Journal
BIOCHEMISTRY
Volume 41, Issue 31, Pages 9776-9786Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi020178t
Keywords
-
Categories
Ask authors/readers for more resources
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 approximate to10- and approximate to30-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 approximate to500-2000-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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available