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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 NmtR

by Pennella, Mario Antonio

Abstract (Summary)
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 N?2 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.
Bibliographical Information:

Advisor:Giedroc, David P.; Kunkel, Gary R.; LiWang, Patricia J.; Scholtz, J. Martin

School:Texas A&M University

School Location:USA - Texas

Source Type:Master's Thesis

Keywords:metal regulation czra and nmtr

ISBN:

Date of Publication:05/01/2003

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