The Heparin Binding Site of Protein C Inhibitor Is Protease-dependent

Abstract

Protein C inhibitor (PCI) is a member of the serpin family of protease inhibitors with many biological functions and broad inhibitory specificity. Its major targets in blood are thrombin and activated protein C (APC), and the inhibition of both enzymes can be accelerated by glycosaminoglycans, including heparin. Acceleration of thrombin and APC inhibition by PCI requires that both protease and inhibitor bind to the same heparin chain to form a bridged Michaelis complex. However, the position of the heparin binding site of APC is opposite to that of thrombin, and formation of the bridged complexes must require either radical reorientation of the proteases relative to PCI or alternate heparin binding modes for PCI. In this study, we investigate how heparin bridges thrombin and APC to PCI by determining the effect of mutations in and around the putative heparin binding site of PCI. We found that heparin binds PCI in a linear fashion along helix H to bridge thrombin, consistent with our recent crystal structure (3B9F), but that it must rotate by ϳ60°to engage Arg-229 to bridge APC. To gain insight into the possible modes of heparin binding to PCI, we solved a crystal structure of cleaved PCI bound to an octasaccharide heparin fragment to 1.55 Å resolution. The structure reveals a binding mode across the N terminus of helix H to engage Arg-229 and align the heparin binding site of APC. A molecular model for the heparin-bridged PCI⅐APC complex was built based on mutagenesis and structural data.

Conserved Amblyomma americanum tick Serpin19, an inhibitor of blood clotting factors Xa and XIa, trypsin and plasmin, has anti-haemostatic functions

“…The serpin protein C inhibitor structure (2OL2) (Li and Huntington, 2008) was retrieved from the Protein Data Bank (PDB) (http://www.rcsb.org) and used as a molecular template for AAS19 modeling based on 30% and 53% sequence identity and similarity, respectively. Sequence alignments were generated using the ClustalW algorithm (Larkin et al, 2007) and used as input in the Modeller 9v14 program (Webb and Sali, 2014).…”

A microscopic view on the renal endothelial glycocalyx

“…First, thrombomodulin can bind chondroitin sulfate, greatly facilitating the interaction with thrombin and the formation of APC (56,116). The activity of APC and thrombin in their turn are negatively regulated by protein C inhibitor, which form together with different HS repeats ternary complexes (54). While very complex and still only partially understood, the intricate interaction of the coagulation system with the glycocalyx demonstrates the importance of the latter as a molecular scaffold.…”

Semi-Rigid Solution Structures of Heparin by Constrained X-ray Scattering Modelling: New Insight into Heparin–Protein Complexes

“…We now survey the heparin linker conformation in 19 heparin-protein crystal structures [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33] and the NMR dp12 solution structure. 12 The two major conformations of six-membered iduronate rings in heparin are the 1 C 4 chair and the 2 S 0 skew boat.…”

“…These include crystal structures for acidic and basic fibroblast growth factors separately; 14,15 the complexes of acidic and basic fibroblast growth factors with their receptors; 16,17 thrombin, 18 antithrombin, and the complex of antithrombin-S195A and coagulation factor Xa; [19][20][21][22] footand-mouth disease virus; 23 neurokinin 1 receptor; 24 annexins V and A2; 25,26 vaccinia complement control protein (VCP); 27 and protein C inhibitor. 28 Many of these crystal structures likewise show extended heparin conformations. At the opposite extreme of structural resolution, macroscopic solution structures for large polydisperse heparin fractions have been studied by X-ray solution scattering and analytical ultracentrifugation (AUC).…”