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 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.

Deciphering complement mechanisms: The contributions of structural biology

“…That the C1r catalytic domain associates as a homodimer in solution has been demonstrated using various techniques (Villiers et al, 1985;Weiss et al, 1986;Lacroix et al, 2001). Likewise, the particular head-totail configuration seen in the structure is consistent with previous chemical cross-linking experiments and with the observation that deletion of the CCP 1 module prevents dimer formation (Lacroix et al, 1997(Lacroix et al, , 2001). It appears very likely, therefore, that this crystal structure is physiologically relevant and corresponds to a resting, thermodynamically stable conformation of the C1r catalytic domain.…”

Characterization of the Interaction Between L-Ficolin/P35 and Mannan-Binding Lectin-Associated Serine Proteases-1 and -2

“…Mass spectrometry analyses were performed using the matrix-assisted laser desorption ionization technique on a Voyager Elite XL instrument (Perseptive Biosystems, Cambridge, MA), under conditions described previously (31).…”

The Cleavage of Two C1s Subunits by a Single Active C1r Reveals Substantial Flexibility of the C1s-C1r-C1r-C1s Tetramer in the C1 Complex

“…Models based on chemical cross-linking and homology modeling (9) suggest that the second complement control protein (CCP) module in C1r and C1s is attached to the serine protease domain while significant flexibility is assumed at the CCP1-CCP2 domaindomain interface. This assumption is in accord with experimentally (nuclear magnetic resonance) observed flexibility between two contiguous CCP modules in a related molecule, human factor H (24).…”

“…The protein-protein interactions between the subcomponents, which regulate the activation of C1, have been extensively studied. The roles of the individual domains of the serine-protease subcomponents have been investigated by limited proteolysis (5)(6)(7)(8)(9), recombinant protein expression (10 -12) and chemical synthesis (13)(14)(15). These experiments provided important information about the structure and function of C1, but our knowledge about the mechanism of the activation process is far from complete.…”