Abstract
Plasminogen activator inhibitor-1 (PAI-1) is the main physiological inhibitor of tissue-type (tPA) and urokinase-type (uPA) plasminogen activators (PAs). Apart from being critically involved in fibrinolysis and wound healing, emerging evidence indicates that PAI-1 plays an important role in many diseases, including cardiovascular disease, tissue fibrosis, and cancer. Targeting PAI-1 is therefore a promising therapeutic strategy in PAI-1 related pathologies. Despite ongoing efforts no PAI-1 inhibitors were approved to date for therapeutic use in humans. A better understanding of the molecular mechanisms of PAI-1 inhibition is therefore necessary to guide the rational design of PAI-1 modulators. Here, we present a 1.9 Å crystal structure of PAI-1 in complex with an inhibitory nanobody VHH-s-a93 (Nb93). Structural analysis in combination with biochemical characterization reveals that Nb93 directly interferes with PAI-1/PA complex formation and stabilizes the active conformation of the PAI-1 molecule.
Highlights
Plasminogen activator inhibitor-1 (PAI-1), a 45-kDa glycoprotein, is the main physiological inhibitor of plasminogen activators (PAs) such as tissue-type PA and urokinase-type PA [1]
The structure of PAI-1 in the complex (Figure 1A) shows the evolutionarily conserved serpin topology consisting of three β-sheets (A–C) and nine α-helices [2]
Comparison of the PAI-1-W175F structure in the complex and the isolated PAI-1-W175F structure (PDB ID 3Q02, chain A) shows that the conformation of PAI-1 is unaltered by interacting with nanobody VHH-s-a93 (Nb93) (Cα RMSD of 1.197 Å, excluding the flexible reactive center loop (RCL) residues 331–356)
Summary
Plasminogen activator inhibitor-1 (PAI-1), a 45-kDa glycoprotein, is the main physiological inhibitor of plasminogen activators (PAs) such as tissue-type PA (tPA) and urokinase-type PA (uPA) [1]. In contrast to other serpins, PAI-1 has the unique ability to spontaneously convert into a stable latent form by inserting the RCL segment N-terminal to the P1-P1 cleavage site (residues 331 to 346, designated as P16-P1) into the core of the protein. This transition occurs with a half-life of approximately two hours at 37 ◦C in vitro but is slightly longer in vivo due to the high-affinity association with vitronectin in plasma and the extracellular matrix [6,7]
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