Sm11p is a dimer in solution: Characterization of denaturation and of recombinant Sm11p

Sm11p is a small 104-amino acid protein from Saccharomyces cerevisiae that binds to the large subunit (Rnr1p) of the ribonucleotide reductase complex (RNR) and inhibits its activity. During DNA damage, S phase, or both, RNR activity must be tightly regulated, since failure to control the cellular level of dNTP pools may lead to genetic abnormalities, such as genome rearrangements, or even cell death. Structural characterization of Sm11p is an important step in understanding the regulation of RNR. Until now the oligomeric state of Sm11p was unknown. Mass spectrometric analysis of wild-type Sm11p revealed an intermolecular disulfide bond involving the cysteine residue at position 14 of the primary sequence. To determine whether disulfide bonding is essential for Sm11p oligomerization, we mutated the Cys14 to serine. Sedimentation equilibrium measurements in the analytical ultracentrifuge show that both wild-type and C14S Sm11p exist as dimers in solution, indicating that the dimerization is not a result of a disulfide bond. Further studies of several truncated Sm11p mutants revealed that the N-terminal 8-20 residues are responsible for dimerization. Unfolding/refolding studies of wild-type and C14S Sm11p reveal that both proteins retold reversibly and have almost identical unfolding/refolding profiles. It appears that Sm11p is a two-domain protein where the N-terminus is responsible for dimerization and the C-terminus for binding and inhibiting Rnr1p activity.