Structure of native protein C inhibitor provides insight into its multiple functions.

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 beta-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.