bi200737a_si_001.pdf (1.03 MB)

Mycobacterium tuberculosis NmtR Harbors a Nickel Sensing Site with Parallels to Escherichia coli RcnR

Download (1.03 MB)
journal contribution
posted on 20.09.2011, 00:00 by Hermes Reyes-Caballero, Chul Won Lee, David P. Giedroc
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: KNi1 = (1.2 ± 0.1) × 1010 M–1, and KNi2 = (0.7 ± 0.4) × 1010 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-NH2-Gly2-His3-Gly4 motif at the N-terminus make significant contributions to the magnitude of KNi. 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 KNi 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. 15N 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.