Investigation of the new substitution glycine to alanine within the Kringle-2 domain of reteplase: a molecular dynamics study.

Abstract

Background: Recombinant plasminogen activator (r-PA) consists of the Kringle-2 and protease domains of human tissue-type plasminogen. It is used clinically to treat coronary artery thrombosis and acute myocardial infarction. However, the expression and production of reteplase (r-PA) are limited due to its susceptibility to proteolysis during manufacturing processes. Therefore, efforts have been made to address this limitation.

Materials and methods: To enhance the conformational stability of r-PA and increase its resistance to proteolysis, we used Gly 6 Ala substitutions in the Kringle-2 domain through in silico . We created an in silico mutant collection with eight structures, incorporating four designated mutations (R103S, G39A, G53A, and G55A). Using MODELLER software and homology modeling, we developed three-dimensional structures for two Kringle-2 and tissue plasminogen activator protease domains, including the wild noncleavable form (R103S) and mutants with all four designated mutations. We assessed protein stability using a dynamic cross-correlation matrix by extracting global properties such as Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF) from trajectory files.

Results: The findings revealed that a single glycine-alanine substitution (G39A) enhanced the conformational stability of r-PA, as evidenced by improvements in RMSD, RMSF, radius of gyration, surface accessibility, hydrogen bond formation, eigenvector projection, and density analysis.

Conclusion: The conformational stability of r-PA conferred by glycine replacement with alanine may decrease the propensity for proteolysis in protease - rich environments across various recombinant systems and potentially enhance its production and expression levels.