Rossi V, Gaboriaud C, Lacroix M, Ulrich J, Fontecilla-Camps J C, Gagnon J, Arlaud G J
Laboratoire d'Enzymologie Moléculaire, Institut de Biologie Structurale Jean-Pierre Ebel (CEA-CNRS), Grenoble, France.
Biochemistry. 1995 Jun 6;34(22):7311-21. doi: 10.1021/bi00022a004.
C1s is a multidomain serine protease that is responsible for the enzymatic activity of C1, the first component of the classical pathway of complement. Its catalytic region (gamma-B) comprises two contiguous complement control protein (CCP) modules, IV and V (about 60 residues each), a 15-residue intermediary segment, and the B chain (251 residues), which is the serine protease domain. With a view to identify domain-domain interactions within this region, the gamma-B fragment of C1s, obtained by limited proteolysis with plasmin, was chemically cross-linked with the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide; then cross-linked peptides were isolated after CNBr cleavage and thermolytic digestion. N-Terminal sequence and mass spectrometry analyses allowed us to identify two cross-links between Lys 405 of module V and Glu 672 of the B chain and between Glu 418 of the intermediary segment and Lys 608 of the B chain. Three-dimensional modeling of the CCP modules IV and V and of the catalytic B chain was also carried out on the basis of their respective homology with the 16th and 5th CCP modules of complement factor H and type I serine proteases. The information provided by both the chemical cross-linking studies and the homology modeling enabled us to construct a three-dimensional model for the assembly of the C-terminal part of the gamma-B region, comprising module V, the intermediary segment, and the B chain. This model shows that module V interacts with the serine protease B chain on the side opposite to both the activation site and the catalytic site. Functional implications of this interaction are discussed in terms of the possible role of module V in the specific recognition and positioning of C4, one of the two substrates of C1s.
C1s是一种多结构域丝氨酸蛋白酶，负责补体经典途径的首个组分C1的酶活性。其催化区域（γ-B）包含两个相邻的补体控制蛋白（CCP）模块，即模块IV和模块V（各约60个残基）、一个15个残基的中间片段以及B链（251个残基），B链即丝氨酸蛋白酶结构域。为了确定该区域内结构域之间的相互作用，用纤溶酶进行有限蛋白酶解获得的C1s的γ-B片段，与水溶性碳二亚胺1-乙基-3-[3-(二甲基氨基)丙基]碳二亚胺进行化学交联；然后在溴化氰裂解和热解消化后分离交联肽。N端序列和质谱分析使我们能够鉴定出模块V的赖氨酸405与B链的谷氨酸672之间以及中间片段的谷氨酸418与B链的赖氨酸608之间的两个交联。还基于CCP模块IV和V以及催化B链分别与补体因子H的第16个和第5个CCP模块以及I型丝氨酸蛋白酶的同源性，对它们进行了三维建模。化学交联研究和同源性建模提供的信息使我们能够构建γ-B区域C端部分组装的三维模型，该部分包括模块V、中间片段和B链。该模型表明，模块V在与激活位点和催化位点相对的一侧与丝氨酸蛋白酶B链相互作用。根据模块V在C1s的两个底物之一C4的特异性识别和定位中的可能作用，讨论了这种相互作用的功能意义。
