Structure of Native Protein C Inhibitor Provides Insight into Its Multiple Functions

Abstract

Protein C inhibitor (PCI) is a multifunctional serpin with wide ranging protease inhibitory functions, unique cofactor binding activities, and potential non-inhibitory functions akin to the hormone-transporting serpins. To gain insight into the molecular mechanisms utilized by PCI we developed a robust expression system in Escherichia coli and solved the crystal structure of PCI in its native state. The five monomers obtained from our two crystal forms provide an NMR-like ensemble revealing regions of inherent flexibility. The reactive center loop (RCL) of PCI is long and highly flexible with no evidence of hinge region incorporation into ␤-sheet A, as seen for other heparin-binding serpins. We adapted an extrinsic fluorescence method for determining dissociation constants for heparin and find that the N-terminal tail of PCI and residues adjacent to helix H are not involved in heparin binding. The minimal heparin length capable of tight binding to PCI was determined to be chains of eight monosaccharide units. A large hydrophobic pocket occupied by hydrophobic crystal contacts was found in an analogous position to the hormone-binding site in thyroxine-binding globulin. In conclusion, the data presented here provide important insights into the mechanisms by which PCI exercises its multiple inhibitory and non-inhibitory functions.

Crystallization and crystallographic studies of kallistatin

“…As serpins are known to adopt different conformations, models of protein C inhibitor Li et al, 2007) and thyroxine-binding globulin (Zhou et al, 2006), representing one relaxed and two stressed serpin conformations ($47% sequence identity; PDB entries 1lq8, 2ce0 and 2hi9), were selected as search models. A clear single solution was obtained from a search using PDB entry 1lq8 as the model with one copy of molecule in the asymmetric unit, indicating a solvent content of $60%.…”

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

“…1B). Although 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). In fact, it takes at least three simultaneous mutations to see an appreciable detrimental effect and removal of all four positively charged residues to knock out heparin acceleration altogether.…”

“…In fact, it takes at least three simultaneous mutations to see an appreciable detrimental effect and removal of all four positively charged residues to knock out heparin acceleration altogether. We have recently shown that the minimum heparin length that fully occupies the heparin binding site of PCI is an octasaccharide, which fits neatly along the length of helix H (22) (Fig. 1C).…”

“…Our recent structures of native PCI (22) and the PCI⅐ thrombin⅐heparin complex (23) demonstrate that heparin accelerates PCI inhibition solely by bridging proteases to PCI (i.e. not an allosteric mechanism), and biochemical studies have shown that the minimum heparin length required for effective bridging to thrombin and APC is around 14 monosaccharide units (24).…”

“…After many screens, we managed to obtain a PCI-heparin co-crystal in a low ionic strength condition but determined by SDS-PAGE that the PCI had been cleaved in the reactive center loop. Because native and cleaved PCI have similar affinities for 8-mer heparin (22,24), we decided to generate cleaved PCI for crystallization trials using porcine pancreatic elastase. With cleaved PCI, we readily obtained high quality isomorphous crystals in complex with 8-mer and 10-mer heparins.…”