An Essential Protein Repair Enzyme: Investigation of the Molecular Recognition Mechanism of Methionine Sulfoxide Reductase A

The amino acid methionine is particularly sensitive to damage by reactive oxygen species. The enzyme methionine sulfoxide reductase A MsrA is capable of repairing oxidized methionines Met-O found in a wide range of damaged substrates, ultimately protecting cells against oxidative damage. How MsrA reverses oxygen modifications to these damaged proteins is well known, but very little is known about how MsrA recognizes the damaged proteins in the first place. Unlike most enzymes which carry out reactions on a single target molecule, MsrA can repair damage to a single methionine, a peptide, or an entire protein. This study focused on understanding how MsrA is able to recognize, then ultimately repair such a range of oxidatively modified substrates. We propose that MsrA functions as a molecular chaperone, recognizing overall characteristics of unfolded proteins due to oxidative damage. Enzyme-ligand interactions were studied between MsrA of E. coli and four target molecules in their normal and oxidized forms staphylococcal nuclease, staphylococcal nuclease T62P which has an unfolded structure due to a mutation, a 9-amino acid peptide KKMVENAKK derived from staphylococcal nuclease, and the non-steroidal anti-inflammatory drug sulindac. Using the hydrophobic marker 8-anilino-1-naphthalene sulfonic acid ANS, changes in ANS fluorescence were used to identify changes in the exposed hydrophobic surface area of the protein-ligand complex. The investigation reveals an MsrA target molecule recognition mechanism of weak, but specific hydrophobic interactions enhanced by the presence of a sulfoxide. This study represents the first investigation of the interaction of MsrA with physiologically relevant ligands and has laid the foundation for a novel method of investigating the hydrophobic recognition mechanism of MsrA as a chaperone for oxidatively modified target molecules.