Crystal structure of mature myroilysin and implication for its activation mechanism

Myroilysin is a novel bacterial member of M12A metalloproteases family with an uncommon “cysteine switch” activation mechanism and a unique “cap” structure. However, activation of pro-myroilysin is elusive. Here, mature myroilysin was obtained for structure determination by treating pro-myroilysin with trypsin. The structure of mature myroilysin showed that the active-site zinc ion of the mature protein is coordinated by three histidine residues, a water molecule, and a tyrosine residue (Tyr208) in the conserved Met-turn motif (SIMHY). The “cap” structure moves away from the active-site to leave the active cleft open; the newly formed N-terminus is deeply buried in myroilysin, and Glu151 forms a salt bridge directly with the first amino acid residue (Gly38), whereas they are far from each other in the pro-myroilysin. The mutation of Tyr208 indicates that Tyr208 plays an important role in activity of myroilysin. The proteolytic activity and thermostability of mutant E151A decreased dramatically, implying that Glu151 is not only important for catalysis, but also crucial for structural stability in myroilysin. Structural comparison also reveals differences existed between myroilysin and astacin. Our biochemical and structural data provide new insights into the activation of myroilysin and functional involvement of crucial residues Tyr208 and Glu151.