Structural insight on the recognition of surface-bound opsonins by the integrin I domain of complement receptor 3

Abstract

Complement receptors (CRs), expressed notably on myeloid and lymphoid cells, play an essential function in the elimination of complement-opsonized pathogens and apoptotic/necrotic cells. In addition, these receptors are crucial for the cross-talk between the innate and adaptive branches of the immune system. CR3 (also known as Mac-1, integrin αMβ2, or CD11b/CD18) is expressed on all macrophages and recognizes iC3b on complement-opsonized objects, enabling their phagocytosis. We demonstrate that the C3d moiety of iC3b harbors the binding site for the CR3 αI domain, and our structure of the C3d:αI domain complex rationalizes the CR3 selectivity for iC3b. Based on extensive structural analysis, we suggest that the choice between a ligand glutamate or aspartate for coordination of a receptor metal ion-dependent adhesion site-bound metal ion is governed by the secondary structure of the ligand. Comparison of our structure to the CR2:C3d complex and the in vitro formation of a stable CR3:C3d:CR2 complex suggests a molecular mechanism for the hand-over of CR3-bound immune complexes from macrophages to CR2-presenting cells in lymph nodes.

Keywords: innate immunity; integrin receptor; phagocytosis; structural biology.

Fig. 1.

SPR analysis of the C3 fragment binding selectivity of the CR3 (A–C) and CR4 I (D–F) domains. The CR3 or CR4 I domain, stabilized by mutagenesis in the open, ligand-binding conformation (50, 51), were injected in concentrations ranging from 250 nM to 100 μM over surfaces coupled with C3b (A and D), iC3b (B and E), or C3d (C and F). The data were analyzed with the EVILFIT algorithm with settings assuming a priori all binding parameters to be equally likely (52, 53). The volume of interactions, indicated with colored contours (in RU as shown by scale bars) was plotted as a function of the dissociation constant (10⁻⁸ M ≤ K_D ≤ 10⁻¹ M) and rate (10⁻³ s⁻¹ ≤ k_d ≤ 10⁰ s⁻¹). Red arrows indicate a population of high-affinity interactions for the CR3 I domain (K_D ∼0.4 μM) shared between iC3b and C3d but not observed for interactions with C3b.

Fig. 2.

The structure of the C3d:I domain complex. (A) The edge of C3d (brown) interacts with the MIDAS (marked by the Ni²⁺ ion) of the CR3 I domain (purple). (B) The Ni²⁺ ion bound in the MIDAS. The electron density contoured at 6 σ obtained from anomalous differences in diffraction data (Table S1) is shown as a mesh around the Ni²⁺ ion. (C) Details of the intermolecular interface with putative hydrogen bonds and electrostatic interactions indicated by dashed lines. C3 labels are underlined in all panels. Fig. S4D shows a stereo view of the interface.

Fig. 3.

ITC studies of the CR3 I domain interaction with iC3b, C3d WT, C3d R1254A, and C3d D1247E mutants. Raw titration isotherms and the integrated peak areas are shown. Dissociation constants calculated according to a simple independent binding site model are displayed.

Fig. 4.

The CR3 I domain discriminates against C3b. (A) The structure of C3b superimposed with the C3d:CR3 I domain complex. The βI domain and the α-chain β-propeller within CR3 are shown schematically. (B) Close-up of the region framed in A. Notice the overlap between the CR3 I domain and the C3b CUB domain in the hypothetical CR3:C3b complex. C3 labels are underlined.

Fig. 5.

The multiple interactions of the TE domain in C3 and its proteolytic degradation products. (A) Surface areas (gray) in the C3 TE domain interacting with the CUB, MG8, and the MG2 domains in native C3 are mapped onto C3d from its CR3 complex; the thioester Gln1013 is shown in red. (B) As in A after a 180° rotation. Surface areas of C3d interacting with CR2 (pink), factor H (green), and CR3 (purple) are indicated. (C) Superposition of the complex between C3d (brown surface) with the CR3 I domain (purple cartoon) with that of the C3d:CR2 complex (21) and the C3d:factor H complex (20). (D) Silver-stained gel after denaturing SDS/PAGE analysis of fractions from the CR3 I domain affinity column. Labels + and − indicate, respectively, the presence or absence of the protein in the pull-down assay at that particular step. From left to right, molecular weight (Mw) marker together with purified CR2 CCP 1-2 (insect cell-expressed) and C3d and CR3 I domain affinity column pull-down using C3d WT showing the flow-through and the wash steps and the final EDTA elution. The same fractions are shown for the C3d D1247A and the D1154A control mutants unable to bind CR3 and CR2, respectively. All the volumes in wash and elution steps were the same. Likewise, the volumes loaded for SDS/PAGE analysis were identical. For comparison, the pull-down was performed with the CR2 expressed in either bacteria or insect cells (Fig. S7). (E) Silver-stained gel after denaturing SDS/PAGE analysis of fractions from analytical size-exclusion chromatography (Fig. S8). The same fractions resulting from the C3d:CR2, C3d:CR3 and CR3:C3d:CR2 runs were analyzed.