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Biochemical Characterization of the Mycobacterium tuberculosis Ni(II) Sensor NmtR and Streptococcus pneumoniae Zn(II) Sensor AdcRReyes Caballero, Hermes 2011 August 1900 (has links)
NmtR and AdcR belong to two structural and functional classes of transcriptional metalloregulators. The present study shows that AdcR is a novel Zn(II) dependent repressor and the first ever metalloregulator of the MarR family. In contrast, NmtR is a repressor that is inactivated by Ni(II) binding.
NmtR is a member of the extensively characterized ArsR/SmtB family. Two Ni(II) ions bind to the NmtR dimer in an octahedral coordination complex with stepwise binding affinities of KNi1 = 1.2 (±0.1) x 10¹⁰ and KNi2 = 0.7 (±0.4) x 10¹⁰ M⁻¹ (pH 7.0). A glutamine scanning mutagenesis approach reveals that Asp 91, His 93, His 104 and His 107 in the [alpha]5 helix and His 3 at the extreme N-terminal contribute to KNi. In contrast residues from the C-terminal tail (H109, D114 and H116), previously implicated in NmtR binding, are characterized by near wild-type KMe and allosteric coupling free energies. However, deletion of most of the C-terminal tail to create Δ111 NmtR reduce Ni(II) binding stoichiometry to one per dimer and greatly reduced Ni(II) responsiveness. H3Q and Δ111 NmtR also show important perturbations in the rank order of metal responsiveness, with both different from wild-type NmtR. The use of both presumably unstructured N- and C- terminal extensions is a unique property relative to other members of the ArsR/SmtB family previously characterized and provides a distinct metal specificities profile.
AdcR binds two regulatory Zn(II) ions per dimer in an unusual five coordinate geometry as determined by X-ray and electronic absorption spectroscopy. Functional characterization of single residue substitution mutants identified His 108 and His 112 in [alpha]5 helix and His 42 in [alpha]2 helix, as residues essential for allosteric activation of DNA operator binding by AdcR as revealed by fluorescence anisotropy experiments. The stability constant for the regulatory site, KZn, is sensitive to pH and range from ~10¹⁰ M⁻¹ at pH 6.0 to ~10¹⁰ M⁻¹ at pH 8.0. Zn(II) binds to an additional one to two pairs of ancillary sites per dimer depending on the pH. A novel feature not shared by other Zn(II) regulators is an apparent reduced metal specificity, since non-cognate metals Mn(II) and Co(II) activate AdcR to the same extent than Zn(II) does. However, each non-cognate metal binds with very low affinity (<̲ 10⁶ M⁻¹ at pH 8.0) and are not inducers in vivo.
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Metal specificity and the mechanism of allosteric regulation in metal-sensing metal-responsive transcriptional repressors Staphylococcus aureus CzrA and Mycobacterium tuberculosis NmtRPennella, Mario Antonio 29 August 2005 (has links)
The metal-responsive transcriptional repressors of the SmtB/ArsR family repress
the expression of their respective operons in the absence of metal and are released from
the operator/promoter region when metal ions bind, thus allowing RNA polymerase to
bind and transcribe the operon, which encodes genes involved in homeostasis and
resistance. To elucidate the determinants of metal ion selectivity, comparative metalbinding
and DNA-binding properties of S. aureus CzrA and M. tuberculosis NmtR were
characterized. The structure of the metal coordination complexes of CzrA and NmtR
reveal that CzrA forms a 4-coordinate, tetrahedral complex with both Zn(II) and Co(II)
potent regulators of czr operator/promoter (O/P) binding in vitro and de-repression in
vivo. In contrast, NmtR adopts 5- or 6-coordinate complexes with Ni(II) and Co(II), the
strongest allosteric regulators of nmt O/P binding in vitro and de-repression in vivo.
Zn(II), a non-inducer in vivo and poor regulator in vitro, binds NmtR with high affinity
and forms a non-native 4-coordinate complex. These studies suggest that metal
coordination geometries (number), not metal binding affinities, are primary determinants
of functionality.
To gain molecular insight into the mechanism of allosteric regulation of O/P
binding by metal ions, NMR and X-ray crystallographic studies of apo- and zinc forms
of CzrA, and another ArsR/SmtB zinc sensor, Synechococcus PCC7942 SmtB, were
performed. These studies showed that formation of the metal chelate drives a quaternary
structural switch mediated by an intersubunit hydrogen-binding network that originates
with the nonliganding Nε2 face of His97 in CzrA (His117 in SmtB) that stabilizes a low
affinity DNA-binding conformation.
Mutagenesis experiments reveal that substitution of D84 and H97 in CzrA,
results in the formation of higher coordination number complexes that are nonfunctional
in driving zinc-mediated allosteric regulation of DNA binding. In contrast, conservative
mutations of H86 and H100 in CzrA bind Co(II) or Zn(II) in a tetrahedral manner, albeit
with greatly reduced affinity, and allosterically regulate O/P binding with significant
lower coupling free energies compared to wild-type CzrA. These findings further
reinforce the notion that metal coordination geometry is the primary determinant for
functional sites in metal-sensing transcriptional repressors.
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