Solution structure of a paradigm ArsR family zinc sensor in the DNA-bound state

Abstract

Staphylococcus aureusCzrA is a zinc-dependent transcriptional repressor from the ubiquitous ArsR family of metal sensor proteins. Zn(II) binds to a pair of intersubunit C-terminal ␣5-sensing sites, some 15 Å distant from the DNA-binding interface, and allosterically inhibits DNA binding. This regulation is characterized by a large allosteric coupling free energy (⌬Gc) of approximately ؉6 kcal mol ؊1 , the molecular origin of which is poorly understood. Here, we report the solution quaternary structure of homodimeric CzrA bound to a palindromic 28-bp czr operator, a structure that provides an opportunity to compare the two allosteric ''end'' states of an ArsR family sensor. Zn ( M etal ion homeostasis is a complex process that involves maintaining a delicate balance between metal uptake/ efflux and other metal storage systems to meet the needs of the cell (1, 2). This process is tightly regulated at the level of transcription by means of specific metal-dependent transcriptional regulators that respond to changes in metal ion concentrations in the host environment (3). In Staphylococcus aureus, the czr operon encodes a CDF antiporter, CzrB, a homolog of Escherichia coli zinc transporter YiiP that confers resistance to Zn(II) and Co(II) (4, 5), and the metal-regulated repressor, CzrA (6, 7). CzrA binds Zn(II) with picomolar affinity and strong negative homotropic cooperativity (8, 9) and is thought to undergo a conformational change that alleviates transcriptional repression of the resistance gene czrB.CzrA belongs to the ubiquitous ArsR (or ArsR/SmtB) family of metalloregulators found in many bacterial genomes that sense a wide variety of metals including biologically essential metals as well as toxic metal pollutants (10, 11). Members of this family appear to adopt a common winged helix-turn-helix homodimeric fold, but have evolved physically and structurally distinct pairs of allosteric metal-sensing sites (2, 12). These sites are thought to have arisen as a result of convergent evolution due to evolutionary pressures (12), a finding consistent with the ''rule of varied allosteric control'' in which protein families evolve seemingly random allosteric control pathways (13). CzrA and its homolog SmtB in Synechococcus (14) are ␣5 sensors that bind Zn(II) ions in two rotationally symmetric tetrahedral coordination sites formed by pairs of metal ligands derived from the ␣5 helix of each subunit (8). The crystal structure of CzrA and SmtB in the apo and the Zn(II)-bound states have been solved (8). Although the structures of CzrA were found to be very similar in these two states, SmtB revealed measurable differences in quaternary structure in which the apo form adopted a comparatively ''flat'' conformation not well suited to interact with canonical B-form DNA (8,15). The structural and thermodynamic underpinnings of metalloregulation for any member of the ubiquitous ArsR family remains poorly understood due to a lack of detailed insight for the DNA operator-bound state (3).We report here the NMR solution structure of ...

Plasmid protein TubR uses a distinct mode of HTH-DNA binding and recruits the prokaryotic tubulin homolog TubZ to effect DNA partition

“…Because all but the N-terminal residues of the TubR recognition helices are buried in the dimer interface, TubR must use a different mode of DNA binding than the ArsR or other HTH containing proteins (23). Examination of surface electrostatics of TubR reveals that one face of the protein is electronegative, whereas the other is strongly electropositive (Fig.…”

Structural basis for regulation of rhizobial nodulation and symbiosis gene expression by the regulatory protein NolR

“…Previous structural studies of ArsR/SmtB proteins largely focused on the role of metal-dependent conformational changes and not protein-DNA interaction, with one exception. NMR and mutagenesis probed residues in CzrA from Staphylococcus aureus for roles in binding to the palindromic 28-bp czr operator (28). This work suggests that CzrA interaction with target DNA increases protein motion in the allosteric sites and showed essential roles for residues analogous to Gln56, Ser57, Ser60, and Gln61 in DNA binding by CzrA, but did not reveal the details of protein-DNA contacts required for phosphate backbone interactions and/or base specificity.…”

“…Sequence and structural comparisons identify NolR as a member of the ArsR/SmtB family of transcription factors (26)(27)(28)(29). NolR shares 22-40% amino acid sequence identity with BigR, HlyU, CadC, CzrA, NmtR, and SmtB (Fig.…”

“…In the ArsR/SmtB proteins, metal binding triggers conformational changes that result in derepression of gene expression by driving repositioning of the helix-turn-helix DNA interaction motif (26)(27)(28)(29)(31)(32)(33). This movement results in a switch from a "closed" DNA-binding structure to an "open" low-affinity conformation of the homodimer.…”

“…S1). Unlike other ArsR/ SmtB transcription factors (26)(27)(28)(29)(31)(32)(33), no global structural Titrations were performed using 22-bp DNA duplexes.…”