Structural insight on the recognition of surface-bound opsonins by the integrin I domain of complement receptor 3 Goran Bajica, Laure Yatimea, Robert B. Simb, Thomas Vorup-Jensenc,1, and Gregers R. Andersena,1 Departments of aMolecular Biology and Genetics and cBiomedicine, Aarhus University, DK-8000 Aarhus, Denmark; and bDepartment of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom Edited by Douglas T. Fearon, University of Cambridge School of Clinical Medicine, Cambridge, United Kingdom, and approved August 28, 2013 (received for review June 13, 2013) Complement receptors (CRs), expressed notably on myeloid and degranulation, and changes in leukocyte cytokine production (2, lymphoid cells, play an essential function in the elimination of 5–7). CR3, and to a lesser degree CR4, are essential for the complement-opsonized pathogens and apoptotic/necrotic cells. In phagocytosis of complement-opsonized particles or complexes addition, these receptors are crucial for the cross-talk between the (6, 8, 9). Complement-opsonized immune complexes are cap- innate andadaptive branches ofthe immune system. CR3 (also known tured in the lymph nodes by CR3-positive subcapsular sinus as Mac-1, integrin α β , or CD11b/CD18) is expressed on all macro- macrophages (SSMs) and conveyed directly to naïve B cells or M 2 γ phages and recognizes iC3b on complement-opsonized objects, en- through follicular dendritic cells (10) using CR1, CR2, and Fc abling their phagocytosis. We demonstrate that the C3d moiety of receptors for antigen capture (11, 12). Hence, antigen-presenting iC3b harbors the binding site for the CR3 αI domain, and our structure cells such as SSMs may act as antigen storage and provide B of the C3d:αI domain complex rationalizes the CR3 selectivity for iC3b. lymphocytes with antigens (10, 12). Based on extensive structural analysis, we suggest that the choice Here, we establish the C3d fragment as the minimal and high- affinity binding partner for the CR3 I domain. By contrast, the between a ligand glutamate or aspartate for coordination of a recep- binding site for the CR4 I domain was located in the C3c frag- tor metal ion-dependent adhesion site–bound metal ion is governed ment by electron microscopy (13). We present the crystal by the secondary structure of the ligand. Comparison of our structure structure of the CR3 I domain in complex with C3d. The classic to the CR2:C3d complex and the in vitro formation of a stable CR3:C3d: observation of CR3 binding to iC3b, but not to its precursor C3b CR2 complex suggests a molecular mechanism for the hand-over of (14), is consistent with our structure. In addition, our structure CR3-bound immune complexes from macrophages to CR2-presenting and functional data suggest simultaneous binding of CR3 and cells in lymph nodes. another complement receptor, CR2, to C3 fragments, which might provide the basis for trafficking of complement-opsonized innate immunity | phagocytosis | integrin receptor | structural biology immune complexes from macrophages to B cells and follicular dendritic cells in lymph nodes. ctivation of complement leads to proteolytic cleavage of the Results Acentral complement component, C3. Its major fragment, C3b, acts as an opsonin and becomes covalently bound to the CR3 and CR4 I Domains Recognize Distinct Binding Sites on iC3b. To activating surface via a reactive thioester located in the thioester quantitatively compare binding properties of the CR3 and CR4 I (TE) domain of nascent C3b (Fig. S1A). Proteolytic processing domain with regard to binding of C3 proteolytic fragments, C3b, by factor I within the CUB domain of C3b leads to the formation fi of iC3b and C3dg. Finally, C3d—which practically corresponds Signi cance to the TE domain present in C3, C3b, and iC3b (Fig. S1 B–G)— is formed by other plasma proteases. These activation products Fragments of complement component C3 tag surfaces such as are ligands for five complement receptors (1), with iC3b being those presented by microbial pathogens or dying host cells for the primary ligand of complement receptors (CRs) CR3 and recognition by cells from the innate immune system. Comple- fi CR4 (also known as CD11c/CD18, p150,95, or integrin αXβ2), ment receptor (CR) 3 enables ef cient binding of complement- which is structurally similar to CR3. tagged surfaces by macrophages and dendritic cells, which Like other integrins, CR3 is a heterodimeric complex of two eventually transport the CR3-bound material into lymph nodes. fi transmembrane proteins, αM and β2. It is abundantly expressed The study identi es in atomic details the fragments of CR3 and on myeloid leukocytes, including neutrophil granulocytes, den- C3 required for such binding. The structural organization per- dritic cells, monocytes, and macrophages and also on lymphoid mits concomitant recognition by another complement receptor, natural killer (NK) cells (2). Most ligands, including iC3b (3), are namely CR2, expressed on cells of the adaptive immune system, bound by the Von Willebrand factor A (VWA) domain in the suggesting a structural rationale for the exchange of antigens α-chain, also referred to as the αI domain owing to its insertion between leukocytes of the innate and adaptive immune sys- in the β-propeller domain. I domain residues coordinate a metal tems critical in the formation of humoral immune responses. ion essential for ligand recognition through a metal ion-dependent adhesion site (MIDAS). Integrins adopt at least three major Author contributions: G.B., L.Y., T.V.-J., and G.R.A. designed research; G.B. and T.V.-J. conformations in the cell membrane. The bent-closed conforma- performed research; R.B.S. contributed new reagents/analytic tools; G.B., L.Y., T.V.-J., tion is inactive in ligand binding, the extended-closed conforma- and G.R.A. analyzed data; and T.V.-J. and G.R.A. wrote the paper. tion has low ligand affinity, and the extended-open conformation The authors declare no conflict of interest. binds ligands with high affinity. The transition from the bent- This article is a PNAS Direct Submission. closed to the open-extended conformation is exerted by a cyto- Data deposition: Crystallography, atomic coordinates, and structure factors have been plasmic force on the leg of the β-subunit, a process usually referred deposited in the Protein Data Bank, www.pdb.org (PDB ID code 4M76). to as the inside-out signaling (4). 1To whom correspondence may be addressed. E-mail: [email protected] Binding of ligands to CR3 leads to conformational changes in or [email protected]. its ectodomain transmitting an outside-in signal through the cell This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. membrane. This may lead to actin remodeling, phagocytosis, 1073/pnas.1311261110/-/DCSupplemental. 16426–16431 | PNAS | October 8, 2013 | vol. 110 | no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1311261110 Downloaded by guest on October 1, 2021 + iC3b, C3c, C3dg, and C3d were immobilized in surface plasmon ion in the MIDAS site (Fig. 2B and Table S1). The ability of Ni2 resonance (SPR) flow cells. For both I domains good binding to stabilize MIDAS interactions with a ligand is well known from signals were observed with C3b, iC3b, and C3c as ligands (Fig. S2 the complement convertases (18). A–C and F–H). Nevertheless, even high concentrations (100 μM) Within the complex, C3d adopts the well-described compact of the CR3 or CR4 I domain did not lead to saturation. This is α-α6 barrel structure (Fig. 2A and Fig. S4C) with only minor consistent with X-ray crystallography (15) and inhibition experi- conformational differences compared with known structures ments showing that both the CR3 and CR4 I domain interact containing C3d (19–21). The α-helix connecting loop regions of weakly (Kd ∼300 μM) with acidic side chains acting as ligand C3d are presented in an alternating fashion at the circumference mimetics (16). The C3d and C3dg-coated surfaces produced of two opposite surfaces: a concave, mainly negatively charged robust SPR signals of ∼1,200 resonance units (RU) and showed surface and a positively charged convex surface. At the rim of the signs of saturation at high CR3 I domain concentrations (Fig. S2 concave surface Asp1247, situated in a loop region connecting D and E). By contrast, the CR4 I domain only poorly bound helices α10 and α11 (Fig. 2C and Fig. S4 C and D), provides the these fragments (Fig. S2 I and J). As detailed in other studies (16, final coordination bond to the divalent cation in the CR3 MI- 17), the binding kinetics of the CR3 and CR4 I domain ligand DAS (Fig. 2B). The quite polar interface between C3d and the I binding are not well-described with simple 1:1 Langmuir iso- domain is modest, with an area of ∼490 Å2, which is smaller but therms. The interactions were quantified by analysis of the sen- comparable to similar I domain:ligand complexes (Table S2). sorgrams with the EVILFIT algorithm, which calculates the Besides Asp1247 on C3d involved in the MIDAS ion interaction, minimal distribution in binding kinetics for the heterogeneous the nearby Asp1245 in C3d engages in a salt bridge with CR3 interactions with ligands (Fig. 1). In general, the modeled dis- Arg208 (Fig. 2C and Fig. S4D). C3d Lys1217 also seems to sta- tribution in kinetics efficiently described the experimental data bilize the interaction, because it is capable of forming salt bridges as reflected in the small rmsds. For the CR3 and CR4 I domain, with CR3 Glu178 and Glu179 and hydrogen bonds with main some of the interactions with C3b, iC3b, and C3c were of modest chain carbonyls of Leu205 and Leu206. Finally, C3d Arg1254 −4 −3 strength with Kd ∼10 to 10 M (Fig.