BlaI family of transcriptional repressors. Sequence alignment of identified and putative members (based on

bioinformatic genome searches) of the ~125-residue MecI/BlaI family. For each member, the SwissProt/TrEMBL access code is provided

(www.expasy.ch). Amino-acid numbering, position and extend of the two subdomains and the regular secondary structure elements

correspond to MecI of S. aureus strain N315 (MRSA). Conserved residues are highlighted in blue. Tr. Rep. stands for (putative)

transcriptional repressor, Pred. for predicted protein.

Fig. 2. In-vitro studies of MecI. (a) Analytical gel-filtration assay showing the evolution of peak profiles as a function of protein

concentration. The concentration of loaded sample was: A, 1.25mg/ml (75 �M); B, 0.62 mg/ml (42 �M); C, 0.31 mg/ml (21 �M); D, 0.15

mg/ml (11 �M); and E, 0.06mg/ml (4.2 �M). (b) Cross-linking analysis of MecI depending of glutaraldehyde (GA) concentration: A, 0 mM;

B, 0.08 mM; C, 0.8 mM; D, 8 mM; and E, 40 mM. (c) Band-shift assay. The non-denaturing 5% polyacrylamide gel was stained

successively with ethidium bromide (left) and Coomassie Blue (right). Lanes show the presence (+) or absence (-) of annealed DNA and

MecI. The left lane only contains the oligonucleotide (control) and the right one the 2:1 protein/DNA complex.

Fig. 3. Structure of methicillin repressor MecI. (a) Initial experimental Fobs-type �a-weighted electron density map after density

modification and averaging, superimposed with the final refined model around helix �5 of each protomer (contour level 1� above average).

(b) C�-plot of MecI. Both molecules within a dimer are shown, in green and white, after optimal superimposition of their DNA-binding

domains (DBDs). The dimerisation domains (DDs) display a certain degree of flexibility. Selected residues are labelled. (c) Ribbon-plot of a

MecI dimer, with DBDs on top and DDs at bottom in frontal view. Regular secondary structure elements are labelled in orange and the

scissile bond (Asn101-Phe102) is indicated by red arrows (see Fig. 1). Close-up view of residues participating in close contacts (below 4 Å)

between the protomers. Some residues are labelled. (d) Same as (c) but after a vertical 90º-rotation (clockwise).

Fig. 4. MecI dimerisation, similarity of MecI with other proteins and working model . (a) Structure of MecI displaying one protomer

superimposed with its Connolly surface coloured according to electrostatic potential ranging from –20 kBT/e (red) to 20 kBT/e (blue) and the

second protomer as green backbone worm in a lateral (left) and a top view (right). (b) Same type of surface as in (a) but displaying a whole

dimer. (c) Superimposition of monomers of S. aureus MecI (yellow C�-trace), E. coli MarR regulator (green; PDB 1jgs; (48)) and

Synechococcus SmtB repressor (magenta; PDB 1smt; (49)). The N- and C-termini of each polypeptide chain are labelled. MecI and MarR

share 79 topologically equivalent C� atoms (deviating less than 3 Å) showing a r.m.s. deviation of 1.42 Å (out of 119 defined C� atoms in

MecI and 138 within MarR). MecI and SmtB have 68 equivalent C� atoms with a r.m.s. deviation of 1.30 Å (98 residues are defined in the

SmtB chain). (d) Superimposition of the methicillin repressor dimer (yellow) with the functional dimer of diphtheria toxin repressor (cyan;

PDB 1ddn; (50)) as observed in its complex with tox operator. (e) Detail of the dimerisation domains with the two cavities encountered


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shown as green (117 Å3) and blue (22 Å3) surfaces, respectively. (f) Proposed working model of a MecI dimer (green and white backbone

worms) in complex with the 24-bp target sequence protected from DNase I including the Z-dyad of the bla divergon (19,26). The coding

strand is shown as a red ribbon, the complementary one in blue, adenines are represented as red tiles, thymines in blue, cytosines in

magenta, and guanines in green. Discussed structural features in the text are the wing (�), N-cap of helix �2 (�), the recognition helix

(�), and the N-terminal segment and N-cap of helix �1 (�).


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Table 1– Crystallographic statistics ________________________________________________________________________________________________________________________ Dataset MecI SeMet MecI (f´´max) SeMet MecI (f´min) SeMet MecI (remote) Space group P212121 P212121 Cell constants (Å) 66.05, 73.36, 74.68 66.95, 73.54, 73.61 Wavelength (Å) 0.97625 0.97915 0.97926 0.96111 No. of measurements 61,498 151,268 30,442 18,555 No. of unique reflections 14,159 9,399 9,090 5,693 Resolution range (Å) (outermost shell) 66.1-2.40 (2.53-2.40) 74.5-2.80 (2.95-2.80) 73.6-2.80 (2.95-2.80) 74.5-3.30 (3.48-3.30)

Completeness (%) 96.6 (84.7) 99.8 (99.5) 97.0 (83.8) 98.0 (91.8) Anomalous completeness (%) - 99.8 (99.7) 91.1 (65.4) 92.0 (71.1) Rmerge (%) a 0.061 (0.506) 0.089 (0.404) 0.048 (0.393) 0.093 (0.417)

Average intensity (<I / �(I)>) 5.6 (1.4) 4.9 (1.8) 7.1 (1.9) 6.6 (1.8)

B-Factor (Wilson) (Å2) 58.3 94.0 92.8 85.4 Average multiplicity 4.3 (2.1) 16.1 (10.2) 3.3 (2.7) 3.3 (2.8) Ranomalous b - 0.050 (0.156) 0.045 (0.269) 0.076 (0.302) Rderi c,d,e - reference 0.051 (0.170) 0.092 (0.366) Resolution range used for phasing (Å) c 49.4-2.90 Mean figure of merit (fom) before/after DM f,c 0.33/0.66 Resolution range used for refinement (Å) 50.0 – 2.40 No. of reflections used (test set) 13,363 (753) Crystallographic Rfactor (free Rfactor) g 0.218 (0.298) No. of protein atoms (asymmetric unit) 2,051 No. of solvent molecules 69 No. of other atoms 14 (2 glycerol, 1 chloride) R.m.s. deviation from target values bonds (Å) 0.018

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angles (°) 1.56 bonded B-factors (Å2) main-chain/side-chain 2.50/4.12 Average B-factors for protein atoms (Å2) 33.7

________________________________________________________________________________________________________________________ a Rmerge= �hkl�i |Ii(hkl) - <I(hkl)>| / �hkl�i Ii(hkl), where Ii(hkl) is the i-th intensity measurement of reflection hkl, including symmetry-related reflections, and <I(hkl)> its average.

b Ranomalous= �hkl|<I(hkl)> - <I(-h-k-l)>| / �hkl(<I(hkl)> + <I(-h-k-l)>). c calculations were performed in P21, assuming two dimers per asymmetric unit. d Rderi= �hkl ||F PH| - |F P|| / �hkl |F P| with respect to the dataset chosen as a reference (f´´max). e The dataset at the absorption peak (f´´max) was taken as reference. f fom = | F (hkl)best | / | F (hkl) | , with F (hkl)best = ��P (�) F hkl(�) / ��P (�). g Rfactor = �hkl ||Fobs| - k |Fcalc|| / �hkl |Fobs|, with Fobs and Fcalc as the observed and calculated structure factor amplitudes; free Rfactor, same for a test set of reflections (> 500) not used during

refinement. Values in parenthesis refer to the outermost resolution shell if not otherwise indicated.


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DNA-binding “winged-helix” domain (1-73) Dimerisation domain (74-123)

10 �� 23 26 �� 38 41 �� 55 57 �� 63 66 �� 71 74 �� 91 95 �� 106 109 �� 121

1 10 20 30 40 50 60 70 80 90 100 110 120 | | | | | | | | | | | | | MecI (S.aureus N315) P26598 -----------------MDNKTYEISSAEWEVMNIIWMKK-YASANNIIEEIQMQK-DWSPKTIRTLITRLYKKGFIDRKKDN-KIFQYYSLVEESDIKYKTSKNFINKVYKGGFNSLVLNFVEKEDLSQDEIEELRNILNKK------- MecI (S.aureus Mu50) Q932L5 -----------------MDNKTYEISSAEWEFMNIIWMKK-YASANNIIEEIQMQK-DWSPKTIRTLITRLYKKGFIDRKKDN-KIFQYYSLVEESDIKYKTSKNFINKVYKGGFNSLVLNFVEKEDLSQDEIEELRNILNKK-------

MecI (S.sciuri) O54285 -----------------MDNKTYEISSAEWEVMNIIWMKK-YASANYMIEEIQMQK-DWSPKTIRTLITRLYKKGFIDRKKDN-KIFQYYSLVEESDIKYKTSKNFINKVYKGGFNSLVLNFVEKEDLSQDEIEELRNILNKK-------

BlaI (S.epidermis) Q8CNN5 -----------------MANKQVEISMAEWDVMNIIWNKK-SVSANEIVVEIQKNK-EVSDKTIRTLITRLYKKEIIKRYKYN-NIYFYSSIIKEDDIKMKTAKTFLNKLYGGDMKSLVLNFAKNEELNNKEIEELRDILNDISKK----

BlaI (S.aureus N315) Q9AC78 -----------------MTNKQVEISMAEWDVMNIIWDKK-SVSANEIVVEIQKYK-EVSDKTIRTLITRLYKKEIIKRYKSE-NIYFYSSNIKEDDIKMKTAKTFLNKLYGGDMKSLVLNFAKNEELNNKEIEELRDILNDISKK----

BlaI (S.haemolyticus) Q9K4N1 -----------------MANKQVEISMAEWDVMNIIWDKK-SVSANEIVVEIQKYK-EVSDKTIRTLITRLYKKEIIKRYKSE-NIYFYSSNIKEDDIKMKTAKTFLNKLYGGDMKSLVLNFAKNEELNNKEIEELRDILNDISKK----

BlaI (B.licheniformis) P06555 ------------------MKKIPQISDAELEVMKVIWKHS-SINTNEVIKELSKTS-TWSPKTIQTMLLRLIKKGALNHHKEG-RVFVYTPNIDESDYIEVKSHSFLNRFYNGTLNSMVLNFLENDQLSGEEINELYQILEEHKNRKKE-

BlaI (C.acetobutilicum) Q97DN4 ------------------MKSPVKISDAEWSVMQALWKHY-PATFSEIVEGLNEEC-EWSPKTVHTLISRLVKKGVVSTIKDT-KHYKYSPLVTEDEMMNLETESFINKIYHGSVNLFVSNFLKKQKLNKNEILELKKILDENMK-----

BlaI (C.crescentus) Q9A7T0 MRNRSLDGNVYVRNRSLMETTSQRISGAESEVMKVLWADS-PKPAEEVLAILAKDH-GWAEGTVKTLLNRLLKKGAIAADKDG-RRFLYRPLIGREDYVDSESQGLLDRLFDGRLAPLVSHFSKREKLNPEDVAELRRLLERIDDGQ---

BlaI (C.crescentus) Q9A2P9 ----------------------MKISAAESVVMEALWRNS-PLTSEAIMSEVCAPQ-DWTEGTVKTLISRLVKKKAVAAKADG-RRYLYSPLLSRRDYVQGESQGLLDRLFDGRLAPLVMHFSDGDKLSDEDVAALKALVERIGK-----

BlaI (C.crescentus) Q9A854 ----------------------MHITAAEAHVMEALWRRS-PLSADELVAEVGAAQ-SWGEATVKTLINRLLKKKAIKSERAE-GKHGYRPLVDRSAYVQAESQGLLDRLFDGQLAPLISHFAQHRPLKPDEVAKLKKLIDEMGE-----

BlaI (X.axonopodis) Q8PH96 ----------------------MPISDAEAVVMHVLWEDA-PRTAEEVIAALAHTG--WAEPTIKTLLNRLLTKGAVAAEKHG-RKYHYAPLMAREQWVQQQSEGLLQRLFGGRVAPLVAHFSERGKLSASDIAELKRLLQELDDAP---

BlaI (X.campestris) Q8P5X7 ----MTGFEITCVIEKNYGCNRTAISESEAIVMQVLWERA-PRTAEEVVAALAHTG--WAEPTIKTLLNRLLNKGAVAAHKDG-RKYHYAPVLLREHWVQQQSEGLLQRVFGGRVAPLVAWMSQRAEVSDTELAELEALVAKLQSQRKED

Pred. (T.tengcongensis) Q8RAW1 ------------------MKKMPKISESELEVMKIIWELK-QASSSEIVERLVKTT-CWKPKTIHTLINRLVEKGALKAEKSKGKAYIYFPVISEEEYRSHASKTFLERVFNGSLSLMLAHFSARGKLSASDLAELKQLIKDLDDAH---

Tr.Reg.(B.thetaiotaomicron) AAO75742 ------------MKQMKRLTVKEEEIMRIFWEHG-PMFVRELLSFYDEPK--PHYNTVSTLVRGLEEKGFVGYKAYG-NTYQYYALVSEKEYKSSALKEVVSQYYNNSYINVVSSFIKEQKVSKEEIEKLKKLLDEEV------

Pred. (B.thetaiotaomicron) AAO79303 ----------------MKGLTVKEEELMGYFWEKG-PLFVKEMLAFYEEPK--PHFNTLSTIVRGLEDKGFLAHHTFG-NTYQYYPVVSEEDFRKGTLRNVISKYFNNSYLSAVSSFIEEEGMSVDELKSLIEYIEQSKKKK---

Tr.Reg.(B.thetaiotaomicron) AAO75919 ---------------MERLTQQEEEVMIYFWKMG-PSFIREIVNEMPEPK--PPYTSVASVVRNLEKKKFLSPFKLG-NSIQYHVLVKESDYKRSFMNGVVSNYFTGSYKEMVSSLVKEEDISLDDLKRLINEVEQAHQK----

Tr.Reg.(B.thetaiotaomicron) AAO79004 ---------------MEKLTIQEEEVMIYIWELQ-DCFVKDIVSKFPQPA--PPYTTVASIVKNLERKGYVKSKHIG-NTYQYTPSIRENEYKRHFMSGVVRNYFENSYKEMVSFFVRDRKISKKELEDLINIIEDEES-----

Pred. (L.monocytogenes) Q8YAA2 -------------------MAIKSISKSELEVMKIIWDFGRAVQYADVAGKLEEKNYSWKKNTVLTFLTRLVEKNLLSVKKVG-RKNEYYALVSENEYLERQTETFVEEIYEGDVKGLITNLVQNDLISPDELEDLQQFWKRMKSPNE--

Pred. (D.hafniense) Q8RPJ5 -------------------MEQYKLGEMEQKFADLIWHHA-PIPSGELVKLCEKEL-SWKKSTTYTMLKRLCDRGIFENQKGR-----VIALMSREDFTAAQGEQFLSETFGGSLPRFFAAFTRRNKLSAKEINELQQLIDQHKEE----

Cdu1 (C.difficile) P94623 -------------------MCLKKLSKLELVIMKFIWNLD-IKTNSYEIIDYMKEE-HNLPEKVALKTLSKLSKKKFLYVQET-GKCMYYTVAIKEKAYLEFISRNVQNLLKNNFIRNLLVSFHEEELTEEKIKSLENWVVNWEEAYV--

Pred. (X.axonopodis) Q8PR94 -------------------MRGKTIGDQELALLQYIEEQR-HASVGEVAAAFGEPR-GLARSTVLTMMERLRAKGFLRRKQVD-GVYRYSATAGQDDVVRGAVGQFVEKTLQGSVSPFVAFFAKDQKISTDDLKDIIELIEKGKEK----

Pred. (X.campestris) Q8PED6 -------------------MRGKTIGDQELALLQHIEEQR-HASVGEVAAAFGEPR-GLARSTVLTMMERLRAKGFLRRKQAG-GMYRYSATAGQDDVVRGAVGQFVEKTLQGSVSPFVAWMSQRAEVSDTELAELEALVAKLQSQRKED

Fig. 1


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Miquel Coll and F. Xavier Gomis-RüthRaquel García-Castellanos, Aniebrys Marrero, Goretti Mallorquí-Fernández, Jan Potempa,

of staphylococcal methicillin resistanceThree-dimensional structure of MecI: Molecular basis for transcriptional regulation

published online July 24, 2003J. Biol. Chem.

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staphylococcus aureus (sa) plays a pathogenic role in nosocomial

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