Abstract
C1, the complex that triggers the classic pathway of complement, is a 790-kDa assembly resulting from association of a recognition protein C1q with a Ca(2+)-dependent tetramer comprising two copies of the proteases C1r and C1s. Early structural investigations have shown that the extended C1s-C1r-C1r-C1s tetramer folds into a compact conformation in C1. Recent site-directed mutagenesis studies have identified the C1q-binding sites in C1r and C1s and led to a three-dimensional model of the C1 complex (Bally, I., Rossi, V., Lunardi, T., Thielens, N. M., Gaboriaud, C., and Arlaud, G. J. (2009) J. Biol. Chem. 284, 19340-19348). In this study, we have used a mass spectrometry-based strategy involving a label-free semi-quantitative analysis of protein samples to gain new structural insights into C1 assembly. Using a stable chemical modification, we have compared the accessibility of the lysine residues in the isolated tetramer and in C1. The labeling data account for 51 of the 73 lysine residues of C1r and C1s. They strongly support the hypothesis that both C1s CUB(1)-EGF-CUB(2) interaction domains, which are distant in the free tetramer, associate with each other in the C1 complex. This analysis also provides the first experimental evidence that, in the proenzyme form of C1, the C1s serine protease domain is partly positioned inside the C1q cone and yields precise information about its orientation in the complex. These results provide further structural insights into the architecture of the C1 complex, allowing significant improvement of our current C1 model.
Highlights
Complement is an essential component of innate immunity due to its ability to recognize pathogens and to limit infection in the vertebrate host
Mutagenesis experiments have recently ruled out this hypothesis [17] and led to a refined three-dimensional model of the C1 complex in which acidic residues involved in the Ca2ϩ-binding sites of the C1r CUB1 and CUB2 and C1s CUB1 modules interact with the C1q stems
We chose to investigate the solvent accessibility of lysines in the free and complexed forms of the tetramer for several reasons: (i) these residues are abundant and evenly distributed in the tetramer; (ii) lysine residues are mostly located on the surface of proteins and are ideal candidates for probing protein-protein interfaces; and (iii) unlike C1q, the tetramer does not have an oligomeric organization, which is expected to facilitate identification of the areas of C1r and C1s involved in conformational changes and/or in binding to C1q
Summary
The classic pathway is triggered by C1, a 790-kDa Ca2ϩ-dependent complex resulting from the association of a recognition protein C1q and a tetramer comprising two copies of the serine proteases C1r and C1s [3,4,5,6]. Recognition of targets such as pathogens or immune complexes by the C1q moiety of C1 elicits self-activation of C1r, which in turn converts C1s into its active form. Our data are consistent with the hypothesis that the C1s interaction domains interact with each other in C1 and provide experimental evidence that the C1s catalytic domains are partly located inside the C1q cone, yielding further insights into C1 architecture
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