title: Mycobacterium tuberculosis NmtR harbors a nickel sensing site with parallels to Escherichia coli RcnR.
abstract: Mycobacterium tuberculosis NmtR is a Ni(II)/Co(II)-sensing metalloregulatory protein from the extensively studied ArsR/SmtB family. Two Ni(II) ions bind to the NmtR dimer to form octahedral coordination complexes with the following stepwise binding affinities: K(Ni1) = (1.2 ± 0.1) × 10(10) M(-1), and K(Ni2) = (0.7 ± 0.4) × 10(10) M(-1) (pH 7.0). A glutamine scanning mutagenesis approach reveals that Asp91, His93, His104, and His107, all contained within the C-terminal α5 helix, and His3 as part of the conserved α-NH(2)-Gly2-His3-Gly4 motif at the N-terminus make significant contributions to the magnitude of K(Ni). In contrast, substitution of residues from the C-terminal region, His109, Asp114, and His116, previously implicated in Ni(II) binding and metalloregulation in cells, gives rise to wild-type K(Ni) and Ni(II)-dependent allosteric coupling free energies. Interestingly, deletion of residues 112-120 from the C-terminal region (Δ111 NmtR) reduces the Ni(II) binding stoichiometry to one per dimer and greatly reduces Ni(II) responsiveness. H3Q and Δ111 NmtRs also show clear perturbations in the rank order of metal responsiveness to Ni(II), Co(II), and Zn(II) that is distinct from that of wild-type NmtR. (15)N relaxation experiments with apo-NmtR reveal that both N-terminal (residues 2-14) and C- terminal (residues 110-120) regions are unstructured in solution, and this property likely dictates the metal specificity profile characteristic of the Ni(II) sensor NmtR relative to other ArsR family regulators.
authors.affiliation: Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102, USA.
authors.forename: Hermes
authors.initials: H
authors.lastname: Reyes-Caballero
authors.forename: Chul Won
authors.initials: CW
authors.lastname: Lee
authors.forename: David P
authors.initials: DP
authors.lastname: Giedroc
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000378:metabolism; D009532:Nickel / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C558390:RcnR protein, E coli; D012097:Repressor Proteins; D009532:Nickel