Distinct recognition of complement iC3b by integrins αXβ2 and αMβ2 Shutong Xua,1, Jianchuan Wangb,1, Jia-Huai Wanga,c,2, and Timothy A. Springerb,2 aDepartment of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215; bProgram in Cellular and Molecular Medicine, Boston Children’s Hospital and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; and cDepartment of Pediatrics, and Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02215 Contributed by Timothy A. Springer, February 2, 2017 (sent for review December 2, 2016; reviewed by Anne Nicholson-Weller and Thomas Vorup-Jensen) α β α β α α β α β Recognition by the leukocyte integrins X 2 and M 2 of complement The Idomainof X 2 and M 2 plays a key role in recognition iC3b-opsonized targets is essential for effector functions including of iC3b by both integrins; however, alternative recognition modes phagocytosis. The integrin-binding sites on iC3b remain incompletely have also been suggested. Mutagenesis and antibody blocking characterized. Here, we describe negative-stain electron microscopy studies on αXβ2 and αMβ2 showed that the αI domain was required and biochemical studies of αXβ2 and αMβ2 in complex with iC3b. De- for binding to iC3b (6, 17, 18). More recently, electron microscopy spite high homology, the two integrins bind iC3b at multiple distinct (EM) studies with the αXβ2 ectodomain revealed binding solely sites. αXβ2 uses the αX αIdomaintobindiC3bonitsC3cmoietyatone through its αI domain to a site at the interface between the mac- of two sites: a major site at the interface between macroglobulin roglobulin (MG) 3 and MG4 domains on the C3c moiety of iC3b (MG) 3 and MG4 domains, and a less frequently used site near the (Fig. 1A) (19). However, stoichiometry suggests multiple binding α β α C345C domain. In contrast, M 2 uses its IdomaintobindiC3batthe sites for the αX αIdomaininiC3b(20).Becauseofthehighsimi- thioester domain and simultaneously interacts through a region near larity between αXβ2 and αMβ2, it was expected that αMβ2 would bind α β β β the M -propeller and 2 I domain with a region of the C3c moiety tothesamesiteoniC3basαXβ2. It was surprising then when a near the C345C domain. Remarkably, there is no overlap between the crystal structure of a complex between the αM αIdomainandTED α β α β primary binding site of X 2 and the binding site of M 2 on iC3b. (C3d) revealed a specific complex between them (21). As the au- α β α β Distinctive binding sites on iC3b by integrins X 2 and M 2 may be thors pointed out, binding of the αM αI domain alone to TED could biologically beneficial for leukocytes to more efficiently capture not explain the specificity of αMβ2 for binding to iC3b and not to BIOCHEMISTRY opsonized pathogens and to avoid subversion by pathogen factors. C3d (21). Moreover, the αM subunit β-propeller domain and the β2 subunit inserted domain (βI) (Fig. 1B) have been suggested to – α β ctivation of mammalian complement is critical for the clearance additionally contribute to iC3b binding (22 24). Both M 2 and α β of pathogens and altered host cells, whereas excessive activation X 2 recognize a variety of unrelated ligands and proteolyzed or A α β results in tissue damage (1). Complement activation can be initiated denatured proteins (25); given this broad specificity, X 2 has been “ ” α α by three distinct pathways with proteolytic cleavage of complement termed a danger receptor (26).Furthermore,theisolated M I component C3 as the pivotal step in each pathway. C3 is cleaved to domain has been crystallized in the open conformation bound to a C3b by C3 convertases. C3b participates in some C3 convertases to neighboring αI domain in a ligand-mimetic lattice contact (27). α α amplify C3 cleavage; however, further cleavage of C3b to iC3b in- Thus, it was interesting to determine whether the M Idomain activates convertase activity. In turn, iC3b can be digested to yield complexwiththeTEDdomainincrystals(21)couldbeex- C3c plus C3dg, and C3dg can be further cleaved to C3d (2) (Fig. 1A). tended to larger integrin and complement fragments and Conversion of C3 to C3b exposes the otherwise buried reactive whether additional contacts might be found. These findings thioesterbondinthethioesterdomain(TED)andenablesittoco- valently attach to hydroxyl and amino groups on pathogenic, im- Significance munogenic, and apoptotic cell surfaces (3). Opsonic fragments of C3 covalently bound through the TED, i.e., C3b, iC3b, and C3dg/C3d Complement is deposited on foreign antigens and particles and serve as ligands for five distinct complement receptors (CRs). Each marks them for recognition by immune cells and removal by CR has been characterized for its preference for specific C3 opsonic phagocytes. Immune cells have two homologous integrins, fragments. CR type 3 (CR3, also known as CD11b/CD18, Mac-1, or αXβ2 and αMβ2, that recognize the complement fragment iC3b. integrin αMβ2) and CR type 4 (CR4, also known as CD11c/CD18, Although it might have been suspected that these integrins p150, 95, or integrin αXβ2) specifically recognize iC3b as shown by would bind to the same site on iC3b, direct comparisons here selective rosette formation with erythrocytes opsonized with iC3b but show they do not. Using negative stain electron microscopy not C3b or C3d (4–6), and specific isolation from cells by affinity and purified integrin fragments and iC3b, we show that αXβ2 α β chromatography on iC3b-Sepharose beads (7). However, at high binds to two distinct sites on iC3b. A single M 2 integrin binds concentrations on opsonized cells, C3d and C3dg can show reactivity to two different sites on iC3b, each of which is distinct from α β with CR3 (8, 9). those to which X 2 binds. Our findings reveal remarkable di- αXβ2 and αMβ2 are heterodimeric proteins belonging to the β2 versity among integrins that recognize complement and sug- integrin subfamily. They are expressed on myeloid cells in- gest possible cooperative responses. cluding neutrophil granulocytes, monocytes, macrophages, and also on activated lymphocytes and lymphoid natural killer cells Author contributions: S.X., J.W., J.-H.W., and T.A.S. designed research; S.X. and J.W. per- – α β α β formed research; S.X., J.W., J.-H.W., and T.A.S. analyzed data; and S.X., J.W., J.-H.W., and (10 12). X 2 and M 2 are essential for recognition and T.A.S. wrote the paper. phagocytosis of pathogens and immune complexes in vivo (13). Reviewers: A.N.-W., Harvard Medical School, Beth Israel Deaconess Medical Center; and Deficiency of αXβ2 and αMβ2 in leukocyte adhesion deficiency T.V.-J., University of Aarhus. results in recurring bacterial infections (14) and deficiency of The authors declare no conflict of interest. α β M 2 alters susceptibility to injury from immune complexes 1S.X. and J.W. contributed equally to this work. β α (15). The 2-associated subunits all contain an inserted do- 2To whom correspondence may be addressed. Email: [email protected] or main (αI domain), which adopts a Rossmann fold with a metal [email protected]. ion-dependent adhesion site (MIDAS) at the ligand-binding This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. site (Fig. 1B) (16). 1073/pnas.1620881114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1620881114 PNAS Early Edition | 1of6 AB CD Fig. 1. Integrins, iC3b, and negative-stain EM of αX I domain and iC3b complexes. (A) Schematic of iC3b. (B) Schematic of domain organization and con- formational states of αI-integrins αXβ2 and αMβ2.(C) Superdex 200 size exclusion chromatography profiles of iC3b in presence or absence of the αX αI F273S/ 2+ F300A mutant in presence of 2 mM Mg .(D) Better resolved class averages of αX αI and iC3b complexes. (Scale bar: 10 nm.) Schematic interpretations of classes are shown to the right. also suggested the importance of further comparisons between and 6) and its lack of defined orientation (absence of density) in αMβ2 and αXβ2, particularly with integrin fragments comple- other class averages (Fig. 1D, 3, 4, 7, and 8). The TED (296 residues) mentary to those previously used in structural studies, i.e., the was clearly distinguished by its stronger density from the smaller αI ectodomain in the case of αXβ2 (19) and the αI domain in the domain (190 residues) (Fig. 1D). case of αMβ2 (21). α β Using negative-stain EM with the αX αI domain and the αMβ2 Interaction of X 2 with C3c. Further cleavage by factor I of iC3b in headpiece here, we have shown that αXβ2 and αMβ2 bind to the CUB domain separates TED (C3dg) from C3c. We prepared α α 2+ different regions of iC3b. Distinct recognition modes toward X I domain complexes with C3c in Mg by gel filtration (Fig. A B A iC3b by two closely related β2 integrins reveal surprising diversity 2 ) and characterized them by EM (Fig. 2 and Fig. S2 ) using that might be important for complement recognition in the the same methods as described above for iC3b. Although some context of the quite different surfaces or antigens on which particles showed C3c alone (Fig. 2B, 1 and 2), most (30 classes, complement can be deposited and enable cooperative rather corresponding to 68% of particles) showed αI bound near the B 3–8 than competitive functions for αXβ2 and αMβ2. MG3-MG4 interface on the MG key ring (Fig. 2 , ). Strik- ingly, at least 8 of 50 class averages showed an additional density Results for αI near the C345C knob (Fig. 2B, 5–8). α Interaction of αXβ2 with iC3b.