NORMAL GENOMIC

• Bahlawane C et al.
• Structural and mechanistic insights into Helicobacter pylori NikRactivation.
• Nucleic Acids Res. 2010; 38: 3106-18
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• NikR is a transcriptional metalloregulator central in the mandatoryresponse to acidity of Helicobacter pylori that controls the expression ofnumerous genes by binding to specific promoter regions. NikR/DNAinteractions were proposed to rely on protein activation by Ni(II) bindingto high-affinity (HA) and possibly secondary external (X) sites. Wedescribe a biochemical characterization of HpNikR mutants that shows thatthe HA sites are essential but not sufficient for DNA binding, while thesecondary external (X) sites and residues from the HpNikR dimer-dimerinterface are important for DNA binding. We show that a second metal isnecessary for HpNikR/DNA binding, but only to some promoters. Small-angleX-ray scattering shows that HpNikR adopts a defined conformation insolution, resembling the cis-conformation and suggests that nickel doesnot trigger large conformational changes in HpNikR. The crystal structuresof selected mutants identify the effects of each mutation on HpNikRstructure. This study unravels key structural features from which wederive a model for HpNikR activation where: (i) HA sites and an hydrogenbond network are required for DNA binding and (ii) metallation of a uniquesecondary external site (X) modulates HpNikR DNA binding to low-affinitypromoters by disruption of a salt bridge.

• Xu Q et al.
• Crystal structure of a novel archaeal AAA+ ATPase SSO1545 from Sulfolobussolfataricus.
• Proteins. 2009; 74: 1041-9

• Liu T et al.
• A Cu(I)-sensing ArsR family metal sensor protein with a relaxed metalselectivity profile.
• Biochemistry. 2008; 47: 10564-75
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• ArsR (or ArsR/SmtB) family metalloregulatory homodimeric repressorscollectively respond to a wide range of metal ion inducers in regulatinghomeostasis and resistance of essential and nonessential metal ions inbacteria. BxmR from the cyanobacterium Osciliatoria brevis is the firstcharacterized ArsR protein that senses both Cu (I)/Ag (I) and divalentmetals Zn (II)/Cd (II) in cells by regulating the expression of a P-typeATPase efflux pump (Bxa1) and an intracellular metallothionein (BmtA). Weshow here that both pairs of predicted alpha3N and alpha5 sites bind metalions, but with distinct physicochemical and functional metalspecificities. Inactivation of the thiophilic alpha3N site via mutation(C77S) abolishes regulation by both Cd (II) and Cu (I), while Zn (II)remains a potent allosteric negative effector of operator/promoter binding(Delta G c >or= +3.2 kcal mol (-1)). In contrast, alpha5 site mutantretains regulation by all four metal ions, albeit with a smaller couplingfree energy (Delta G c approximately +1.7 (+/-0.1) kcal mol (-1)). Unlikethe other metals ions, the BxmR dimer binds 4 mol equiv of Cu (I) to forman alpha3N binuclear Cu (I) 2S 4 cluster by X-ray absorption spectroscopy.BxmR is thus distinguishable from other closely related ArsR familysensors, in having evolved a metalloregulatory alpha3N site that can adoptan expanded range of coordination chemistries while maintaining redundancyin the response to Zn (II). The evolutionary implications of thesefindings for the ArsR metal sensor family are discussed.

• Reddy MC, Gokulan K, Jacobs WR Jr, Ioerger TR, Sacchettini JC
• Crystal structure of Mycobacterium tuberculosis LrpA, a leucine-responsiveglobal regulator associated with starvation response.
• Protein Sci. 2008; 17: 159-70
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• The bacterial leucine-responsive regulatory protein (Lrp) is a globaltranscriptional regulator that controls the expression of many genesduring starvation and the transition to stationary phase. TheMycobacterium tuberculosis gene Rv3291c encodes a 150-amino acid protein(designated here as Mtb LrpA) with homology with Escherichia coli Lrp. Thecrystal structure of the native form of Mtb LrpA was solved at 2.1 A. TheMtb LrpA structure shows an N-terminal DNA-binding domain with ahelix-turn-helix (HTH) motif, and a C-terminal regulatory domain. Incomparison to the complex of E. coli AsnC with asparagine, theeffector-binding pocket (including loop 100-106) in LrpA appears to belargely preserved, with hydrophobic substitutions consistent with itsspecificity for leucine. The effector-binding pocket is formed at theinterface between adjacent dimers, with an opening to the core of theoctamer as in AsnC, and an additional substrate-access channel opening tothe outer surface of the octamer. Using electrophoretic mobility shiftassays, purified Mtb LrpA protein was shown to form a protein-DNA complexwith the lat promoter, demonstrating that the lat operon is a directtarget of LrpA. Using computational analysis, a putative motif isidentified in this region that is also present upstream of other operonsdifferentially regulated under starvation. This study provides insightsinto the potential role of LrpA as a global regulator in the transition ofM. tuberculosis to a persistent state.

• Malgieri G et al.
• The prokaryotic Cys2His2 zinc-finger adopts a novel fold as revealed bythe NMR structure of Agrobacterium tumefaciens Ros DNA-binding domain.
• Proc Natl Acad Sci U S A. 2007; 104: 17341-6
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• The first putative prokaryotic Cys(2)His(2) zinc-finger domain has beenidentified in the transcriptional regulator Ros from Agrobacteriumtumefaciens, indicating that the Cys(2)His(2) zinc-finger domain,originally thought to be confined to the eukaryotic kingdom, could bewidespread throughout the living kingdom from eukaryotic, both animal andplant, to prokaryotic. In this article we report the NMR solutionstructure of Ros DNA-binding domain (Ros87), providing 79 structuralcharacterization of a prokaryotic Cys(2)His(2) zinc-finger domain. The NMRstructure of Ros87 shows that the putative prokaryotic Cys(2)His(2)zinc-finger sequence is indeed part of a significantly larger zinc-bindingglobular domain that possesses a novel protein fold very different fromthe classical fold reported for the eukaryotic classical zinc-finger. TheRos87 globular domain consists of 58 aa (residues 9-66), is arranged in abetabetabetaalphaalpha topology, and is stabilized by an extensive15-residue hydrophobic core. A backbone dynamics study of Ros87, based on(15)N R(1), (15)N R(2), and heteronuclear (15)N-{(1)H}-NOE measurements,has further confirmed that the globular domain is uniformly rigid andflanked by two flexible tails. Mapping of the amino acids necessary forthe DNA binding onto Ros87 structure reveals the protein surface involvedin the DNA recognition mechanism of this new zinc-binding protein domain.

• 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.

• Aishima J, Wolberger C
• Insights into nonspecific binding of homeodomains from a structure ofMATalpha2 bound to DNA.
• Proteins. 2003; 51: 544-51
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• The 2.1-A resolution crystal structure of the MATalpha2 homeodomain boundto DNA reveals the unexpected presence of two nonspecifically bound alpha2homeodomains, in addition to the two alpha2 homeodomains bound tocanonical alpha2 binding sites. One of the extra homeodomains makes fewbase-specific contacts, while the other extra homeodomain binds to DNA ina previously unobserved manner. This unusually bound homeodomain isrotated on the DNA, making possible major groove contacts by side-chainsthat normally do not contact the DNA. This alternate docking may representone way in which homeodomains sample nonspecific DNA sequences.

• 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.

• Laity JH, Dyson HJ, Wright PE
• DNA-induced alpha-helix capping in conserved linker sequences is adeterminant of binding affinity in Cys(2)-His(2) zinc fingers.
• J Mol Biol. 2000; 295: 719-27
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• High-affinity, sequence-specific DNA binding by Cys(2)-His(2) zinc fingerproteins is mediated by both specific protein-base interactions andnon-specific contacts between charged side-chains and the phosphatebackbone. In addition, in DNA complexes of multiple zinc fingers,protein-protein interactions between the finger units contribute to thebinding affinity. We present NMR evidence for another contribution tohigh- affinity binding, a highly specific DNA-induced helix cappinginvolving residues in the linker sequence between fingers. Capping at theC terminus of the alpha-helix in each zinc finger, incorporating aconsensus TGEKP linker sequence that follows each finger, providessubstantial binding energy to the DNA complexes of zinc fingers 1-3 ofTFIIIA (zf1-3) and the four zinc fingers of the Wilms' tumor suppressorprotein (wt1-4). The same alpha-helix C-capping motif is observed in theX-ray structures of four other protein-DNA complexes. The structures ofeach of the TGEKP linkers in these complexes can be superimposed on thelinker sequences in the zf1-3 complex, revealing a remarkable similarityin both backbone and side-chain conformations. The canonical linkerstructures from the zinc-finger-DNA complexes have been compared to theNMR structure of the TGEKP linker connecting fingers 1 and 2 in zf1-3 inthe absence of DNA. This comparison reveals that additional stabilizationlikely arises in the DNA complexes from hydrogen bonding between thebackbone amide of E3 and the side-chain O(gamma) of T1 in the linker. Wesuggest that these DNA-induced C-capping interactions provide a meanswhereby the multiple-finger complex, which must necessarily bedomain-flexible in the unbound state as it searches for the correct DNAsequence, can be "snap-locked" in place once the correct DNA sequence isencountered. These observations provide a rationale for the highconservation of the TGEKP linker sequences in Cys(2)-His(2) zinc fingerproteins.

• Jin C, Marsden I, Chen X, Liao X
• Dynamic DNA contacts observed in the NMR structure of winged helixprotein-DNA complex.
• J Mol Biol. 1999; 289: 683-90
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• Genesis is an HNF-3/fkh homologous protein. By using multi-dimensional NMRtechniques, we have obtained the solution structure and backbone dynamicsof Genesis complexed with a 17 base-pair DNA. Our results indicate thatboth the local folding and dynamic properties of Genesis are perturbedwhen it binds to the DNA site. Our data show that a conserved flexibleamino acid sequence (wing 1) makes dynamic contacts to DNA in the complexand a short helix is induced by Genesis-DNA interactions. Our dataindicate that, unlike the HNF-3gamma/DNA complex, a magnesium ion is notrequired in forming the stable Genesis-DNA complex.

• Wang G, Wylie GP, Twigg PD, Caspar DL, Murphy JR, Logan TM
• Solution structure and peptide binding studies of the C-terminal srchomology 3-like domain of the diphtheria toxin repressor protein.
• Proc Natl Acad Sci U S A. 1999; 96: 6119-24
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• The diphtheria toxin repressor (DtxR) is the best-characterized member ofa family of homologous proteins that regulate iron uptake and virulencegene expression in the Gram-positive bacteria. DtxR contains two domainsthat are separated by a short, unstructured linker. The N-terminal domainis structurally well-defined and is responsible for Fe2+ binding,dimerization, and DNA binding. The C-terminal domain adopts a fold similarto eukaryotic Src homology 3 domains, but the functional role of theC-terminal domain in repressor activity is unknown. The solution structureof the C-terminal domain, consisting of residues N130-L226 plus a13-residue N-terminal extension, has been determined by using NMRspectroscopy. Residues before A147 are highly mobile and adopt a randomcoil conformation, but residues A147-L226 form a single structured domainconsisting of five beta-strands and three helices arranged into apartially orthogonal, two-sheet beta-barrel, similar to the structureobserved in the crystalline Co2+ complex of full-length DtxR. Chemicalshift perturbation studies demonstrate that a proline-rich peptidecorresponding to residues R125-G139 of intact DtxR binds to the C-terminaldomain in a pocket formed by residues in beta-strands 2, 3, and 5, andhelix 3. Binding of the proline-rich peptide by the C-terminal domain ofDtxR presents an example of peptide binding by a prokaryotic Src homology3-like protein. The results of this study, combined with previous x-raystudies of intact DtxR, provide insights into a possible biologicalfunction of the C-terminal domain in regulating repressor activity.

• Marsden I, Jin C, Liao X
• Structural changes in the region directly adjacent to the DNA-bindinghelix highlight a possible mechanism to explain the observed changes inthe sequence-specific binding of winged helix proteins.
• J Mol Biol. 1998; 278: 293-9
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• The hepatocyte nuclear factor 3 (HNF-3)/fork head (fkh) family contains alarge number of transcription factors and folds into a winged helix motif.Despite having almost invariable amino acid sequences in their principalDNA-binding helices, HNF-3/fkh proteins show a wide diversity ofsequence-specific binding. Previous studies of chimeric HNF-3/fkh proteinsdemonstrated that the binding specificity is primarily influenced by aregion directly adjacent to the binding helix. We report our findings ofan NMR structural study performed on an HNF-3/fkh family member (Genesis,formerly HFH-2) and compare it to that of another family member(HNF-3gamma) complexed to DNA and determined by X-ray crystallography. Itis found that in comparison to HNF-3gamma, Genesis contains an extra smallhelix directly prior to the N terminus of the primary DNA contact helix.Due to the insertion of this helix, a shorter and slightly re-positionedprimary DNA contact helix is observed, which we believe leads to theDNA-binding specificity differences among family members.

• Cai M, Zheng R, Caffrey M, Craigie R, Clore GM, Gronenborn AM
• Solution structure of the N-terminal zinc binding domain of HIV-1integrase.
• Nat Struct Biol. 1997; 4: 567-77
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• The solution structure of the N-terminal zinc binding domain (residues1-55; IN1-55) of HIV-1 integrase has been solved by NMR spectroscopy.IN1-55 is dimeric, and each monomer comprises four helices with the zinctetrahedrally coordinated to His 12, His 16, Cys 40 and Cys 43. IN1-55exists in two interconverting conformational states that differ withregard to the coordination of the two histidine side chains to zinc. Thedifferent histidine arrangements are associated with large conformationaldifferences in the polypeptide backbone (residues 9-18) around thecoordinating histidines. The dimer interface is predominantly hydrophobicand is formed by the packing of the N-terminal end of helix 1, and helices3 and 4. The monomer fold is remarkably similar to that of a number ofhelical DNA binding proteins containing a helix-turn-helix (HTH) motifwith helices 2 and 3 of IN1-55 corresponding to the HTH motif. In contrastto the DNA binding proteins where the second helix of the HTH motif isemployed for DNA recognition, IN1-55 uses this helix for dimerization.

• Newman M, Strzelecka T, Dorner LF, Schildkraut I, Aggarwal AK
• Structure of restriction endonuclease bamhi phased at 1.95 A resolution byMAD analysis.
• Structure. 1994; 2: 439-52
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• BACKGROUND: Type II restriction endonucleases recognize DNA sequences thatvary between four to eight base pairs, and require only Mg2+ as a cofactorto catalyze the hydrolysis of DNA. Their protein sequences display asurprising lack of similarity, and no recurring structural motif analogousto the helix-turn-helix or the zinc finger of transcription factors, hasyet been discovered. RESULTS: We have determined the crystal structure ofrestriction endonuclease BamHI at 1.95 A resolution. The structure wassolved by combining phase information derived from multi-wavelength X-raydata by algebraic and maximum likelihood methods. The BamHI subunitconsists of a central beta-sheet with alpha-helices on both sides. Thedimer configuration reveals a large cleft which could accommodate B-formDNA. Mutants of the enzyme that are deficient in cleavage are located ator near the putative DNA-binding cleft. BamHI and endonuclease EcoRI sharea common core motif (CCM) consisting of five beta-strands and two helices.It remains to be determined if other restriction enzymes also contain theCCM. CONCLUSIONS: The structure of BamHI provides the first clear evidencethat there may be substantial structural homology amongst restrictionenzymes, even though it is undetectable at the sequence level.

• Fogh RH, Ottleben G, Ruterjans H, Schnarr M, Boelens R, Kaptein R
• Solution structure of the LexA repressor DNA binding domain determined by1H NMR spectroscopy.
• EMBO J. 1994; 13: 3936-44
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• The structure of the 84 residue DNA binding domain of the Escherichia coliLexA repressor has been determined from NMR data using distance geometryand restrained molecular dynamics. The assignment of the 1H NMR spectrumof the molecule, derived from 2- and 3-D homonuclear experiments, is alsoreported. A total of 613 non-redundant distance restraints were used togive a final family of 28 structures. The structured region of themolecule consisted of residues 4-69 and yielded a r.m.s. deviation from anaverage of 0.9 A for backbone and 1.6 A for all heavy atoms. The structurecontains three regular alpha-helices at residues 6-21 (I), 28-35 (II) and41-52 (III), and an antiparallel beta-sheet at residues 56-58 and 66-68.Helices II and III form a variant helix-turn-helix DNA binding motif, withan unusual one residue insert at residue 38. The topology of the LexA DNAbinding domain is found to be the same as for the DNA binding domains ofthe catabolic activator protein, human histone 5, the HNF-3/fork headprotein and the Kluyveromyces lactis heat shock transcription factor.

• Zhao D, Arrowsmith CH, Jia X, Jardetzky O
• Refined solution structures of the Escherichia coli trp holo- andaporepressor.
• J Mol Biol. 1993; 229: 735-46
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• The solution structures of the trp-repressor from Escherichia coli in boththe liganded (holo-) and unliganded (apo-) form, have been refined byrestrained molecular dynamics with simulated annealing using the programXPLOR and additional experimental constraints. The ensemble of refinedholorepressor structures have a root-mean-square deviation (r.m.s.d.) of0.8 A relative to the average structure for the backbone of the dimer core(helices A, B, C, A', B', C') and 2.5 A for the helix-turn-helixDNA-binding domain (helices D and E). The corresponding values for theaporepressor are 0.9 A for the backbone of the ABC-dimer core and 3.2 Afor the DE helix-turn-helix. The r.m.s.d. of the average structures fromthe corresponding crystal structures are 2.3 A for the holorepressor ABCcore and 4.2 A for its DE region; 2.3 A for the aporepressor core and 5.5A for its DE region. The relative disorder of the DNA-binding domain isreflected in a number of experimental parameters including substantiallymore rapid backbone proton exchange rates, exchange-limited relaxationtimes and crystallographic B-factors. The stabilizing effect of the L-Trpligand is evident in these measurements, as it is in the higher precisionof the holorepressor structure.

• Clark KL, Halay ED, Lai E, Burley SK
• Co-crystal structure of the HNF-3/fork head DNA-recognition motifresembles histone H5.
• Nature. 1993; 364: 412-20
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• The three-dimensional structure of an HNF-3/fork head DNA-recognitionmotif complexed with DNA has been determined by X-ray crystallography at2.5 A resolution. This alpha/beta protein binds B-DNA as a monomer,through interactions with the DNA backbone and through both direct andwater-mediated major and minor groove base contacts, inducing a 13 degreesbend. The transcription factor fold is very similar to the structure ofhistone H5. In its amino-terminal half, three alpha-helices adopt acompact structure that presents the third helix to the major groove. Theremainder of the protein includes a twisted, antiparallel beta-structureand random coil that interacts with the minor groove.