X-ray Structure of the Ca2+-binding Interaction Domain of C1s

Abstract

C1, the complex that triggers the classical pathway of complement, is assembled from two modular proteases C1r and C1s and a recognition protein C1q. The N-terminal CUB1-EGF segments of C1r and C1s are key elements of the C1 architecture, because they mediate both Ca 2؉ -dependent C1r-C1s association and interaction with C1q. The crystal structure of the interaction domain of C1s has been solved and refined to 1.5 Å resolution. The structure reveals a head-to-tail homodimer involving interactions between the CUB1 module of one monomer and the epidermal growth factor (EGF) module of its counterpart. A Ca 2؉ ion is bound to each EGF module and stabilizes both the intra-and inter-monomer interfaces. Unexpectedly, a second Ca 2؉ ion is bound to the distal end of each CUB1 module, through six ligands contributed by Glu 45 , Asp 53 , Asp 98 , and two water molecules. These acidic residues and Tyr 17 are conserved in approximately two-thirds of the CUB repertoire and define a novel, Ca 2؉ -binding CUB module subset. The C1s structure was used to build a model of the C1r-C1s CUB1-EGF heterodimer, which in C1 connects C1r to C1s and mediates interaction with C1q. A structural model of the C1q/C1r/C1s interface is proposed, where the rod-like collagen triple helix of C1q is accommodated into a groove along the transversal axis of the C1r-C1s heterodimer.

The X-ray Structure of Human Mannan-binding Lectin-associated Protein 19 (MAp19) and Its Interaction Site with Mannan-binding Lectin and L-ficolin

“…In contrast, as discussed previously (22), the Ca 2ϩ -binding site observed in the rat MASP-2 structure only involves five coordination ligands (21). As observed in the C1s structure (22), Asn 143 lacks ␤-hydroxylation, providing further evidence that post-translational modification of this residue to erythro-␤-hydroxyasparagine is not achieved in baculovirus/insect cell systems and is not essential for Ca 2ϩ binding.…”

“…Overall, the assembly is reminiscent of those observed for the CUB1-EGF segments of human C1s and rat MASP-2 (21,22). However, a detailed comparison of the three structures reveals differences in the relative positioning of their CUB1 and EGF modules.…”

“…Loop 10, which is disordered in the rat MASP-2 structure (21), is structurally well defined in MAp19 and exhibits a rather extended conformation (Fig. 1B), different from that of the corresponding loop of C1s (22). The remainder of the MAp19, C1s, and rat MASP-2 EGF modules show root mean square deviation values of 0.61-0.69 Å, indicative of high structural homology.…”

“…A comparison with the homologous site previously observed in C1s (22) On the other hand, as observed in C1s, the Ca 2ϩ ion is the central element of a network of interactions that connect together loops L3, L5, and L9, thereby extensively stabilizing the distal end of the MAp19 CUB1 module. In this respect, it is interesting to note that Tyr 24 is H-bonded to the side chain carboxyl of Asp 60 and the main chain nitrogen of Glu 52 , and thereby stabilizes loop L3 in the same way as does its counterpart Tyr 17 in C1s (22).…”

“…The rotational and translational searches were carried out using the program AMORE (26). Whereas initial searches using the human C1s CUB1-EGF structure (22) were unsuccessful, a molecular replacement solution was obtained using the CUB1-EGF fragment of rat MASP-2 in the dimeric form (Protein Data Bank accession code 1NT0) (21). Rigid body refinement with the program CNS (27) further improved the orientation and position of each of the domains of the search model.…”