Perkins S J
Department of Biochemistry and Chemistry, Royal Free Hospital School of Medicine, London, U.K.
Behring Inst Mitt. 1993 Dec(93):63-80.
New data on the structure of C1- inhibitor have become available as the result of the completion of its amino acid and nucleic acid sequences and its carbohydrate content, together with data from neutron scattering, crystal structure determinations, 1H NMR spectroscopy and Fourier transform infrared spectroscopy. C1- inhibitor is a two-domain protein. It is constructed as a heavily glycosylated N-terminal domain with 113 amino acids and 3 N-linked and 7 O-linked oligosaccharides, and a C-terminal domain with 365 amino acids and 3 N-linked oligosaccharides. Its molecular weights is calculated as 71,100, rather than the apparent value of 105,000 commonly reported from SDS-PAGE. Neutron scattering shows that C1- inhibitor possesses a distinct two domain structure in which an extended N-terminal domain with extended carbohydrate structures is attached to the C-terminal SERPIN domain. The length of C1- inhibitor is 18 nm, rather than that of 33 nm originally proposed from electron microscopy. Microcalorimetry supports the concept of two independently-folded domains in the C1- inhibitor structure. Combination of the C1- inhibitor sequence with crystal structure determinations of proteins belonging to the SERPIN superfamily of serine protease inhibitors shows that the protein structure of the C-terminal domain is well-described in terms of this structure. Comparison of the exon structure of C1- inhibitor with its presumed protein crystal structure shows that the 7 exons are distributed throughout the SERPIN fold with no obvious segregation into structural elements. Native C1- inhibitor is cleaved at its reactive centre loop to release a more stable cleaved form that is no longer active. 1H NMR spectroscopy shows that structures involved in hydrophobic interactions within the core of the SERPIN fold are preserved to within 0.05 nm before and after the transition. Fourier transform infrared spectroscopy shows the presence of alpha-helix and beta-sheet in both forms. Large changes are detectable in both the alpha-helix and beta-sheet contents of the SERPIN fold, and these changes are matched by a greater rate of 1H-2H exchange in 2H2O solvents in the native form than in the cleaved form. These data show that changes in the whole secondary structure and not just in the reactive site loop are involved in SERPIN inactivation.
由于完成了C1抑制因子的氨基酸和核酸序列及其碳水化合物含量的测定，以及来自中子散射、晶体结构测定、1H核磁共振光谱和傅里叶变换红外光谱的数据，关于C1抑制因子结构的新数据已经可用。C1抑制因子是一种双结构域蛋白。它由一个高度糖基化的N端结构域和一个C端结构域组成，N端结构域有113个氨基酸以及3个N-连接和7个O-连接的寡糖，C端结构域有365个氨基酸以及3个N-连接的寡糖。其分子量经计算为71,100，而非SDS-PAGE通常报道的105,000的表观值。中子散射表明，C1抑制因子具有独特的双结构域结构，其中带有延伸碳水化合物结构的延伸N端结构域连接到C端丝氨酸蛋白酶抑制剂（SERPIN）结构域。C1抑制因子的长度为18纳米，而非最初电子显微镜提出的33纳米。微量量热法支持C1抑制因子结构中存在两个独立折叠结构域的概念。将C1抑制因子序列与属于丝氨酸蛋白酶抑制剂SERPIN超家族的蛋白质的晶体结构测定结果相结合表明，C端结构域的蛋白质结构可以根据该结构得到很好的描述。将C1抑制因子的外显子结构与其推测的蛋白质晶体结构进行比较表明，7个外显子分布在整个SERPIN折叠结构中，没有明显地分隔成结构元件。天然的C1抑制因子在其反应中心环处被切割，以释放出一种更稳定的切割形式，这种形式不再具有活性。1H核磁共振光谱表明，在转变前后，SERPIN折叠结构核心内参与疏水相互作用的结构保持在0.05纳米以内。傅里叶变换红外光谱表明两种形式中均存在α-螺旋和β-折叠。在SERPIN折叠结构的α-螺旋和β-折叠含量中均可检测到较大变化，并且与天然形式相比，天然形式在2H2O溶剂中的1H-2H交换速率更高，这些变化与切割形式相匹配。这些数据表明，SERPIN失活涉及整个二级结构的变化，而不仅仅是反应位点环的变化。
