NORMAL GENOMIC

• Madoori PK, Agustiandari H, Driessen AJ, Thunnissen AM
• Structure of the transcriptional regulator LmrR and its mechanism ofmultidrug recognition.
• EMBO J. 2009; 28: 156-66
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• LmrR is a PadR-related transcriptional repressor that regulates theproduction of LmrCD, a major multidrug ABC transporter in Lactococcuslactis. Transcriptional regulation is presumed to follow a drug-sensitiveinduction mechanism involving the direct binding of transporter ligands toLmrR. Here, we present crystal structures of LmrR in an apo state and intwo drug-bound states complexed with Hoechst 33342 and daunomycin. LmrRshows a common topology containing a typical beta-winged helix-turn-helixdomain with an additional C-terminal helix involved in dimerization. Itsdimeric organization is highly unusual with a flat-shaped hydrophobic poreat the dimer centre serving as a multidrug-binding site. The drugs bind ina similar manner with their aromatic rings sandwiched in between theindole groups of two dimer-related tryptophan residues. Multidrugrecognition is facilitated by conformational plasticity and the absence ofdrug-specific hydrogen bonds. Combined analyses using site-directedmutagenesis, fluorescence-based drug binding and protein-DNA gel shiftassays reveal an allosteric coupling between the multidrug- andDNA-binding sites of LmrR that most likely has a function in the inductionmechanism.

• Kosinski J, Plotz G, Guarne A, Bujnicki JM, Friedhoff P
• The PMS2 subunit of human MutLalpha contains a metal ion binding domain ofthe iron-dependent repressor protein family.
• J Mol Biol. 2008; 382: 610-27
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• DNA mismatch repair (MMR) is responsible for correcting replicationerrors. MutLalpha, one of the main players in MMR, has been recently shownto harbor an endonuclease/metal-binding activity, which is important forits function in vivo. This endonuclease activity has been confined to theC-terminal domain of the hPMS2 subunit of the MutLalpha heterodimer. Inthis work, we identify a striking sequence-structure similarity of hPMS2to the metal-binding/dimerization domain of the iron-dependent repressorprotein family and present a structural model of the metal-binding domainof MutLalpha. According to our model, this domain of MutLalpha comprisesat least three highly conserved sequence motifs, which are also present inmost MutL homologs from bacteria that do not rely on the endonucleaseactivity of MutH for strand discrimination. Furthermore, based on ourstructural model, we predict that MutLalpha is a zinc ion binding proteinand confirm this prediction by way of biochemical analysis of zinc ionbinding using the full-length and C-terminal domain of MutLalpha. Finally,we demonstrate that the conserved residues of the metal ion binding domainare crucial for MMR activity of MutLalpha in vitro.

• Gu R et al.
• Conformational change of the AcrR regulator reveals a possible mechanismof induction.
• Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008; 64: 584-8
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• The Escherichia coli AcrR multidrug-binding protein repressestranscription of acrAB and is induced by many structurally unrelatedcytotoxic compounds. The crystal structure of AcrR in space group P222(1)has been reported previously. This P222(1) structure has provided directinformation about the multidrug-binding site and important residues fordrug recognition. Here, a crystal structure of this regulator in spacegroup P3(1) is presented. Comparison of the two AcrR structures revealspossible mechanisms of ligand binding and AcrR regulation.

• Wang S, Engohang-Ndong J, Smith I
• Structure of the DNA-binding domain of the response regulator PhoP fromMycobacterium tuberculosis.
• Biochemistry. 2007; 46: 14751-61
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• The PhoP-PhoR two-component signaling system from Mycobacteriumtuberculosis is essential for the virulence of the tubercle bacillus. Theresponse regulator, PhoP, regulates expression of over 110 genes. In orderto elucidate the regulatory mechanism of PhoP, we determined the crystalstructure of its DNA-binding domain (PhoPC). PhoPC exhibits a typical foldof the winged helix-turn-helix subfamily of response regulators. Thestructure starts with a four-stranded antiparallel beta-sheet, followed bya three-helical bundle of alpha-helices, and then a C-terminalbeta-hairpin, which together with a short beta-strand between the firstand second helices forms a three-stranded antiparallel beta-sheet.Structural elements are packed through a hydrophobic core, with the firsthelix providing a scaffold for the rest of the domain to pack. The secondand third helices and the long, flexible loop between them form thehelix-turn-helix motif, with the third helix being the recognition helix.The C-terminal beta-hairpin turn forms the wing motif. The molecularsurfaces around the recognition helix and the wing residues show strongpositive electrostatic potential, consistent with their roles in DNAbinding and nucleotide sequence recognition. The crystal packing of PhoPCgives a hexamer ring, with neighboring molecules interacting in ahead-to-tail fashion. This packing interface suggests that PhoPC couldbind DNA in a tandem association. However, this mode of DNA binding islikely to be nonspecific because the recognition helix is partiallyblocked and would be prevented from inserting into the major groove ofDNA. Detailed structural analysis and implications with respect to DNAbinding are discussed.

• Gorman J, Shapiro L
• Crystal structures of the tryptophan repressor binding protein WrbA andcomplexes with flavin mononucleotide.
• Protein Sci. 2005; 14: 3004-12
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• The tryptophan repressor binding protein WrbA binds to the tryptophanrepressor protein TrpR. Although the biological role of WrbA remainsunclear, it has been proposed to function in enhancing the stability ofTrpR-DNA complexes. Sequence database analysis has identified WrbA as afounding member of a flavodoxin-like family of proteins. Here we presentcrystal structures of WrbA from Deinococcus radiodurans and Pseudomonasaeruginosa and their complexes with flavin mononucleotide. The protomerstructure is similar to that of previously determined long-chainflavodoxins; however, each contains a conserved inserted region unique tothe WrbA family. Interestingly, each WrbA protein forms a homotetramerwith 222 symmetry, unique among flavodoxin-like proteins, in which eachprotomer binds one flavin mononucleotide cofactor molecule.

• Park SY, Chao X, Gonzalez-Bonet G, Beel BD, Bilwes AM, Crane BR
• Structure and function of an unusual family of protein phosphatases: thebacterial chemotaxis proteins CheC and CheX.
• Mol Cell. 2004; 16: 563-74
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• In bacterial chemotaxis, phosphorylated CheY levels control the sense offlagella rotation and thereby determine swimming behavior. In E. coli,CheY dephosphorylation by CheZ extinguishes the switching signal. But,instead of CheZ, many chemotactic bacteria contain CheC, CheD, and/orCheX. The crystal structures of T. maritima CheC and CheX reveal a commonfold unlike that of any other known protein. Unlike CheC, CheX dimerizesvia a continuous beta sheet between subunits. T. maritima CheC, as well asCheX, dephosphorylate CheY, although CheC requires binding of CheD toachieve the activity of CheX. Structural analyses identified one conservedactive site in CheX and two in CheC; mutations therein reduceCheY-phosphatase activity, but only mutants of two invariant asparagineresidues are completely inactive even in the presence of CheD. Ourstructures indicate that the flagellar switch components FliY and FliMresemble CheC more closely than CheX, but attribute phosphatase activityonly to FliY.

• Rumpel S et al.
• Structure and DNA-binding properties of the cytolysin regulator CylR2 fromEnterococcus faecalis.
• EMBO J. 2004; 23: 3632-42
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• Enterococcus faecalis is one of the major causes for hospital-acquiredantibiotic-resistant infections. It produces an exotoxin, calledcytolysin, which is lethal for a wide range of Gram-positive bacteria andis toxic to higher organisms. Recently, the regulation of the cytolysinoperon was connected to autoinduction by a quorum-sensing mechanisminvolving the CylR1/CylR2 two-component regulatory system. We report herethe crystal structure of CylR2 and its properties in solution asdetermined by heteronuclear NMR spectroscopy. The structure reveals arigid dimer containing a helix-turn-helix DNA-binding motif as part of afive-helix bundle that is extended by an antiparallel beta-sheet. We showthat CylR2 is a DNA-binding protein that binds specifically to a 22 bpfragment of the cytolysin promoter region. NMR chemical shift perturbationexperiments identify surfaces involved in DNA binding and are in agreementwith a model for the CylR2/DNA complex that attributes binding specificityto a complex network of CylR2/DNA interactions. Our results propose amechanism where repression is achieved by CylR2 obstruction of thepromoter preventing biosynthesis of the cytolysin operon transcript.

• Lee EH, Rouquette-Loughlin C, Folster JP, Shafer WM
• FarR regulates the farAB-encoded efflux pump of Neisseria gonorrhoeae viaan MtrR regulatory mechanism.
• J Bacteriol. 2003; 185: 7145-52
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• The farAB operon of Neisseria gonorrhoeae encodes an efflux pump whichmediates gonococcal resistance to antibacterial fatty acids. It waspreviously observed that expression of the farAB operon was positivelyregulated by MtrR, which is a repressor of the mtrCDE-encoded efflux pumpsystem (E.-H. Lee and W. M. Shafer, Mol. Microbiol. 33:839-845, 1999).This regulation was believed to be indirect since MtrR did not bind to thefarAB promoter. In this study, computer analysis of the gonococcal genomesequence database, lacZ reporter fusions, and gel mobility shift assayswere used to elucidate the regulatory mechanism by which expression of thefarAB operon is modulated by MtrR in gonococci. We identified a regulatoryprotein belonging to the MarR family of transcriptional repressors andfound that it negatively controls expression of farAB by directly bindingto the farAB promoter. We designated this regulator FarR to signify itsrole in regulating the farAB operon. We found that MtrR binds to the farRpromoter, thereby repressing farR expression. Hence, MtrR regulates farABin a positive fashion by modulating farR expression. This MtrR regulatorycascade seems to play an important role in adjusting levels of the FarABand MtrCDE efflux pumps to prevent their excess expression in gonococci.

• Davidson AL
• Structural biology. Not just another ABC transporter.
• Science. 2002; 296: 1038-40

• Xu HE et al.
• Structural basis for antagonist-mediated recruitment of nuclearco-repressors by PPARalpha.
• Nature. 2002; 415: 813-7
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• Repression of gene transcription by nuclear receptors is mediated byinteractions with co-repressor proteins such as SMRT and N-CoR, which inturn recruit histone deacetylases to the chromatin. Aberrant interactionsbetween nuclear receptors and co-repressors contribute towards acutepromyelocytic leukaemia and thyroid hormone resistance syndrome. Thebinding of co-repressors to nuclear receptors occurs in the unligandedstate, and can be stabilized by antagonists. Here we report the crystalstructure of a ternary complex containing the peroxisomeproliferator-activated receptor-alpha ligand-binding domain bound to theantagonist GW6471 and a SMRT co-repressor motif. In this structure, theco-repressor motif adopts a three-turn alpha-helix that prevents thecarboxy-terminal activation helix (AF-2) of the receptor from assuming theactive conformation. Binding of the co-repressor motif is furtherreinforced by the antagonist, which blocks the AF-2 helix from adoptingthe active position. Biochemical analyses and structure-based mutagenesisindicate that this mode of co-repressor binding is highly conserved acrossnuclear receptors.

• Biswas I et al.
• Disruption of the helix-u-turn-helix motif of MutS protein: loss ofsubunit dimerization, mismatch binding and ATP hydrolysis.
• J Mol Biol. 2001; 305: 805-16
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• The DNA mismatch repair protein, MutS, is a dimeric protein thatrecognizes mismatched bases and has an intrinsic ATPase activity. Here, aseries of Taq MutS proteins having C-terminal truncations in the vicinityof a highly conserved helix-u-turn-helix (HuH) motif are assessed forsubunit oligomerization, ATPase activity and DNA mismatch binding. Thoseproteins containing an intact HuH region are dimers; those without the HuHregion are predominantly monomers in solution. Steady-state kinetics oftruncated but dimeric MutS proteins reveals only modest decreases in theirATPase activity compared to full-length protein. In contrast, disruptionof the HuH region results in a greatly attenuated ATPase activity. Inaddition, only dimeric MutS proteins are proficient for mismatch binding.Finally, an analysis of the mismatch repair competency of truncatedEscherichia coli MutS proteins in a rifampicin mutator assay confirms thatthe HuH region is critical for in vivo function. These findings indicatethat dimerization is critical for both the ATPase and DNA mismatch bindingactivities of MutS, and corroborate several key features of the MutSstructure recently deduced from X-ray crystallographic studies.

• Sauer RT
• Lac repressor at last.
• Structure. 1996; 4: 219-22
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• Crystal and cocrystal structures of the LacI and PurR repressors reveal anovel use of hinge alpha helices which bind in the minor groove of theoperator and mediate transmission of the allosteric signals that modulateDNA-binding activity.

• Artymiuk PJ, Rice DW, Poirrette AR, Willet P
• A tale of two synthetases.
• Nat Struct Biol. 1994; 1: 758-60