The Journal of Immunology Stable Coordination of the Inhibitory Ca 2+ Ion at the Metal Ion-Dependent Adhesion Site in Integrin CD11b/CD18 by an Antibody-Derived Ligand Aspartate: Implications for Integrin Regulation and Structure-Based Drug Design Bhuvaneshwari Mahalingam,* ,† Kaouther Ajroud, †,1 Jose ´ Luis Alonso, † Saurabh Anand, † Brian D. Adair,* ,† Alberto L. Horenstein, ‡ Fabio Malavasi, ‡ Jian-Ping Xiong,* ,† and M. Amin Arnaout* ,† A central feature of integrin interaction with physiologic ligands is the monodentate binding of a ligand carboxylate to a Mg 2+ ion hexacoordinated at the metal ion-dependent adhesion site (MIDAS) in the integrin A domain. This interaction stabilizes the A domain in the high-affinity state, which is distinguished from the default low-affinity state by tertiary changes in the domain that culminate in cell adhesion. Small molecule ligand-mimetic integrin antagonists act as partial agonists, eliciting similar activating conformational changes in the A domain, which has contributed to paradoxical adhesion and increased patient mortality in large clinical trials. As with other ligand-mimetic integrin antagonists, the function-blocking mAb 107 binds MIDAS of integrin CD11b/ CD18 A domain (CD11bA), but in contrast, it favors the inhibitory Ca 2+ ion over the Mg 2+ ion at MIDAS. We determined the crystal structures of the Fab fragment of mAb 107 complexed to the low- and high-affinity states of CD11bA. Favored binding of the Ca 2+ ion at MIDAS is caused by the unusual symmetric bidentate ligation of a Fab-derived ligand Asp to a heptacoordinated MIDAS Ca 2+ ion. Binding of the Fab fragment of mAb 107 to CD11bA did not trigger the activating tertiary changes in the domain or in the full-length integrin. These data show that the denticity of the ligand Asp/Glu can modify the divalent cation selectivity at MIDAS and hence integrin function. Stabilizing the Ca 2+ ion at MIDAS by bidentate ligation to a ligand Asp/Glu may provide one approach for designing pure integrin antagonists. The Journal of Immunology, 2011, 187: 6393–6401. I ntegrins are a/b heterodimeric adhesion receptors that couple the extracellular matrix or counter-receptors on other cells with the contractile cytoskeleton, transducing mecha- nochemical signals across the plasma membrane that regulate most cellular functions (1). Deregulation of integrin functions, how- ever, plays critical roles in a diverse range of diseases, including in- flammatory and vascular diseases and tumor metastasis, establish- ing integrins as potential therapeutic targets (2–4). Small molecule antagonists developed based on the structures of natural integrin ligands display agonist-like activities (5–7), which have contributed to adverse autoimmune reactions and to paradoxical increased mor- tality in treated patients (4, 8, 9), limiting their use and reflecting the need for a better understanding of the structure–activity relation- ships in these conformationally dynamic receptors. At the core of integrin interaction with physiologic ligands is a force-bearing Asp (or Glu)–Mg 2+ ion bond (10), with Asp/Glu derived from the ligand and the metal ion from a GTPase-like von Willebrand factor type A domain present in the integrin a (aA or I domain) and/or b (bA or I-like domain) subunits (Fig. 1) (11). In solved structures of complexes of integrins with natural li- gands, ligand mimetics, or pseudo-ligands (12–18), the metal ion is coordinated at the metal ion-dependent adhesion site (MIDAS), which replaces the catalytic site of GTPases. Side chain oxygen atoms from three surface loops in the A domain coordinate the MIDAS metal ion, with the ligand-derived Asp/Glu binding mo- nodentately to complete the hexacoordinated Mg 2+ ion (19–21); it is replaced by a water molecule in the unliganded structure (Fig. 1B,1C). Formation of the Asp/Glu–Mg 2+ bond in aA domains is coupled mechanically to a conformational switch of the domain from the default low-affinity (closed) state to the high-affinity (open) state, which includes a 180˚ flip of a conserved Gly 243 , leading to the downward axial displacement of the C-terminal a7 helix on the opposite pole of MIDAS (Fig. 1A). This movement enables aA to engage the bA MIDAS through an invariant Glu at the C terminus of the a7 helix (22), thus translating ligand occupancy in aA into quaternary changes downstream, leading to outside-in signaling and cell adhesion (23). In the aA-lacking integrin subgroup, extrinsic ligands directly bind the Mg 2+ ion *Structural Biology Program, Division of Nephrology, Massachusetts General Hos- pital, Harvard Medical School, Charlestown, MA, 02129; † Leukocyte Biology and Inflammation Program, Division of Nephrology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, 02129; and ‡ Laboratory of Immunoge- netics, University of Torino Medical School, Torino, Italy 1 Current address: Department of Neurology and Clinical Neurosciences, Northwest- ern University Feinberg School of Medicine, Chicago, IL. Received for publication August 18, 2011. Accepted for publication October 19, 2011. This work was supported by grants from the National Institutes of Health (National Institute of Diabetes and Digestive and Kidney Diseases). The atomic coordinates and structure factors presented in this article have been de- posited in the Protein Data Bank (http://www.pdb.org) under accession codes 3Q3G for the low-affinity CD11bA/Fab 107 complex and 3QA3 for the high-affinity CD11bA/mAb107 complex. Address correspondence and reprint requests to Prof. M. Amin Arnaout, Division of Nephrology, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129. E-mail address: aarnaout1@partners.org Abbreviations used in this article: CD11bA, integrin CD11b/CD18 A domain; Fab 107, Fab fragment of mAb 107; MIDAS, metal ion-dependent adhesion site; RMSD, root-mean-square deviation; RU, resonance unit; scFv, single-chain variable frag- ment; WT, wild-type. Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102394