Site in Integrin Cd11b/CD18 by an Antibody-Derived Ligand Aspartate: Implications for Integrin Regulation and Structure-Based Drug Design
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Published November 16, 2011, doi:10.4049/jimmunol.1102394 The Journal of Immunology Stable Coordination of the Inhibitory Ca2+ 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 Mg2+ 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 Ca2+ ion over the Mg2+ 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 Ca2+ ion at MIDAS is caused by the unusual symmetric bidentate ligation of a Fab-derived ligand Asp to a heptacoordinated MIDAS Ca2+ 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 Ca2+ 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: 000–000. ntegrins are a/b heterodimeric adhesion receptors that antagonists developed based on the structures of natural integrin couple the extracellular matrix or counter-receptors on other ligands display agonist-like activities (5–7), which have contributed I cells with the contractile cytoskeleton, transducing mecha- to adverse autoimmune reactions and to paradoxical increased mor- nochemical signals across the plasma membrane that regulate tality in treated patients (4, 8, 9), limiting their use and reflecting the most cellular functions (1). Deregulation of integrin functions, how- need for a better understanding of the structure–activity relation- ever, plays critical roles in a diverse range of diseases, including in- ships in these conformationally dynamic receptors. flammatory and vascular diseases and tumor metastasis, establish- At the core of integrin interaction with physiologic ligands is ing integrins as potential therapeutic targets (2–4). Small molecule a force-bearing Asp (or Glu)–Mg2+ 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 (aAor *Structural Biology Program, Division of Nephrology, Massachusetts General Hos- I domain) and/or b (bA or I-like domain) subunits (Fig. 1) (11). † pital, Harvard Medical School, Charlestown, MA, 02129; Leukocyte Biology and In solved structures of complexes of integrins with natural li- Inflammation Program, Division of Nephrology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, 02129; and ‡Laboratory of Immunoge- gands, ligand mimetics, or pseudo-ligands (12–18), the metal ion netics, University of Torino Medical School, Torino, Italy is coordinated at the metal ion-dependent adhesion site (MIDAS), 1Current address: Department of Neurology and Clinical Neurosciences, Northwest- which replaces the catalytic site of GTPases. Side chain oxygen ern University Feinberg School of Medicine, Chicago, IL. atoms from three surface loops in the A domain coordinate the Received for publication August 18, 2011. Accepted for publication October 19, MIDAS metal ion, with the ligand-derived Asp/Glu binding mo- 2011. nodentately to complete the hexacoordinated Mg2+ ion (19–21); it This work was supported by grants from the National Institutes of Health (National is replaced by a water molecule in the unliganded structure (Fig. Institute of Diabetes and Digestive and Kidney Diseases). 1B,1C). Formation of the Asp/Glu–Mg2+ bond in aA domains is 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 coupled mechanically to a conformational switch of the domain for the low-affinity CD11bA/Fab 107 complex and 3QA3 for the high-affinity from the default low-affinity (closed) state to the high-affinity CD11bA/mAb107 complex. (open) state, which includes a 180˚ flip of a conserved Gly243, Address correspondence and reprint requests to Prof. M. Amin Arnaout, Division leading to the downward axial displacement of the C-terminal a7 of Nephrology, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129. E-mail address: [email protected] helix on the opposite pole of MIDAS (Fig. 1A). This movement Abbreviations used in this article: CD11bA, integrin CD11b/CD18 A domain; Fab enables aA to engage the bA MIDAS through an invariant Glu 107, Fab fragment of mAb 107; MIDAS, metal ion-dependent adhesion site; RMSD, at the C terminus of the a7 helix (22), thus translating ligand root-mean-square deviation; RU, resonance unit; scFv, single-chain variable frag- occupancy in aA into quaternary changes downstream, leading to ment; WT, wild-type. outside-in signaling and cell adhesion (23). In the aA-lacking 2+ Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 integrin subgroup, extrinsic ligands directly bind the Mg ion www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102394 2 ACa2+ AT MIDAS IN CD11bA/Fab COMPLEX at the bA MIDAS (19), initiating similar activating conforma- Formation of CD11bA/Fab 107 complexes tional changes. Human low-affinity (closed) CD11bA was mixed with Fab 107 in a 1.5:1.0 In addition to the role of the above conformational changes in molar ratio for 15 min on ice in 20 mM Tris buffer (pH 8.2) containing 2 mM integrin affinity modulation, it also is established that integrin– CaCl2. High-affinity CD11bA was incubated with Fab 107 in the same ligand interactions are critically dependent on the nature of the molar ratio for 60 min on ice in 20 mM Tris buffer (pH 8.2) containing 5 divalent cation at MIDAS. Solved crystal structures of closed (24) mM MgCl2. Each mixture then was applied to a Superdex 200 10/300 GL column (Pharmacia Biotech) using a BioLogic DuoFlow FPLC system and liganded (12–14) aA domains and of integrin ectodomains (Bio-Rad) at a flow rate of 0.4 ml/min at 4˚C. The elution profiles were complexed to natural ligands or ligand mimetics (16, 17, 19, 21) monitored in-line by UV absorbance at 280 nm. The eluted peaks were confirmed the presence of Mg2+ (or Mn2+) but not Ca2+ at MIDAS, analyzed by 12% SDS-PAGE under nonreducing conditions, followed by although Mg2+ and Ca2+ are present in equimolar concentrations in Coomassie staining, and fractions containing CD11bA/Fab 107 complex were pooled, concentrated, and used for protein crystallization. circulating plasma. This preference is related to the octahedral environment at MIDAS that favors Mg2+ over Ca2+ (25), ac- Crystallography, structure determination, and refinement counting for the critical dependence of integrin–ligand inter- 2+ The low-affinity CD11bA/Fab 107 complex (at 10 mg/ml) in 20 mM Tris actions on Mg at MIDAS (26–29). All of the previous studies in buffer (pH 8.2) containing 2 mM CaCl2 and the high-affinity CD11bA/Fab integrins have emphasized the charge of the ligand Asp/Glu as 107 complex (at 5 mg/ml) in 20 mM Tris buffer (pH 8.2) containing 5 mM a critical contributor in metal binding and selectivity at MIDAS. MgCl2 with 1 mM PMSF were crystallized at room temperature by vapor However, the Asp or Glu side chains are unique among the natural diffusion using the hanging drop method. Crystals used for data collection were grown from reservoir solutions containing 15% polyethylene glycol amino acids in possessing a carboxylate group that can ligate the 4000, 50 mM Tris buffer (pH 8.2), and 0.3 M NaCl (for the low-affinity metal ion via one or both of the carboxylate oxygen atoms. CD11bA/Fab 107 complex) and 13% polyethylene glycol 8000, 50 mM However, despite this unique feature, the possibility that the den- Tris buffer (pH 8.2), 0.25 M NaCl, and 10 mM CaCl2 (for the high-affinity ticity of the ligand Asp/Glu also may modulate metal ion selec- CD11bA/Fab 107 complex). Crystals were cryoprotected in reservoir so- lution containing 24% glycerol. Data were collected at the Advanced tivity and function in integrins has not been considered previously. Photon Source (Chicago, IL) at beam line 19ID and processed using The primate-specific and function-blocking mAb 107 binds HKL2000 (38). A low-resolution model of the low-affinity CD11bA/Fab with nanomolar affinity to isolated CD11bA in solution or in the 107 complex that was available in our laboratory was used for molecular context of the full-length CD11b/CD18 integrin in leukocytes replacement using Phaser (39). Rigid-body refinement was followed by the (30). Like ligand-mimetic antagonists, mAb 107 binds at MIDAS autobuild procedure in Phenix (40), with noncrystallographic symmetry 2+ 2+ averaging for the four molecules in the asymmetric unit. The final refined of CD11bA, but in contrast it favors Ca over Mg there (31).