The Cyanobacterial Repressor SmtB Is Predominantly a Dimer and Binds Two Zn²⁺ Ions per Subunit

Abstract

The Synechococcus PCC7942 metallothionein repressor gene smtB has been cloned into a high expression vector and the protein purified to near homogeneity (>/=98%). Analytical ultracentrifugation studies demonstrate that the protein is predominantly dimeric in 0.1 M NaCl, pH 7.4, and 22 degrees C, exhibiting a monomer-dimer-tetramer equilibrium. The monomer-dimer (Ka(1,2)) and the dimer-tetramer (Ka(2,4)) association constants are 3.24 x 10(5) and 9.90 x 10(2) M-1, respectively. The repressor binds two Zn2+ ions per subunit with an overall Kd of 3.49 x 10(-6) M. In the presence of Zn2+, Ka(1, 2) increases by 2 orders of magnitude to 1.25 x 10(7) M-1 and the apparent weight-averaged sedimentation coefficient increases from 2. 00 to 2.22 S. The fact that the increase in sedimentation coefficient is greater than that predicted by increased dimerization is interpreted as caused by compaction of the structure in the presence of metal ions. At pH 6.0, 0.1 M NaCl, and 22 degrees C, the protein exhibits only a monomer-dimer equilibrium, with Ka(1,2) = 1.52 x 10(7) M-1 which is almost identical to that seen upon binding Zn2+ at pH 7.4. The compaction and conformational change in SmtB caused by Zn2+ is consistent with a role for this altered quaternary state in derepression of smtA in Synechococcus challenged with heavy metal ions.

A Metal–Ligand-mediated Intersubunit Allosteric Switch in Related SmtB/ArsR Zinc Sensor Proteins

“…For example, in this superposition (Figure 1(c)), the Ser74 C a atom in the aR helix of the subunit shown on the left moves by 4.8 Å , or an average of < 2.4 Å in each subunit within the dimer; this reduces the pointto-point distance between Ser74 C a atoms on opposite subunits by 3.6 Å , from 51.7 Å to 48.1 Å . Thus, the homodimer becomes more globally compact on Zn(II) binding, in qualitative agreement with the results of previous sedimentation equilibrium experiments; 27 this results in significant movements of both the HTH motif and b-hairpin wings in opposite subunits relative to one another.…”

Role of Cysteinyl Residues in Sensing Pb(II), Cd(II), and Zn(II) by the Plasmid pI258 CadC Repressor

“…CadC purified as a homodimer, and no evidence of monomer was observed upon molecular sieve chromatography (data not shown). This is consistent with the reported dimerization of the homologous ArsR (25) and SmtB (26). Upon SDS-polyacrylamide gel electrophoresis, CadC was present as both monomer and dimer (data not shown).…”

The SmtB/ArsR family of metalloregulatory transcriptional repressors: structural insights into prokaryotic metal resistance

“…6) [23,32,39,55], with the monomer^dimer equilibrium linked to site-speci¢c DNA binding equilibria. SmtB is thus far unique in that a monomer^dimer^tet-ramer equilibrium better describes the self-association of this protein [55]. The monomer is assumed not to have appreciable DNA binding activity, although an assembly or cooperative, monomer binding model cannot yet be rigorously ruled out.…”