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.
Li
Wei
W
Department of Haematology, Division of Structural Medicine, Thrombosis Research Unit, Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, United Kingdom.
Adams
Ty E
TE
Kjellberg
Margareta
M
Stenflo
Johan
J
Huntington
James A
JA
D001665:Binding Sites / Q000502:physiology; D018360:Crystallography, X-Ray; D006493:Heparin / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding / Q000502:physiology; D018046:Protein C Inhibitor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D013975:Thyroxine-Binding Proteins / Q000737:chemistry
D018046:Protein C Inhibitor; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D013975:Thyroxine-Binding Proteins; D006493:Heparin