Flexibility in monomeric Cu,Zn superoxide dismutase detected by limited proteolysis and molecular dynamics simulation

Limited proteolysis by trypsin of monomeric Cu,Zn superoxide dismutase from Escherichia coli induces a specific cleavage of the polypeptide chain at the level of Lys60 located in the S-S subloop of loop 6,5 where, when compared to the eukaryotic enzyme, a seven-residues insertion, completely exposed to the solvent, is observed. This result suggests that this subloop is disordered and flexible, thus enabling binding and adaptation to the active site of the proteolytic enzyme. Indeed, molecular dynamics simulation indicates that the S-S subloop undergoes high fluctuations and that its high flexibility coupled to an high solvent accessibility can explain the specific bond selection of the protease. As a matter of fact, of the possible 14 solvent accessible proteolytic sites only the Lys60 flexible site is cleaved. High flexibility and solvent exposure are confirmed by the short water residence time for the residues corresponding to the cleavage site evaluated by molecular dynamics simulation. These experiments demonstrate that molecular dynamics simulation and limited proteolysis are complementary and unambiguous tools to identify flexible sites in proteins.