Metalloregulatory proteins control the expression of genes that allow organisms to quickly adapt to chronic toxicity or deprivation of both biologically essential metal ions and heavy metal pollutants found in their microenvironment. Emerging evidence suggests that metal ion homeostasis and resistance defines an important tug-of-war in human host-bacterial pathogen interactions. This adaptive response originates with the formation of "metal receptor" complexes of exquisite selectivity. In this perspective, we summarize consensus structural features of metal sensing coordination complexes and the evolution of distinct metal selectivities within seven characterized metal sensor protein families. In addition, we place recent efforts to understand the structural basis of metal-induced allosteric switching of these metalloregulatory proteins in a thermodynamic framework, and review the degree to which coordination chemistry drives changes in protein structure and dynamics in selected metal sensor systems. New insights into how metal sensor proteins function in the complex intracellular milieu of the cytoplasm of cells will require a more sophisticated understanding of the "metallome" and will benefit greatly from ongoing collaborative efforts in bioinorganic, biophysical and analytical chemistry, structural biology and microbiology.

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 ...

The initial high affinity binding of single-stranded DNA (ssDNA) by replication protein A (RPA) is involved in the tandem domains in the central region of the RPA70 subunit (RPA70AB). However, it was not clear whether the two domains, RPA70A and RPA70B, bind DNA simultaneously or sequentially. Here, using primarily heteronuclear NMR complemented by fluorescence spectroscopy, we have analyzed the binding characteristics of the individual RPA70A and RPA70B domains and compared them with the intact RPA70AB. NMR chemical shift comparisons confirmed that RPA70A and RPA70B tumble independently in solution in the absence of ssDNA. NMR chemical shift perturbations showed that all ssDNA oligomers bind to the same sites as observed in the x-ray crystal structure of RPA70AB complexed to d(C) 8 . Titrations using a variety of 5-mer ssDNA oligomers showed that RPA70A has a 5-10-fold higher affinity for ssDNA than RPA70B. Detailed analysis of ssDNA binding to RPA70A revealed that all DNA sequences interact in a similar mode. Fluorescence binding measurements with a variety of 8 -10-mer DNA sequences showed that RPA70AB interacts with DNA with ϳ100-fold higher affinity than the isolated domains. Calculation of the theoretical "linkage effect" from the structure of RPA70AB suggests that the high overall affinity for ssDNA is a byproduct of the covalent attachment of the two domains via a short flexible tether, which increases the effective local concentration. Taken together, our data are consistent with a sequential model of DNA binding by RPA according to which RPA70A binds the majority of DNA first and subsequent loading of RPA70B domain is facilitated by the linkage effect.The fundamental genetic processes of DNA replication, recombination, and repair are carried out by large multiprotein assemblies. Single-stranded DNA-binding proteins are required to organize and protect exposed ssDNA 1 in all of these pathways. The single-stranded DNA-binding protein function in eukaryotes is carried out by the heterotrimeric replication protein A (RPA). The three subunits (RPA70, RPA32, RPA14), named according to their respective molecular weights, are highly conserved, and all are required for function (1, 2). Threedimensional structures determined by NMR and x-ray crystallography have been obtained for all RPA domains, but the overall quaternary structure of the trimer is not yet known. A schematic representation of the domain organization of RPA is presented in Fig. 1. The N-and C-terminal regions of RPA70 (RPA70 1-110 , RPA70N; RPA70 436 -616 , RPA70C), the central region of RPA70 (RPA70 181-422 , RPA70AB), the central domain of RPA32 (RPA32 43-171 , RPA32D), and RPA14 all adopt an oligonucleotide/oligosaccharide binding fold, a structure common to other known single-stranded DNA-binding proteins (3-7). The C-terminal region of RPA32 (RPA32 205-270 , RPA32C) contains a winged helix-loop-helix domain (8). Binding of ssDNA is carried out by RPA70A (RPA70 181-291 ), RPA70B (RPA70 298 -422 ), RPA70C, and RPA32D. RPA70N and RPA32C do not...

Simian virus 40 (SV40) provides a model system for the study of eukaryotic DNA replication, in which the viral protein, large T antigen (Tag), marshals human proteins to replicate the viral minichromosome. SV40 replication requires interaction of Tag with the host single-stranded DNA-binding protein, replication protein A (hRPA). The C-terminal domain of the hRPA32 subunit (RPA32C) facilitates initiation of replication, but whether it interacts with Tag is not known. Affinity chromatography and NMR revealed physical interaction between hRPA32C and the Tag origin DNA-binding domain, and a structural model of the complex was determined. Point mutations were then designed to reverse charges in the binding sites, resulting in substantially reduced binding affinity. Corresponding mutations introduced into intact hRPA impaired initiation of replication and primosome activity, implying that this interaction has a critical role in assembly and progression of the SV40 replisome.