Structure of native protein C inhibitor provides insight into its multiple functions
Structure of native protein C inhibitor provides insight into its multiple functions
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DOI：
10.1074/jbc.m701074200
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发表时间：
2007-05-04
影响因子：
4.8
通讯作者：
Huntington, James A.
中科院分区：
文献类型：
--
作者：
Li, Wei;Adams, Ty E.;Huntington, James A.
蛋白C抑制因子(Protein C Inhibitor，PCI)是一种多功能的丝蛋白，具有广泛的蛋白酶抑制功能、独特的辅因子结合活性和潜在的非抑制功能，类似于激素转运的丝蛋白。为了深入了解pCI的分子机制，我们在大肠杆菌中建立了一个健壮的表达系统，并解决了pCI在天然状态下的晶体结构。从我们的两种晶型获得的五种单体提供了类似于核磁共振的系综，揭示了固有的灵活性区域。PCI的反应中心环(RCL)是长的和高度灵活的，没有证据表明铰链区整合到β-折叠A中，就像其他肝素结合的蛇毒一样。我们采用外源荧光法测定肝素的解离常数，发现PCIN-末端和螺旋H附近的残基不参与肝素的结合。能够与PCI紧密结合的最小肝素长度被确定为由8个单糖单元组成的链。在与甲状腺激素结合球蛋白的激素结合位置相似的位置上，发现了一个由疏水晶体接触占据的大的疏水口袋。总而言之，本文提供的数据为了解经皮冠状动脉介入治疗行使其多重抑制和非抑制功能的机制提供了重要的见解。
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.