Publications (8)85.18 Total impact
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Article: Allosteric inhibition of a zinc-sensing transcriptional repressor: Insights into the arsenic repressor (ArsR) family.
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ABSTRACT: The molecular basis of allosteric regulation remains a subject of intense interest. Staphylococcus aureus CzrA is a member of the ubiquitous arsenic repressor (ArsR) family of bacterial homodimeric metal sensing proteins, and has emerged as a model system for understanding allosteric regulation of operator DNA binding by transition metal ions. Using unnatural amino acid substitution and a standard linkage analysis, we show that a His97' NHε2•••O=C-His67 quaternary structural hydrogen bond is an energetically significant contributor to the magnitude of the allosteric coupling free energy, ∆G(c). A "cavity" introduced just beneath this hydrogen bond in V66A/L68V CzrA results in a dramatic loss of regulation by Zn(II) despite adopting a wild-type global structure and Zn(II) binding and DNA binding affinities only minimally affected from wild-type. The energetics of Zn(II) binding and heterotropic coupling free energies (∆H(c), -T∆S(c)) of the double mutant are also radically altered and suggest that increased internal dynamics leads to poorer allosteric negative regulation in V66A/L68V CzrA. A statistical coupling analysis of 3000 ArsR proteins reveals a sector that links the DNA-binding determinants and the α5 Zn(II) sensing sites through V66/L68 in CzrA. We propose that distinct regulatory sites uniquely characteristic of individual ArsR proteins results from evolution of distinct connectivities to this sector, each capable of driving the same biological outcome, transcriptional derepression.Journal of Molecular Biology 01/2013; · 4.00 Impact Factor -
Article: Control of Copper Resistance and Inorganic Sulfur Metabolism by Paralogous Regulators in Staphylococcus aureus
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ABSTRACT: All strains of Staphylococcus aureus encode a putative copper-sensitive operon repressor (CsoR) and one other CsoR-like protein of unknown function. We show here that NWMN_1991 encodes a bona fide Cu(I)-inducible CsoR of a genetically unlinked copA-copZ copper resistance operon in S. aureus strain Newman. In contrast, an unannotated open reading frame found between NWMN_0027 and NWMN_0026 (denoted NWMN_0026.5) encodes a CsoR-like regulator that represses expression of adjacent genes by binding specifically to a pair of canonical operator sites positioned in the NWMN_0027–0026.5 intergenic region. Inspection of these regulated genes suggests a role in assimilation of inorganic sulfur from thiosulfate and vectorial sulfur transfer, and we designate NWMN_0026.5 as CstR (CsoR-like sulfur transferase repressor). Expression analysis demonstrates that CsoR and CstR control their respective regulons in response to distinct stimuli with no overlap in vivo. Unlike CsoR, CstR does not form a stable complex with Cu(I); operator binding is instead inhibited by oxidation of the intersubunit cysteine pair to a mixture of disulfide and trisulfide linkages by a likely metabolite of thiosulfate assimilation, sulfite. CsoR is unreactive toward sulfite under the same conditions. We conclude that CsoR and CstR are paralogs in S. aureus that function in the same cytoplasm to control distinct physiological processes.Journal of Biological Chemistry 04/2011; 286(15):13522-13531. · 4.77 Impact Factor -
Article: Control of copper resistance and inorganic sulfur metabolism by paralogous regulators in Staphylococcus aureus.
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ABSTRACT: All strains of Staphylococcus aureus encode a putative copper-sensitive operon repressor (CsoR) and one other CsoR-like protein of unknown function. We show here that NWMN_1991 encodes a bona fide Cu(I)-inducible CsoR of a genetically unlinked copA-copZ copper resistance operon in S. aureus strain Newman. In contrast, an unannotated open reading frame found between NWMN_0027 and NWMN_0026 (denoted NWMN_0026.5) encodes a CsoR-like regulator that represses expression of adjacent genes by binding specifically to a pair of canonical operator sites positioned in the NWMN_0027-0026.5 intergenic region. Inspection of these regulated genes suggests a role in assimilation of inorganic sulfur from thiosulfate and vectorial sulfur transfer, and we designate NWMN_0026.5 as CstR (CsoR-like sulfur transferase repressor). Expression analysis demonstrates that CsoR and CstR control their respective regulons in response to distinct stimuli with no overlap in vivo. Unlike CsoR, CstR does not form a stable complex with Cu(I); operator binding is instead inhibited by oxidation of the intersubunit cysteine pair to a mixture of disulfide and trisulfide linkages by a likely metabolite of thiosulfate assimilation, sulfite. CsoR is unreactive toward sulfite under the same conditions. We conclude that CsoR and CstR are paralogs in S. aureus that function in the same cytoplasm to control distinct physiological processes.Journal of Biological Chemistry 02/2011; 286(15):13522-31. · 4.77 Impact Factor -
Article: Unnatural amino acid substitution as a probe of the allosteric coupling pathway in a mycobacterial Cu(I) sensor.
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ABSTRACT: The Cu(I) sensor Mycobacterium tuberculosis CsoR is a founding member of a new metalloregulatory protein family. Here we show that two "atom" substitutions of the Nepsilon2 face of a Cu(I) coordinating histidine-61 allosterically uncouple Cu(I) and DNA binding, with no effect on Cu(I) binding affinity and coordination structure. A model analogous to the allosteric switch mechanism in Staphylococcus aureus CzrA, a zinc sensor protein with a completely different fold, is proposed.Journal of the American Chemical Society 11/2009; 131(50):18044-5. · 9.91 Impact Factor -
Article: Coordination chemistry of bacterial metal transport and sensing.
Chemical Reviews 09/2009; 109(10):4644-81. · 40.20 Impact Factor -
Article: Molecular insights into the metal selectivity of the copper(I)-sensing repressor CsoR from Bacillus subtilis.
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ABSTRACT: Bacillus subtilis CsoR (Bsu CsoR) is a copper-sensing transcriptional repressor that regulates the expression of the copZA operon encoding a copper chaperone and a Cu efflux P-type ATPase, respectively. Bsu CsoR is a homologue of Mycobacterium tuberculosis CsoR (Mtb CsoR), representative of a large Cu(I)-sensing regulatory protein family. We show here that Bsu CsoR binds approximately 1 mol equiv of Cu(I) per monomer in vitro with an affinity >or=10(21) M(-1). X-ray absorption spectroscopy shows Cu(I) adopts a trigonal S(2)N coordination like Mtb CsoR. Both apo and Cu(I)-bound Bsu CsoR are stable tetramers in the low micromolar monomer concentration range by sedimentation velocity and equilibrium ultracentrifugation. Apo-Bsu CsoR binds to a pseudopalindromic 30 bp copZA operator-promoter DNA with a stoichiometry of two tetramers per DNA and stepwise affinities of K(1)(apo) = 3.1(+/-0.8) x 10(7) M(-1) and K(2)(apo) = 8.3 (+/-2.2) x 10(7) M(-1) (0.4 M NaCl, 25 degrees C, pH 6.5). Cu(I) Bsu CsoR binds to the same DNA with greatly reduced affinities, K(1)(Cu) = 2.9(+/-0.4) x 10(6) M(-1) and K(2)(Cu) <or= 1.0 x 10(5) M(-1) consistent with a copper-dependent derepression model. This Cu-dependent regulation is abrogated by a "second shell" Glu90-to-Ala substitution. Bsu CsoR binds Ni(II) with very high affinity but forms a non-native coordination geometry, as does Co(II) and likely Zn(II); none of these metals strongly regulates copZA operator DNA binding in vitro. The implications of these findings on the specificity of metal-sensing sites in CsoR/RcnR proteins are discussed.Biochemistry 02/2009; 48(15):3325-34. · 3.42 Impact Factor -
Article: A Cu(I)-sensing ArsR family metal sensor protein with a relaxed metal selectivity profile.
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ABSTRACT: ArsR (or ArsR/SmtB) family metalloregulatory homodimeric repressors collectively respond to a wide range of metal ion inducers in regulating homeostasis and resistance of essential and nonessential metal ions in bacteria. BxmR from the cyanobacterium Osciliatoria brevis is the first characterized ArsR protein that senses both Cu (I)/Ag (I) and divalent metals Zn (II)/Cd (II) in cells by regulating the expression of a P-type ATPase efflux pump (Bxa1) and an intracellular metallothionein (BmtA). We show here that both pairs of predicted alpha3N and alpha5 sites bind metal ions, but with distinct physicochemical and functional metal specificities. 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 mutant retains regulation by all four metal ions, albeit with a smaller coupling free energy (Delta G c approximately +1.7 (+/-0.1) kcal mol (-1)). Unlike the other metals ions, the BxmR dimer binds 4 mol equiv of Cu (I) to form an alpha3N binuclear Cu (I) 2S 4 cluster by X-ray absorption spectroscopy. BxmR is thus distinguishable from other closely related ArsR family sensors, in having evolved a metalloregulatory alpha3N site that can adopt an expanded range of coordination chemistries while maintaining redundancy in the response to Zn (II). The evolutionary implications of these findings for the ArsR metal sensor family are discussed.Biochemistry 10/2008; 47(40):10564-75. · 3.42 Impact Factor -
Article: CsoR is a novel Mycobacterium tuberculosis copper-sensing transcriptional regulator.
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ABSTRACT: Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. Here, we identify a new copper-specific repressor (CsoR) of a copper-sensitive operon (cso) in Mycobacterium tuberculosis (Mtb) that is representative of a large, previously uncharacterized family of proteins (DUF156). Electronic and X-ray absorption spectroscopies reveal that CsoR binds a single-monomer mole equivalent of Cu(I) to form a trigonally coordinated (S(2)N) Cu(I) complex. The 2.6-A crystal structure of copper-loaded CsoR shows a homodimeric antiparallel four-helix bundle architecture that represents a novel DNA-binding fold. The Cu(I) is coordinated by Cys36, Cys65' and His61' in a subunit bridging site. Cu(I) binding negatively regulates the binding of CsoR to a DNA fragment encompassing the operator-promoter region of the Mtb cso operon; this results in derepression of the operon in Mtb and the heterologous host Mycobacterium smegmatis. Substitution of Cys36 or His61 with alanine abolishes Cu(I)- and CsoR-dependent regulation in vivo and in vitro. Potential roles of CsoR in Mtb pathogenesis are discussed.Nature Chemical Biology 02/2007; 3(1):60-8. · 14.69 Impact Factor
Top co-authors
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Institutions
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2009–2013
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Indiana University Bloomington
- Department of Chemistry
Bloomington, IN, USA
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2007–2009
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Texas A&M University
- Department of Biochemistry/Biophysics
College Station, TX, USA
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