Metal specificity and the mechanism of allosteric regulation in metal-sensing metal-responsive transcriptional repressors Staphylococcus aureus CzrA and Mycobacterium tuberculosis NmtR

摘要

The metal-responsive transcriptional repressors of the SmtB/ArsR family repressthe expression of their respective operons in the absence of metal and are released fromthe operator/promoter region when metal ions bind, thus allowing RNA polymerase tobind and transcribe the operon, which encodes genes involved in homeostasis andresistance. To elucidate the determinants of metal ion selectivity, comparative metalbindingand DNA-binding properties of S. aureus CzrA and M. tuberculosis NmtR werecharacterized. The structure of the metal coordination complexes of CzrA and NmtRreveal 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 invivo. In contrast, NmtR adopts 5- or 6-coordinate complexes with Ni(II) and Co(II), thestrongest 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 affinityand forms a non-native 4-coordinate complex. These studies suggest that metalcoordination geometries (number), not metal binding affinities, are primary determinantsof functionality.To gain molecular insight into the mechanism of allosteric regulation of O/Pbinding by metal ions, NMR and X-ray crystallographic studies of apo- and zinc formsof CzrA, and another ArsR/SmtB zinc sensor, Synechococcus PCC7942 SmtB, wereperformed. These studies showed that formation of the metal chelate drives a quaternarystructural switch mediated by an intersubunit hydrogen-binding network that originateswith the nonliganding Nε2 face of His97 in CzrA (His117 in SmtB) that stabilizes a lowaffinity 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 nonfunctionalin driving zinc-mediated allosteric regulation of DNA binding. In contrast, conservativemutations of H86 and H100 in CzrA bind Co(II) or Zn(II) in a tetrahedral manner, albeitwith greatly reduced affinity, and allosterically regulate O/P binding with significantlower coupling free energies compared to wild-type CzrA. These findings furtherreinforce the notion that metal coordination geometry is the primary determinant forfunctional sites in metal-sensing transcriptional repressors.