Structure Insight Into The Activation Mechanism Of Myroilysin

Myroilysin,a newly found bacterial family M12A metalloproteases,could digest elastin and many other substrates.In the former study of our lab,we had solved the structure of the pro-myroilysin.The structure of pro-myroilysin revealed a unique "cap"structure tops the active site cleft and an uncomnon "cysteine switch" activation mechanism.However,we failed to obtain diffractable mature myroilysin crystals and the activation mechanism of myroilysin is still unclear.In this study,the amino acids sequence analysis of myroilysin was performed and the result showed that there is a tryptic like cleavage site present between Arg3 7 and Gly3 8 and Gly38 in fact is the first amino acid residue in the mature myroilysin.The mature myroilysin was then obtained by the treatment of trypsin to remove the pro-peptide of the pro-myroilysin.The protease activity assay showed that the mature myroilysin has a pretty high activity.The mature myroilysin was then applied for the crystallization and diffractable mature myroilysin crystals were obtained in 0.1 M sodium acetate:acetic acid pH 4.5,1 M ammonium phosphate dibasic condition.A 2.6 A data set was collected and the structure of the mature myroilysin was then determined by using the molecular replacement with the solved structure of pro-myroilysin as the template.The structure of the myroilysin showed that the active site zinc ion of the mature myroilysin is coordinated by three histidine residues(Hisl40,Hisl44,His150);a glutamate(Glul41)bound water molecule and a tyrosine residue(Tyr208)in the conserved Met-turn motif(SIMHY).Compared to the pro-myroilysin,the "cap" structure of mature myroilysin moves away to exposes the active site,the Glu151 also forms salt bridge with the first amino acid residue of the mature myroilysin(Gly3 8).Moreover,the Tyr208 is engaged in zinc ion binding after the activation.To explain the real role of the Tyr208 in myroilysin,mutants Y208F and Y208A were eonstructed.The protease activity assay showed that the mutant Y208F only retained about 1%residual protease activity of the wild-type myroilysin and the protease activity of mutant Y208A is even lower than that of mutant Y208F(about 50%of Y208F).This implies that Tyr208 plays a very important role in the catalytic mechanism of mature myroilysin.Aside from the differences between myroilysin and astaicn we had observed,such as the unique "cap" structure opens for the substrate binding in mature myroilysin and the the inhibition mechanism,the activation mechanism of myroilysin is also different from that of astacin.The activation of pro-astacin requires two-step limited proteolysis,while activation of pro-myroilysin just occurs through one-step limited proteolysis.This different activation mechanism makes the N-terminus of mature myroilysin is longer and buried deeper than that of astacin,therefore the formed interactions are more stable than that of astacin.Further thermostability assays showed that both of the catalytic activity and thermostability of the mutant E151A are much lower than that of the myroilysin,while the E103A of astacin still retains high catalytic activity.This result indicate that the Glu-151 is more crucial to the myroilysin rather than Glu103 to the astacin.In sunmary,though myroilysin was classified as a M12A member,it has many-unique features and is different from other astacins.Considering the myroilysin is the first structure solved M12A family protease from the bacterium and all other structure solved M12A family proteases are mainly from animals,we proposed that myroilysin may be classified as anew bacterial subfamily of M12.