Allosteric Negative Regulation of smt O/P Binding of the Zinc Sensor, SmtB, by Metal Ions: A Coupled Equilibrium Analysis

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Abstract
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 (Kint = 2.9 × 109 M-1) with the affinities of the third and fourth dimers lower by ≈10- and ≈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 (P2)·D and (P2)2·D SmtB dimer−DNA complexes with the intrinsic affinity of all SmtB homodimers for DNA greatly reduced by ≈500−2000-fold. Substitution or derivatization of cysteines which comprise the α3N 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 α5 metal binding site, is refractory to zinc-induced disassembly of SmtB−DNA complexes. The α5 metal binding sites are therefore regulatory for Zn(II) sensing in vitro and in vivo, while the high-affinity α3N 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.