Biochemical and structural analyses suggest that plasminogen activators coevolved with their cognate protein substrates and inhibitors

Abstract

Edited by Joseph M. JezProtein sequences of members of the plasminogen activation system are present throughout the entire vertebrate phylum. This important and well-described proteolytic cascade is governed by numerous protease-substrate and protease-inhibitor interactions whose conservation is crucial to maintaining unchanged protein function throughout evolution. The pressure to preserve protein-protein interactions may lead to either coconservation or covariation of binding interfaces. Here, we combined covariation analysis and structure-based prediction to analyze the binding interfaces of urokinase (uPA):plasminogen activator inhibitor-1 (PAI-1) and uPA:plasminogen complexes. We detected correlated variation between the S3-pocket-lining residues of uPA and the P3 residue of both PAI-1 and plasminogen. These residues are known to form numerous polar interactions in the human uPA:PAI-1 Michaelis complex. To test the effect of mutations that correlate with each other and have occurred during mammalian diversification on protein-protein interactions, we produced uPA, PAI-1, and plasminogen from human and zebrafish to represent mammalian and nonmammalian orthologs. Using single amino acid point substitutions in these proteins, we found that the binding interfaces of uPA:plasminogen and uPA:PAI-1 may have coevolved to maintain tight interactions. Moreover, we conclude that although the interaction areas between protease-substrate and protease-inhibitor are shared, the two interactions are mechanistically different. Compared with a protease cleaving its natural substrate, the interaction between a protease and its inhibitor is more complex and involves a more fine-tuned mechanism. Understanding the effects of evolution on specific protein interactions may help further pharmacological interventions of the plasminogen activation system and other proteolytic systems. 2 The abbreviations used are: uPA, urokinase; PAI-1, plasminogen activator inhibitor-

Between Protein Fold and Nucleophile Identity: Multiscale Modeling of the TEV Protease Enzyme–Substrate Complex

“…Both methods work since the physicochemical interactions that drive protein folding and protein–peptide interactions are the same . Protease binding sites′ evolve to better recognize alien substrate loops and cognate substrate loops coevolve with their respective proteases. − …”

“… 76 Protease binding sites′ evolve to better recognize alien substrate loops and cognate substrate loops coevolve with their respective proteases. 77 − 79 …”