Mapping surface accessibility of the C1r/C1s tetramer by chemical modification and mass spectrometry provides new insights into assembly of the human C1 complex

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.

FIGURE 1.

Overview of the experimental approach used in this study. Experimental details are described under “Experimental Procedures.”

FIGURE 2.

Effect of C1q binding on the activation state of the C1s-C1r-C1r-C1s tetramer. A, modular structure of C1r and C1s. Both proteases are activated through cleavage of an Arg–Ile bond (represented by a black arrow) located in their SP domain. The only disulfide bridge shown is the one connecting the activation peptide to the SP domain. N-Linked oligosaccharides are represented by open circles. B, MALDI-TOF mass spectra of the isolated tetramer under reducing conditions. In the absence of C1q, a small fraction of the tetramer appears activated, as indicated by the peaks corresponding to the heavy chains of C1r and C1s. C, MALDI-TOF mass spectra of the reconstituted C1 complex under reducing conditions. The peaks at m/z 48114.6 (1), 49695.5 (2), and 51317.8 (3) correspond to random associations of the C1q chains (43).

FIGURE 3.

Modification of the C1r solvent accessibility upon association of the C1s-C1r-C1r-C1s tetramer with C1q. A, amino acid sequence of C1r showing the 36 lysine-containing peptic fragments (in red, bold type, and underlined) selected for quantitative analysis. Lysines that could not be recovered are shown in black, bold type, and underlined. The catalytic residues His⁴⁸⁵, Asp⁵⁴⁰, and Ser⁶³⁷ and the Arg-Ile cleavage site are highlighted in magenta and shown in blue, respectively. C1r residues interacting with C1q (17) are highlighted in yellow. B, effect of C1q binding on the solvent accessibility of residues Lys⁷ (CUB₁), Lys²⁹¹/Lys²⁹⁶ (CCP₁), and Lys⁴⁵²/Lys⁴⁵⁴ (SP domain). Each box-and-whisker plot compares the statistical distribution of the unmodified fraction of a given C1r peptide in the presence (C1) or absence (tetramer) of C1q. Q1, Q2, and Q3 correspond to the lower, median (red bar), and third quartiles, respectively. The largest (Max) and smallest (Min) non-outlier observations are marked with a small black vertical line (whiskers). Data points lying above the upper whisker or below the lower whisker are considered as outliers and indicated by an open circle. C, structure of the zymogen CCP₁-CCP₂-SP C1r catalytic domain (10) showing the position of lysine residues. The catalytic triad (His⁴⁸⁵, Asp⁵⁴⁰, and Ser⁶³⁷) is represented by three magenta spheres. Lysine residues are color-coded as follows: blue, no modification of surface accessibility upon C1 assembly; red, decreased surface accessibility; yellow, decreased and/or unmodified surface accessibility; and black, no data available.

FIGURE 4.

Modification of the C1s solvent accessibility upon interaction of the C1s-C1r-C1r-C1s tetramer with C1q. A, amino acid sequence of C1s showing the 30 lysine-containing peptic fragments used for quantitative analysis. The color coding used is the same as stated in the legend to Fig. 3. B, effect of C1q binding on the surface accessibility of residues Lys⁹⁰ (CUB₁), K¹⁹⁵ (CUB₂), and residues Lys⁴⁸⁴, Lys⁴⁸⁶, Lys⁵⁰⁰, Lys⁵⁸⁴, Lys⁵⁸⁷, Lys⁶⁰⁸, and Lys⁶¹⁴ of the SP domain. Each box-and-whisker plot compares the statistical distribution of the unmodified fraction of a given C1s peptide in the presence (C1) or absence (tetramer) of C1q. C, structures of the C1s CUB₁-EGF (16) and CCP₂-SP regions (33) showing the position of lysine residues. The Ca²⁺ ions bound to CUB₁ (site I) and EGF (site II) are represented by yellow spheres, and the catalytic triad is shown as three magenta spheres. Orange dots correspond to residues not defined in the C1s CCP₂-SP x-ray structure. Lysine residues are color-coded as follows: blue, no modification of solvent accessibility inside the C1 complex; red, decreased accessibility; green, increased accessibility; and black, no data available.

FIGURE 5.

A, space-filling representation of the head-to-tail C1r/C1s CUB₁-EGF heterodimer. One C1s monomer (gray) was used as a template to position and visualize the lysine residues identified in the C1r CUB₁ module, based on the sequence alignment of the CUB₁ modules of C1r and C1s (supplemental Table S1). Lysines are color-coded as defined in Fig. 3. C1r and C1s residues interacting with C1q are colored green (17). B, space-filling representation of the C1s CCP₂-SP region illustrating the position of the lysine residues (in red) showing reduced solvent accessibility upon C1q binding, except for Lys⁴⁸⁴, which lies in a region not defined in the crystal structure (33). Lysines undergoing no modification of solvent accessibility within C1 are all located on the same face, opposite to the one harboring Lys⁴³², Lys⁴⁸⁶, Lys⁶⁰⁸, and Lys⁶¹⁴.

FIGURE 6.

A, space-filling representation of the assembly of the C1r/C1s CUB₁-EGF-CUB₂ interaction domains as proposed to occur in the C1 complex (17) (top view). C1r and C1s are shown in yellow and gray, respectively. The color coding used is the same as stated in the legend to Fig. 3. Residues interacting with C1q are colored green. The six collagen triple helices of C1q are shown as magenta spheres. B and C, side and bottom views of the whole C1 complex highlighting the positioning of the C1s SP domains with respect to the remainder of the complex. Both C1r monomers are in yellow, whereas C1s molecules are shown in cyan and magenta. The color coding used for lysine residues is the same as stated in the legend to Fig. 3.