NORMAL GENOMIC

• 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.

• Schelert J, Dixit V, Hoang V, Simbahan J, Drozda M, Blum P
• Occurrence and characterization of mercury resistance in thehyperthermophilic archaeon Sulfolobus solfataricus by use of genedisruption.
• J Bacteriol. 2004; 186: 427-37
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• Mercury resistance mediated by mercuric reductase (MerA) is widespreadamong bacteria and operates under the control of MerR. MerR represents aunique class of transcription factors that exert both positive andnegative regulation on gene expression. Archaea and bacteria areprokaryotes, yet little is known about the biological role of mercury inarchaea or whether a resistance mechanism occurs in these organisms. Thearchaeon Sulfolobus solfataricus was sensitive to mercuric chloride, andlow-level adaptive resistance could be induced by metal preconditioning.Protein phylogenetic analysis of open reading frames SSO2689 and SSO2688clarified their identity as orthologs of MerA and MerR. Northern analysisestablished that merA transcription responded to mercury challenge, sincemRNA levels were transiently induced and, when normalized to 7S RNA,approximated values for other highly expressed transcripts. Primerextension analysis of merA mRNA predicted a noncanonical TATA box withnonstandard transcription start site spacing. The functional roles of merAand merR were clarified further by gene disruption. The merA mutantexhibited mercury sensitivity relative to wild type and was defective inelemental mercury volatilization, while the merR mutant was mercuryresistant. Northern analysis of the merR mutant revealed merAtranscription was constitutive and that transcript abundance was atmaximum levels. These findings constitute the first report of an archaealheavy metal resistance system; however, unlike bacteria the level ofresistance is much lower. The archaeal system employs a divergent MerRprotein that acts only as a negative transcriptional regulator of merAexpression.

• Busenlehner LS, Apuy JL, Giedroc DP
• Characterization of a metalloregulatory bismuth(III) site inStaphylococcus aureus pI258 CadC repressor.
• J Biol Inorg Chem. 2002; 7: 551-9
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• Staphylococcus aureus pI258 CadC is a metal sensor protein that regulatesthe expression of the cad operon which encodes metal ion resistanceproteins involved in the efficient efflux of Cd(II), Pb(II), Zn(II) and,according to one report, Bi(III) ions. In this paper, direct evidence ispresented that Bi(III) binds to CadC and negatively regulates cadoperator/promoter (O/P) binding. Optical absorption spectroscopy revealsthat dimeric CadC binds approximately 0.8 mol equivalents of Bi(III) perCadC monomer to form a coordination complex characterized by threeS(-)-->Bi(III) ligand-to-metal charge transfer transitions, with thelongest wavelength absorption band centered at 415 nm (epsilon(415)=4000M(Bi)(-1) cm(-1)). UV-Vis absorption spectra of wild-type and mutantCys-->Gly (Ser) substitution CadC mutants compared to [Bi(DTT)(2)],[Bi(GSH)(3)] and [Bi(NAC)](3) model complexes reveal that Cys7, Cys11,Cys60 and Cys58 directly coordinate Bi(III) in a tetrathiolatecoordination complex. The apparent affinity derived from aBi(III)-displacement optical titration with Cd(II) is estimated to beK(Bi)< or =10(12) M(-1). Apo-CadC binds with high affinity [K(a)=1.1(+/-0.3)x10(9) M(-1); 0.40 M NaCl, pH 7.0, 25 degrees C] to a5'-fluorescein-labeled cad O/P oligonucleotide,while the binding of onemolar equivalent of Bi(III) per CadC monomer (Bi(1)-CadC) reduces theaffinity by approximately 170-fold. Strikingly, Bi(III)-responsivenegative regulation of cad O/P binding is abrogated for Bi(1)-C60G CadCand severely disrupted in Bi(1)-C7G CadC, whose relative affinity isreduced only 10-fold. The mechanism of Bi(III)-responsivemetalloregulation is discussed, based on the findings presented here.Electronic supplementary material to this paper can be obtained by usingthe Springer Link server located athttp://dx.doi.org/10.1007/s00775-001-0336-9.

• Gillette WK, Martin RG, Rosner JL
• Probing the Escherichia coli transcriptional activator MarA usingalanine-scanning mutagenesis: residues important for DNA binding andactivation.
• J Mol Biol. 2000; 299: 1245-55
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• The MarA transcriptional activator binds to a 20 bp asymmetric degeneratesequence (marbox) located at different positions and orientations withinthe promoters of the genes of the Escherichia coli mar regulon. Solutionof the MarA-marbox X-ray crystallographic structure suggested the presenceof base-specific and non-specific interactions between the marbox and twohelix-turn-helix (HTH) motifs on the monomeric MarA. Here, we usealanine-scanning mutagenesis and DNA retardation analysis to: (i) evaluatethe contacts between MarA and the marboxes of five differently configuredmar regulon promoters; (ii) assess the role of conserved hydrophobic aminoacid residues for MarA activity; and (iii) identify residues required forRNA polymerase activation. These analyses revealed that thephosphate-backbone contacts and hydrogen bonds with the bases of themarbox are more significant for DNA binding than are the van der Waalsinteractions. While both N and C-terminal HTH motifs make essentialcontributions to binding site affinity, MarA is more sensitive toalterations in the N-terminal HTH. In a similar way, the activity of MarAis more sensitive to alterations in the hydrophobic core of this HTH.Solvent-exposed amino acid residues located at many positions on the MarAsurface are important for activity. Some of these residues affect activityon all promoters and thus, are implicated in maintaining MarA structurewhereas several solvent-exposed amino acids not involved in DNA bindingwere important for MarA activity on specific promoters. The pattern ofactivation defects defined a class II promoter-specific activating region.However, a localized class I activating region was not apparent. Theseresults suggest that MarA activates transcription by at least two distinctmechanisms. Furthermore, the important role of phosphate contacts inmarbox affinity suggests that indirect readout contributes to binding siterecognition by MarA.

• Colpas GJ, Brayman TG, Ming LJ, Hausinger RP
• Identification of metal-binding residues in the Klebsiella aerogenesurease nickel metallochaperone, UreE.
• Biochemistry. 1999; 38: 4078-88
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• The urease accessory protein encoded by ureE from Klebsiella aerogenes isproposed to bind intracellular Ni(II) for transfer to urease apoprotein.While native UreE possesses a histidine-rich region at its carboxylterminus that binds several equivalents of Ni, the Ni-binding sitesassociated with urease activation are internal to the protein as shown bystudies involving truncated H144UreE [Brayman and Hausinger (1996) J.Bacteriol. 178, 5410-5416]. Nine potential Ni-binding residues (five His,two Cys, one Asp, and one Tyr) within H144UreE were independentlysubstituted by mutagenesis to determine their roles in metal binding andurease activation. In vivo effects of these substitutions on ureaseactivity were measured in Escherichia coli strains containing the K.aerogenes urease gene cluster with the mutated ureE genes. Severalmutational changes led to reductions in specific activity, withsubstitution of His96 producing urease activity below the level obtainedfrom a ureE deletion mutant. The metal-binding properties of purifiedvariant UreE proteins were characterized by a combination of equilibriumdialysis and UV/visible, EPR, and hyperfine-shifted 1H NMR spectroscopicmethods. Ni binding was unaffected for most H144UreE variants, but mutantproteins substituted at His110 or His112 exhibited greatly reducedaffinity for Ni and bound one, rather than two, metal ions per dimer.Cys79 was identified as the Cu ligand responsible for the previouslyobserved charge-transfer transition at 370 nm, and His112 also was shownto be associated with this chromophoric site. NMR spectroscopy providedclear evidence that His96 and His110 serve as ligands to Ni or Co. Theresults from these and other studies, in combination with priorspectroscopic findings for metal-substituted UreE [Colpas et al. (1998) J.Biol. Inorg. Chem. 3, 150-160], allow us to propose that the homodimericprotein possesses two nonidentical metal-binding sites, each symmetricallylocated at the dimer interface. The first equivalent of added Ni or Cobinds via His96 and His112 residues from each subunit of the dimer, andtwo other N or O donors. Asp111 either functions as a ligand or may affectthis site by secondary interactions. The second equivalent of Ni or Cobinds via the symmetric pair of His110 residues as well as four other N orO donors. In contrast, the first equivalent of Cu binds via the His110pair and two other N/O donors, while the second equivalent of Cu binds viathe His112 pair and at least one Cys79 residue. UreE sequence comparisonsamong urease-containing microorganisms reveal that residues His96 andAsp111, associated with the first site of Ni binding, are highlyconserved, while the other targeted residues are missing in many cases.Our data are most compatible with one Ni-binding site per dimer beingcritical for UreE's function as a metallochaperone.

• Veals SA, Santa Maria T, Levy DE
• Two domains of ISGF3 gamma that mediate protein-DNA and protein-proteininteractions during transcription factor assembly contribute toDNA-binding specificity.
• Mol Cell Biol. 1993; 13: 196-206
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• Alpha interferon (IFN-alpha) induces the transcription of a large set ofgenes through activation of multimeric transcription factor ISGF3. Thisfactor can be dissociated into two protein components, termed ISGF3 gammaand ISGF3 alpha. ISGF3 gamma is a 48-kDa protein related at the aminoterminus to members of the IFN-regulatory factor (IRF) and Myb families ofDNA-binding proteins; ISGF3 alpha consists of three polypeptides of 84,91, and 113 kDa that self-assemble to form an activated component inresponse to IFN-alpha. DNA-binding studies indicated that ISGF3 gammabinds DNA alone, recognizing the IFN-stimulated response element, whilethe ISGF3 alpha polypeptides alone display no specific interactions withDNA. A complex between ISGF3 gamma and activated ISGF3 alpha binds theIFN-stimulated response element with much greater affinity than does the48-kDa ISGF3 gamma protein alone. The DNA-binding domain of ISGF3 gammaand regions responsible for protein-protein interaction with ISGF3 alphawere identified by using deleted forms of ISGF3 gamma expressed in vitro.The amino-terminal region of ISGF3 gamma homologous to the IRF and Mybproteins was sufficient for interaction with DNA and displayed the bindingspecificity of the intact protein; phosphorylation of this region wasnecessary for activity. A second region of 160 amino acids separated fromthe DNA-binding domain by over 100 amino acids contained a domain capableof associating with ISGF3 alpha and was sufficient to confer specificISGF3 alpha interaction to a heterologous protein. Interaction of theISGF3 alpha component with the protein interaction domain of ISGF3 gammaaltered the DNA-binding specificity of the resulting complex, suggestingthat one or more of the ISGF3 alpha polypeptides make base-specificcontacts with DNA. This interaction defines a mechanism through whichIRF-like proteins complexed with regulatory components can display novelDNA-binding specificities.

• Summers AO
• Untwist and shout: a heavy metal-responsive transcriptional regulator.
• J Bacteriol. 1992; 174: 3097-101

• Wang Y, Moore M, Levinson HS, Silver S, Walsh C, Mahler I
• Nucleotide sequence of a chromosomal mercury resistance determinant from aBacillus sp. with broad-spectrum mercury resistance.
• J Bacteriol. 1989; 171: 83-92
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• A 13.5-kilobase HindIII fragment, bearing an intact mercury resistance(mer) operon, was isolated from chromosomal DNA of broad-spectrummercury-resistant Bacillus sp. strain RC607 by using as a probe a clonecontaining the mercury reductase (merA) gene. The new clone, pYW33,expressed broad-spectrum mercury resistance both in Escherichia coli andin Bacillus subtilis, but only in B. subtilis was the mercuric reductaseactivity inducible. Sequencing of a 1.8-kilobase mercuryhypersensitivity-producing fragment revealed four open reading frames(ORFs). ORF1 may code for a regulatory protein (MerR). ORF2 and ORF4 wereassociated with cellular transport function and the hypersensitivityphenotype. DNA fragments encompassing the merA and the merB genes weresequenced. The predicted Bacillus sp. strain RC607 MerA (mercuricreductase) and MerB (organomercurial lyase) were similar to thosepredicted from Staphylococcus aureus plasmid pI258 (67 and 73% amino acididentities, respectively); however, only 40% of the amino acid residues ofRC607 MerA were identical to those of the mercuric reductase fromgram-negative bacteria. A 69-kilodalton polypeptide was isolated andidentified as the merA gene product by examination of its amino-terminalsequence.