Structure and assembly of the catalytic region of human complement protease C(1)over-barr: A three-dimensional model based on chemical cross-linking and homology modeling

被引:42
作者
Lacroix, M
Rossi, V
Gaboriaud, C
Chevallier, S
Jaquinod, M
Thielens, NM
Gagnon, J
Arlaud, GJ
机构
[1] CNRS, CEA, INST BIOL STRUCT JEAN PIERRE EBEL, LAB ENZYMOL MOL, F-38027 GRENOBLE 1, FRANCE
[2] CNRS, CEA, INST BIOL STRUCT JEAN PIERRE EBEL, LAB CRISTALLOGENESE & CRISTALLOG PROT, F-38027 GRENOBLE 1, FRANCE
[3] CNRS, CEA, INST BIOL STRUCT JEAN PIERRE EBEL, LAB SPECTROMETRIE MASSE PROT, F-38027 GRENOBLE 1, FRANCE
关键词
D O I
10.1021/bi962719i
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 C(1) over barr$ (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 Lys(426) of module V and the C-terminal Asp(688) of the serine protease B domain and one intermonomer cross-link between the N-terminal Gly(280) of fragment gamma and Glu(493) 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 Arg(446)-Ile(447) 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.
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页码:6270 / 6282
页数:13
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