Structure and Assembly of the Catalytic Region of Human Complement Protease C1̄r: A Three-Dimensional Model Based on Chemical Cross-Linking and Homology Modeling

Abstract

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 γ-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 C1̄r (γ-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 γ-B intra and γ-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 γ 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 γ-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 (γ-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.