Structural and Functional Characterization of Mycobacterium tuberculosis CmtR, a PbII/CdII-Sensing SmtB/ArsR Metalloregulatory Repressor

Wang, Yun; Hemmingsen, Lars; Giedroc, David P.

doi:10.1021/bi050094v

Cited by 62 publications

(110 citation statements)

References 51 publications

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“…1C shows the slope of signal disappearance, which is consistent with the exclusive formation of three protein dimers per DNA fragment at the present concentrations. This is not inconsistent with the previous proposal of 1:1, 1:2 and 1:3 species at much lower concentrations (21). The disappearance of the NH signals is due to the formation of the large molecular mass DNA-protein complex, as previously observed by electrophoretic mobility shift assays (16) and by fluorescence anisotropy at elevated protein concentrations (21), and/or due to the occurrence of chemical exchange phenomena, which leads to line broadening.…”

Section: N{supporting

confidence: 84%

“…Protein Characterization-CmtR is a cadmium-detecting transcriptional repressor of 118 residues with a molecular mass of 12.5 kDa that forms homodimers (21). IC-mass spectrometry shows that cadmium-CmtR has a mass of 25,211 under native conditions with volatile buffers, matching a dimer plus two cadmium atoms.…”

Section: Resultsmentioning

confidence: 99%

“…We previously established that CmtR responds in vivo to cadmium and lead to modulate production of a toxic metal-exporting P 1 -type ATPase (16). Residues Cys-57 and Cys-61 plus Cys-102 have been implicated in cadmium perception by a combination of in vivo and in vitro studies (16,21). CmtR has a relatively weak affinity for DNA, in the region of 10 Ϫ7 M, even in the apo form (22).…”

mentioning

confidence: 99%

“…S6). Although Cys-57 and Cys-61 thiols are required for CmtR to bind cadmium in vitro, the Cys-102 thiol is not, with loss of this side group only reducing the cadmium-affinity by one order of magnitude (21). Thus the Cys-102 thiol presumably is an obligatory ligand for the molecular mechanism but optional for cadmium binding.…”

mentioning

confidence: 99%

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NMR Structural Analysis of Cadmium Sensing by Winged Helix Repressor CmtR

Banci

Bertini

Cantini

et al. 2007

Journal of Biological Chemistry

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CmtR from Mycobacterium tuberculosis is a winged helical DNA-binding repressor of the ArsR-SmtB metal-sensing family that senses cadmium and lead. Cadmium-CmtR is a dimer with the metal bound to Cys-102 from the C-terminal region of one subunit and two Cys associated with helix ␣R from the other subunit, forming a symmetrical pair of cadmium-binding sites. This is a significant novelty in the ArsR-SmtB family. The structure of the dimer could be solved at 312 K. The apoprotein at the same temperature is still a dimer, but it experiences a large conformational exchange at the dimer interface and within each monomer. This is monitored by an overall decrease of the number of nuclear Overhauser effects and by an increase of H 2 O-D 2 O exchange rates, especially at the dimeric interface, in the apo form with respect to the cadmium-bound state. The C-terminal tail region is completely unstructured in both apo and cadmium forms but becomes less mobile in the cadmium-bound protein due to the recruitment of Cys-102 as a metal-ligand. DNA binds to the apo dimer with a ratio 1:3 at millimolar concentration. Addition of cadmium to the apo-CmtR-DNA complex causes DNA detachment, restoring the NMR spectrum of free cadmium-CmtR. Cadmium binding across the dimer interface impairs DNA association by excluding the apo-conformers suited to bind DNA.Life depends on multiple metals (1). Iron-, copper-, and zincresponsive transcriptional regulators are known in yeast (AFT1, MAC1, ACE1, and ZAP1) (2) and higher eukaryotes (MTF1) (3, 4). In bacteria, multiple families of metal-responsive transcriptional regulators have been described, including ArsR/ SmtB-like DNA-binding repressors (5-8). These are a subgroup of winged helix repressors. We previously discovered the cellular zinc sensor SmtB (5). In elevated zinc, SmtB repression of the smtA gene is alleviated to allow transcription of a metallothionein gene encoding a protein, which sequesters the surplus metal atoms (9, 10). In contrast, the related ArsR sensor responds to arsenite to regulate production of an arsenicaltranslocating ATPase (6). The location of the metal-sensing site, the complement of ligands and site geometry, vary in different members of this family of regulators (8,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). There is considerable interest in understanding how metal binding to metal sensors is transduced into altered levels of transcription of their target genes.Mycobacterium tuberculosis contains ten genes encoding ArsR/SmtB sensors making it a useful model organism for studies of these regulators. It is plausible that fluctuations in metal concentrations within macrophages have selected for multiple genes encoding such regulators in this pathogen. One of the M. tuberculosis sensors, CmtR, was chosen for structural and functional studies of a sensory mechanism. We previously established that CmtR responds in vivo to cadmium and lead to modulate production of a toxic metal-exporting P 1 -type ATPase (16). Residues Cys-57 and Cys-61 plus Cys-102 have been implicated ...

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Section: N{supporting

confidence: 84%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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NMR Structural Analysis of Cadmium Sensing by Winged Helix Repressor CmtR

Banci

Bertini

Cantini

et al. 2007

Journal of Biological Chemistry

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“…KmtR is responsive to nickel and cobalt and represses transcription of Rv0826 and Rv2025, encoding a hypothetical protein and a deduced transmembrane cation diffusion facilitator family metal ion transporter, respectively (11). NmtR also senses nickel and cobalt but regulates expression of nmtA, encoding a predicted P 1 -type ATPase (13,67). Finally, CmtR is responsive to cadmium (and possibly lead) and regulates expression of its own operon, which includes downstream genes Rv1993c and cmtA, encoding a predicted P1-type ATPase (12,14).…”

Section: Vol 193 2011 Regulation Of M Tuberculosis Rv0081-rv0088 Lmentioning

confidence: 99%

Components of the Rv0081-Rv0088 Locus, Which Encodes a Predicted Formate Hydrogenlyase Complex, Are Coregulated by Rv0081, MprA, and DosR in Mycobacterium tuberculosis

Bretl

Penoske

et al. 2011

J Bacteriol

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Mycobacterium tuberculosis, the etiological agent of tuberculosis, remains a significant cause of morbidity and mortality throughout the world despite a vaccine and cost-effective antibiotics. The success of this organism can be attributed, in part, to its ability to adapt to potentially harmful stress within the host and establish, maintain, and reactivate from long-term persistent infection within granulomatous structures. The DosRSDosT/DevRS-Rv2027c, and MprAB two-component signal transduction systems have previously been implicated in aspects of persistent infection by M. tuberculosis and are known to be responsive to conditions likely to be found within the granuloma. Here, we describe initial characterization of a locus (Rv0081-Rv0088) encoding components of a predicted formate hydrogenylase enzyme complex that is directly regulated by DosR/DevR and MprA, and the product of the first gene in this operon, Rv0081. In particular, we demonstrate that Rv0081 negatively regulates its own expression and that of downstream genes by binding an inverted repeat element in its upstream region. In contrast, DosR/DevR and MprA positively regulate Rv0081 expression by binding to recognition sequences that either partially or completely overlap that recognized by Rv0081, respectively. Expression of Rv0081 initiates from two promoter elements; one promoter located downstream of the DosR/DevR binding site but overlapping the sequence recognized by both Rv0081 and MprA and another promoter downstream of the DosR/DevR, Rv0081, and MprA binding sites. Interestingly, Rv0081 represses Rv0081 and downstream determinants following activation of DosRS-DosT/DevRS-Rv2027c by nitric oxide, suggesting that expression of this locus is complex and subject to multiple levels of regulation. Based on this and other published information, a model is proposed detailing Rv0081-Rv0088 expression by these transcription factors within particular growth environments.

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Perturbed Angular Correlations of γ‐rays (PAC) Spectroscopy

Hemmingsen¹,

Butz²

2005

Encyclopedia of Inorganic Chemistry

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In this article, we give an elementary introduction to the theory and applications of perturbed angular correlation of γ‐rays (PAC) spectroscopy. In most applications in bioinorganic and inorganic chemistry the nuclear quadrupole interaction (NQI) is measured, providing a spectroscopic fingerprint of the local electronic and molecular structure and dynamics at the PAC probe site. The focus is on selected illustrative applications of PAC spectroscopy in bioinorganic chemistry: (i) local structure at metal ion binding sites in the bacterial mercury sensor MerR; de novo designed proteins; alcohol dehydrogenase–pH dependence; carboxypeptidase A—crystalline state versus solution; and carboxypeptidase A–during the hydrolysis of substrate; (ii) trapping intermediates in reactions using rapid freeze quenching; (iii) in vivo experiments on Cd(II) binding to biomolecules in cyanobacteria; (iv) cooperativity of metal ion binding in multinuclear sites; (v) dynamics of protein folding; (vi) rotational diffusion of biomolecules as a tool to monitor protein–protein interactions; (vii) exchange dynamics; and (viii) internal dynamics in proteins. Examples from inorganic chemistry and solid state physics are included for cases where the information derived is different from that already presented in the examples from bioinorganic chemistry: (i) magnetic properties of metals; (ii) defects trapped by the impurity PAC probe atom; (iii) temperature and pressure dependence of the EFG in metals; (iv) amorphous metals, alloys and internal oxidation; (v) metal surfaces and interfaces; (vi) semiconductors; (vii) phase transitions; (viii) charge density waves in transition metal dichalcogenides; (ix) intercalation reactions in 2H‐TaS 2 ; (x) structure and bonding; (xi) single crystal experiments.

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Structural and Functional Characterization of Mycobacterium tuberculosis CmtR, a Pb^II/Cd^II-Sensing SmtB/ArsR Metalloregulatory Repressor

Cited by 62 publications

References 51 publications

NMR Structural Analysis of Cadmium Sensing by Winged Helix Repressor CmtR

NMR Structural Analysis of Cadmium Sensing by Winged Helix Repressor CmtR

Components of the Rv0081-Rv0088 Locus, Which Encodes a Predicted Formate Hydrogenlyase Complex, Are Coregulated by Rv0081, MprA, and DosR in Mycobacterium tuberculosis

Perturbed Angular Correlations of γ‐rays (PAC) Spectroscopy

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