Lacroix M, Rossi V, Gaboriaud C, Chevallier S, Jaquinod M, Thielens N M, Gagnon J, Arlaud G J
Laboratoire d'Enzymologie Moléculaire, Institut de Biologie Structurale Jean-Pierre Ebel (CEA-CNRS), Grenoble, France.
Biochemistry. 1997 May 27;36(21):6270-82. doi: 10.1021/bi962719i.
C1r is the modular serine protease responsible for autocatalytic activation of C1, the first component of the complement classical pathway. Its catalytic region is a noncovalent homodimer of two gamma-B monomers, each comprising two contiguous complement control protein (CCP) modules, IV and V [also known as short consensus repeats (SCRs)], a 15-residue intermediary segment, and the serine protease B domain. With a view to gain insight into domain-domain interactions within this region, fragment C1r (gamma-B)2, obtained by autolytic proteolysis of the active protease, was cross-linked with the water-soluble reagent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Cross-linked species gamma-B intra and gamma-B inter, containing intra- and intermonomer cross-links, respectively, were isolated and then fragmented by CNBr cleavage and trypsin digestion. N-Terminal sequence and mass spectrometry analyses of the resulting cross-linked peptides allowed us to identify one intramonomer cross-link between Lys426 of module V and the C-terminal Asp688 of the serine protease B domain and one intermonomer cross-link between the N-terminal Gly280 of fragment gamma and Glu493 of the B domain. Three-dimensional homology modeling of the CCP modules IV and V and of the B domain was also performed. The complementary information provided by chemical cross-linking and homology modeling studies was used to construct a three-dimensional model of the gamma-B monomer, in which module V interacts with the serine protease on the side opposite to both the active site and the Arg446-Ile447 activation site. Also, a tentative three-dimensional model of the (gamma-B)2 dimer was built, indicating a loose "head to tail" association of the monomers, with the active sites facing opposite directions toward the outside of the dimer. The latter model is compared with available low-resolution structural data, and its functional implications are discussed in terms of the conformational changes occurring during C1r activation.
C1r是一种模块化丝氨酸蛋白酶，负责补体经典途径的首个成分C1的自催化激活。其催化区域是由两个γ-B单体组成的非共价同源二聚体，每个γ-B单体包含两个相邻的补体控制蛋白（CCP）模块，即IV和V [也称为短共有重复序列（SCR）]、一个15个残基的中间片段以及丝氨酸蛋白酶B结构域。为了深入了解该区域内的结构域-结构域相互作用，通过活性蛋白酶的自溶蛋白水解获得的片段C1r（γ-B）2与水溶性试剂1-乙基-3-[3-（二甲基氨基）丙基]碳二亚胺进行交联。分别含有单体内部和单体间交联的交联产物γ-B内部和γ-B间被分离出来，然后通过溴化氰裂解和胰蛋白酶消化进行片段化。对所得交联肽进行N端序列和质谱分析，使我们能够鉴定出模块V的Lys426与丝氨酸蛋白酶B结构域的C端Asp688之间的一个单体内部交联，以及片段γ的N端Gly280与B结构域的Glu493之间的一个单体间交联。还对CCP模块IV和V以及B结构域进行了三维同源性建模。化学交联和同源性建模研究提供的互补信息被用于构建γ-B单体的三维模型，其中模块V在与活性位点和Arg446-Ile447激活位点相对的一侧与丝氨酸蛋白酶相互作用。此外，还构建了（γ-B）2二聚体的初步三维模型，表明单体之间存在松散的“头对尾”关联，活性位点朝着二聚体外部的相反方向。将后一个模型与现有的低分辨率结构数据进行比较，并根据C1r激活过程中发生的构象变化讨论其功能意义。
