ABSTRACT
The origin of metal ion selectivity by members of the SmtB/ArsR family of bacterial metal-sensing transcriptional repressors and the mechanism of negative allosteric regulation of DNA binding is poorly understood. Here, we report that two homologous zinc sensors, Staphylococcus aureus CzrA and cyanobacterial SmtB, are "winged" helix homodimeric DNA-binding proteins that bind Zn(II) to a pair of tetrahedral, interhelical binding sites, with two ligands derived from the alpha5 helix of one subunit, Asp84 O(delta1) (Asp104 in SmtB), His86 N(delta1) (His106), and two derived from the alpha5 helix of the other, His97' N(delta1) (His117') and His100' N(epsilon2) (Glu120'). Formation of the metal chelate drives a quaternary structural switch mediated by an intersubunit hydrogen-binding network that originates with the non-liganding N(epsilon2) face of His97 in CzrA (His117 in SmtB) that stabilizes a low-affinity, DNA-binding conformation. The structure of the Zn(1) SmtB homodimer shows that both metal-binding sites of the dimer must be occupied for the quaternary structural switch to occur. Thus, a critical zinc-ligating histidine residue obligatorily couples formation of the metal-sensing coordination chelate to changes in the conformation and dynamics of the putative DNA-binding helices.
Subject(s)
Bacterial Proteins/chemistry , DNA-Binding Proteins/chemistry , Repressor Proteins/chemistry , Trans-Activators/chemistry , Zinc/chemistry , Allosteric Regulation , Amino Acid Sequence , Bacterial Proteins/metabolism , Binding Sites , Crystallography, X-Ray , Cyanobacteria/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dimerization , Hydrogen Bonding , Ligands , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Quaternary , Protein Subunits/chemistry , Protein Subunits/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Sequence Alignment , Staphylococcus aureus/metabolism , Trans-Activators/metabolismABSTRACT
SmtB is required for Synechococcus to effect a response to toxic concentrations of Zn(II) and other heavy metals. Direct binding of inducing metal ions to SmtB transcriptionally derepresses the expression of SmtA, a prokaryotic class II metallothionein. Homodimeric SmtB binds one Zn(II) or Co(II) per monomer in a cysteine thiolate-containing site in a tetrahedral coordination geometry [VanZile, M. L., et al. (2000) Biochemistry 39, 11818-11829]. In this report, characterization of a set of cysteine substitution mutants of SmtB reveals that SmtB homodimer binds Zn(II) or Co(II) in one of two mutually exclusive metal binding sites, termed alpha3N and alpha5, with very high equilibrium affinities. Both sites are characterized by similar affinities for Co(II) (K(Co) approximately equal to 2-5 x 10(9) M(-1)), while the Zn(II) affinities are at least 20-fold different (K(Zn)(alpha)(3N) > or = 10(13) M(-1); K(Zn)(alpha)(5) approximately equal to 5 x 10(11) M(-1)). Co(II) bound exclusively at the alpha5 sites is capable of rapid equilibration between the alpha3N and alpha5 sites upon reduction of the mixed disulfides in S-methylated SmtB. These results suggest that the alpha3N or alpha5 metal sites might play distinct roles in this Zn(II)-sensing protein, systematically investigated in the following paper [VanZile, M. L., Chen, X., and Giedroc, D. P. (2002) Biochemistry 41, 9776-9786]. Since both the alpha3N and alpha5 sites are present in many members of the SmtB/ArsR family of metal sensor proteins, the presence of these two metal binding sites may explain some of the functional diversity in metal responses across this family of proteins.
Subject(s)
Bacterial Proteins , Cyanobacteria/chemistry , DNA-Binding Proteins/chemistry , Repressor Proteins/chemistry , Zinc/chemistry , Amino Acid Sequence , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Sequence Homology, Amino Acid , Spectrometry, Fluorescence , Spectrophotometry, UltravioletABSTRACT
The Synechococcus PCC 7942 smt operon is responsible for cellular resistance to excess zinc and consists of two divergently transcribed genes, smtB and smtA. SmtB is the Zn(II)-sensing metal-regulated repressor of the system and binds to a 12-2-12 imperfect inverted repeat in the smtA O/P region. Using fluorescence anisotropy to monitor SmtB-smt O/P multiple equilibria, we show that four SmtB homodimers bind to a 40 bp oligonucleotide containing a single 12-2-12 inverted repeat. The binding affinities of the first two dimers are very tight (K(int) = 2.9 x 10(9) M(-1)) with the affinities of the third and fourth dimers lower by approximately 10- and approximately 30-fold, respectively. A single monomer equivalent of Zn(II), Cd(II), or Co(II) promotes disassembly of the oligomeric complex to a mixture of (P(2)).D and (P(2))(2).D SmtB dimer-DNA complexes with the intrinsic affinity of all SmtB homodimers for DNA greatly reduced by approximately 500-2000-fold. Substitution or derivatization of cysteines which comprise the alpha3N metal binding site (Cys14 and Cys61) [VanZile, M. L., et al. (2002) Biochemistry 41, 9765-9775] has no effect on allosteric negative regulation by Zn(II); in contrast, H106Q SmtB, harboring a single zinc-liganding substitution in the alpha5 metal binding site, is refractory to zinc-induced disassembly of SmtB-DNA complexes. The alpha5 metal binding sites are therefore regulatory for Zn(II) sensing in vitro and in vivo, while the high-affinity alpha3N sites play some other role. This finding for SmtB is the opposite of that previously determined for Staphylococcus aureus pI258 CadC, a Pb(II)/Cd(II)/Bi(III) sensor [Busenlehner, L. S., et al. (2002) J. Mol. Biol. 319, 685-701], thus providing insight into the origin of functional metal ion selectivity in this family of metal sensor proteins.
