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

TY - JOUR

T1 - Biochemical and structural analyses suggest that plasminogen activators coevolved with their cognate protein substrates and inhibitors

AU - Jendroszek, Agnieszka

AU - Madsen, Jeppe B

AU - Chana-Muñoz, Andrés

AU - Dupont, Daniel M

AU - Christensen, Anni

AU - Panitz, Frank

AU - Füchtbauer, Ernst-Martin

AU - Lovell, Simon C

AU - Jensen, Jan K

N1 - © 2019 Jendroszek et al.

PY - 2019/3/8

Y1 - 2019/3/8

N2 - Protein 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 co-conservation 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.

AB - Protein 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 co-conservation 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.

KW - Amino Acid Sequence

KW - Animals

KW - Evolution, Molecular

KW - Humans

KW - Models, Molecular

KW - Plasminogen Activator Inhibitor 1/metabolism

KW - Plasminogen Activators/antagonists & inhibitors

KW - Protein Binding

KW - Protein Conformation

KW - Urokinase-Type Plasminogen Activator/metabolism

UR - http://www.scopus.com/inward/record.url?scp=85062630785&partnerID=8YFLogxK

U2 - 10.1074/jbc.RA118.005419

DO - 10.1074/jbc.RA118.005419

M3 - Journal article

C2 - 30651349

SN - 0021-9258

VL - 294

SP - 3794

EP - 3805

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 10

ER -