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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 approximately 60 degrees 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 angstroms 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 PCI.APC complex was built based on mutagenesis and structural data.
Highlights
According to our mutagenesis data, thrombin and activated protein C (APC) are bridged to Protein C inhibitor (PCI) using two alternate heparin binding modes
Heparin acceleration of APC inhibition is extremely sensitive to the R229E mutation, implying a heparin binding mode that lies across helix H and contacts Arg-229
We showed that the cofactor effect of TM was similar to that of heparin in forming a bridge by binding to both thrombin and PCI
Summary
Is not involved, but that helix H (along the back of the serpin if shown in the classic orientation) is likely to be the principal interaction site [18] (Fig. 1A). The issue is complicated by the presence of an N-terminal extension containing several basic residues, the so-called Aϩ-helix [19], which has been shown to have some influence on heparin affinity, and the presence of a large basic patch adjacent to helix H [20] (Fig. 1B) It appears as if the H helix is the principal GAG binding site, mutation of the basic residues on helix H only marginally affects heparin affinity or heparin acceleration of protease inhibition [18, 21, 22]. We previously proposed a co-occupation mechanism for the PCI1⁄7APC1⁄7heparin complex in which PCI and APC share a common site on heparin to relieve the electrostatic repulsion between the proteases and inhibitor [20] This mechanism requires heparin to bind across the H helix and would involve the adjacent basic site.
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