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, José Luis Alonso, Saurabh Anand, Brian D. Adair, Alberto L. Horenstein, Fabio Malavasi, Jian-Ping Xiong and M. Amin Arnaout
J Immunol December 15, 2011, 187 (12) 6393-6401; DOI: https://doi.org/10.4049/jimmunol.1102394

Abstract

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.

Integrins are α/β heterodimeric adhesion receptors that couple the extracellular matrix or counter-receptors on other cells with the contractile cytoskeleton, transducing mechanochemical signals across the plasma membrane that regulate most cellular functions (1). Deregulation of integrin functions, however, plays critical roles in a diverse range of diseases, including inflammatory and vascular diseases and tumor metastasis, establishing 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 mortality in treated patients (4, 8, 9), limiting their use and reflecting the need for a better understanding of the structure–activity relationships in these conformationally dynamic receptors.

At the core of integrin interaction with physiologic ligands is 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 α (αA or I domain) and/or β (βA or I-like domain) subunits (Fig. 1) (11). In solved structures of complexes of integrins with natural ligands, 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 monodentately to complete the hexacoordinated Mg2+ ion (19–21); it is replaced by a water molecule in the unliganded structure (Fig. 1B, 1C). Formation of the Asp/Glu–Mg2+ bond in αA 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 Gly243, leading to the downward axial displacement of the C-terminal α7 helix on the opposite pole of MIDAS (Fig. 1A). This movement enables αA to engage the βA MIDAS through an invariant Glu at the C terminus of the α7 helix (22), thus translating ligand occupancy in αA into quaternary changes downstream, leading to outside-in signaling and cell adhesion (23). In the αA-lacking integrin subgroup, extrinsic ligands directly bind the Mg2+ ion at the βA MIDAS (19), initiating similar activating conformational changes.

In addition to the role of the above conformational changes in integrin affinity modulation, it also is established that integrin–ligand interactions are critically dependent on the nature of the divalent cation at MIDAS. Solved crystal structures of closed (24) and liganded (12–14) αA domains and of integrin ectodomains complexed to natural ligands or ligand mimetics (16, 17, 19, 21) confirmed the presence of Mg2+ (or Mn2+) but not Ca2+ at MIDAS, although Mg2+ and Ca2+ are present in equimolar concentrations in circulating plasma. This preference is related to the octahedral environment at MIDAS that favors Mg2+ over Ca2+ (25), accounting for the critical dependence of integrin–ligand interactions on Mg2+ at MIDAS (26–29). All of the previous studies in integrins have emphasized the charge of the ligand Asp/Glu as a critical contributor in metal binding and selectivity at MIDAS. However, the Asp or Glu side chains are unique among the natural amino acids in possessing a carboxylate group that can ligate the metal ion via one or both of the carboxylate oxygen atoms. However, despite this unique feature, the possibility that the denticity of the ligand Asp/Glu also may modulate metal ion selectivity and function in integrins has not been considered previously.

The primate-specific and function-blocking mAb 107 binds with nanomolar affinity to isolated CD11bA in solution or in the context of the full-length CD11b/CD18 integrin in leukocytes (30). Like ligand-mimetic antagonists, mAb 107 binds at MIDAS of CD11bA, but in contrast it favors Ca2+ over Mg2+ there (31). To elucidate the structural basis for this unusual metal ion selectivity and its potential impact on integrin conformation and function, we cocrystallized the Fab fragment of mAb 107 (Fab 107) complexed with the low- (closed) (32) or high-affinity (open) (12) forms of CD11bA. Both structures reveal stable coordination of a Ca2+ ion at MIDAS, the result of an unexpected bidentate binding by a ligand-mimetic Asp derived from Fab 107, with the absence of ligand-induced conformational changes. Consistently, Fab 107 abolished agonist-induced conformational changes in native CD11b/CD18 in neutrophils in physiologic metal ion conditions. The significance of these findings is discussed.

Materials and Methods

Reagents, cells, and culture

Restriction and modification enzymes were obtained from New England Biolabs, Life Technologies, or Fisher Scientific. K562 cells (American Type Culture Collection) stably expressing wild-type (WT) (low-affinity) or high-affinity CD11b/CD18 [induced by an Ile316Gly mutation in the α7 helix (31, 33)] were generated as described (34). All of the cell culture reagents were from Invitrogen. Fab 107 was generated by digesting affinity-purified mAb 107 (IgG1) with immobilized papain, the immobilized enzyme then was removed, and the Fab-containing supernatant was applied to protein A to remove undigested material. The single-chain variable fragment (scFv) form of mAb 107 was generated as described (31). The CD11b/CD18 heterodimer-specific mAb IB4 (IgG2a) (35) was obtained from the American Type Culture Collection, and the β2 integrin activation reporter mAb 24 (IgG1), which binds to βA when ligated by αA (36, 37), was from Abcam. Isotype control Abs MOPC-21 (IgG1) and MOPC-173 (IgG2a) were from BD Pharmingen, and allophycocyanin-conjugated goat anti-mouse Fc-specific Ab was from Jackson ImmunoResearch Laboratories.

Site-directed mutagenesis

Amino acid substitutions were generated using PCR-based mutagenesis, and authenticity was confirmed by DNA sequencing. The rat-to-human knock-in (Asp178/Glu178, Gln279-Arg282/Lys279-Gln282, Arg203-Asn206-Lys210/Thr203-Leu206-His210, and Asp178-Arg203-Asn206-Lys210/Glu178-Thr203-Leu206-His210) chimeras were generated in cDNA encoding WT rat CD11bA. Asp107Gly and Glu101Gly substitutions were generated in cDNA encoding WT scFv 107.

Formation of CD11bA/Fab 107 complexes

Human low-affinity (closed) CD11bA was mixed with Fab 107 in a 1.5:1.0 molar ratio for 15 min on ice in 20 mM Tris buffer (pH 8.2) containing 2 mM CaCl2. High-affinity CD11bA was incubated with Fab 107 in the same molar ratio for 60 min on ice in 20 mM Tris buffer (pH 8.2) containing 5 mM MgCl2. Each mixture then was applied to a Superdex 200 10/300 GL column (Pharmacia Biotech) using a BioLogic DuoFlow FPLC system (Bio-Rad) at a flow rate of 0.4 ml/min at 4°C. The elution profiles were monitored in-line by UV absorbance at 280 nm. The eluted peaks were analyzed by 12% SDS-PAGE under nonreducing conditions, followed by Coomassie staining, and fractions containing CD11bA/Fab 107 complex were pooled, concentrated, and used for protein crystallization.

Crystallography, structure determination, and refinement

The low-affinity CD11bA/Fab 107 complex (at 10 mg/ml) in 20 mM Tris buffer (pH 8.2) containing 2 mM CaCl2 and the high-affinity CD11bA/Fab 107 complex (at 5 mg/ml) in 20 mM Tris buffer (pH 8.2) containing 5 mM MgCl2 with 1 mM PMSF were crystallized at room temperature by vapor diffusion using the hanging drop method. Crystals used for data collection were grown from reservoir solutions containing 15% polyethylene glycol 4000, 50 mM Tris buffer (pH 8.2), and 0.3 M NaCl (for the low-affinity CD11bA/Fab 107 complex) and 13% polyethylene glycol 8000, 50 mM Tris buffer (pH 8.2), 0.25 M NaCl, and 10 mM CaCl2 (for the high-affinity CD11bA/Fab 107 complex). Crystals were cryoprotected in reservoir solution containing 24% glycerol. Data were collected at the Advanced Photon Source (Chicago, IL) at beam line 19ID and processed using HKL2000 (38). A low-resolution model of the low-affinity CD11bA/Fab 107 complex that was available in our laboratory was used for molecular replacement using Phaser (39). Rigid-body refinement was followed by the autobuild procedure in Phenix (40), with noncrystallographic symmetry averaging for the four molecules in the asymmetric unit. The final refined model was obtained after iterative rounds of model building in Coot (41), followed by refinement in Buster (42). The refined low-affinity CD11bA/Fab 107 complex was used as a starting model for the refinement of the high-affinity CD11bA/Fab 107 complex. Each of the four molecules in this complex also contained a Ca2+ ion at MIDAS. Ca2+ rather than Mg2+ was placed at MIDAS in this case, although both metal ions were present in the crystallization buffer, because placing Mg2+ instead of Ca2+ resulted in much lower B factors for Mg2+ than the surrounding atoms lying within a radius of 10 Å.

Surface plasmon spectroscopy

Surface plasmon resonance spectroscopy (BIAcore AB, Uppsala, Sweden) was used to measure the binding parameters for the interaction of human or rat WT (low-affinity) CD11bA, high-affinity CD11bA, and rat-to-human CD11bA chimeras with the IgG or scFv forms of mAb 107. mAb 107 was coupled covalently via primary amines to the dextran matrix of a CM5 sensor chip as directed by the manufacturer. Chips treated in the same way with BSA (Sigma-Aldrich) or with no protein were used as negative controls. CD11bA in various concentrations was passed over the CM5 sensor chips at a flow rate of 5 μl/min at room temperature in TBS containing 1 mM CaCl2, 1 mM MgCl2, and 0.005% P20. The chip surface was regenerated by 10 mM HCl. All of the data were analyzed using the BIAcore T100 evaluation program, after subtracting binding to the BSA-coupled flow cell to discount the minimal nonspecific binding. For each sensorgram, the peak response in the steady-state region was plotted against the analyte concentration, and the plot was fitted to a single-site binding equation (Langmuir isotherm) to determine the association and dissociation rate constants.

mAb binding to recombinant CD11b/CD18

K562 cells stably expressing low- or high-affinity (Ile316Gly) CD11b/CD18 were maintained in IMDM plus 10% heat-inactivated FBS, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 0.5–1.0 mg/ml G418. Low- or high-affinity CD11b/CD18-expressing K562 cells (1 × 106 cells) were stained in suspension with mAbs IB4 or 24 (at 20 μg/ml) in 100 μl TBS containing 1 mM CaCl2 and 1 mM MgCl2 in 1% globulin-free BSA for 30 min at room temperature or 37°C. After being washed, cells were incubated with the fluorophore-conjugated anti-mouse Fc Ab (for 30 min at 0°C), washed, and fixed with 1% formaldehyde, and fluorescence was measured by a FACSCalibur flow cytometer (BD Biosciences), and mean fluorescence intensity was determined using the BD Cellquest program. Binding of mAb 24 was presented as a percentage of mAb IB4 binding. To assess the effect of mAb 107 on mAb 24 binding, K562 cells expressing WT (low-affinity) or constitutively active (high-affinity) CD11b/CD18 were preincubated with a saturating concentration of the unlabeled Fab 107 (75 μg/ml) for 30 min at 37°C followed by the addition of mAb 24 IgG for an extra 20 min at the same temperature. Cells then were washed and stained with the secondary Fc-specific fluorophore-labeled Ab, and fluorescence was measured by a FACSCalibur flow cytometer.

mAb binding to human neutrophils

Purified human neutrophils were isolated as described (30) from normal volunteers through an Institutional Review Board-approved human subjects protocol. Neutrophils were resuspended at a concentration of 1 × 107 cells/ml in HBSS medium (Life Technologies) containing 1% globulin-free BSA (HBSS-BSA). A total of 1 × 106 cells in 100 μl HBSS-BSA buffer containing 1 mM each of CaCl2 and MgCl2 were left untreated or treated with FMLF (5 × 10−7 M final concentration) for 10 min at 37°C. Unlabeled Fab 107 was added to one half of the replicate tubes for an additional 30 min, followed by the addition of mAb 24 to all of the tubes and incubation for an extra 20 min. Cells then were washed once, stained with the Fc-specific fluorophore-labeled Ab (for 30 min at 0°C), washed again, then fixed with 1% formaldehyde in PBS, and analyzed by flow cytometry as described above.

Results

Crystal structure of Fab 107 in complex with low-affinity CD11bA

The crystallographic data and refinement statistics for the low-affinity CD11bA/Fab 107 complex are given in Table I. Each of the four molecules in the asymmetric unit of the complex contained a Ca2+ ion at MIDAS. The positional root-mean-square deviation (RMSD) between the main chains of all four CD11bA/Fab complexes is small, being 0.14–0.18 Å for CD11bA, 0.45–0.99 and 0.70–0.76 Å for the Fab L and H chains, respectively, and 0.59–1.34 Å for the complex.

View this table:
Table I. Statistics of X-ray diffraction data collection and structure refinement

When compared with natural ligands and ligand mimetics (Fig. 1C), a novel feature of the interaction of Fab 107 with low-affinity CD11bA is that a Ca2+ ion is present at MIDAS, coordinated by seven oxygen atoms forming a pentagonal bipyramid, with five oxygen atoms lying in the equatorial plane and two oxygen atoms in the axial (apical) positions (Fig. 2). Two equatorial oxygen atoms belong to a bidentate carboxylate from Asp107 of the VH chain 3 (VH3) loop (Fig. 2B). This bidentate binding mode by the ligand Asp was not associated with the activating conformational changes normally triggered by the ligation of MIDAS in A domains (Fig. 2A) and contrasts with all of the previous integrin structures where the ligand Asp/Glu binds MIDAS metal monodentately and elicits activating conformational changes in the integrin.

FIGURE 1.
FIGURE 1.

Structures of low- and high-affinity forms of human CD11bA and the corresponding changes in metal ion coordination at MIDAS. A, Ribbon diagram of the superimposed crystal structures of low- (cyan) (1jlm.pdb) and high-affinity (gray) (1ido.pdb) forms of CD11bA with the MIDAS metal ion shown in the respective colors. The conformational switch from the low- to high-affinity state, which reshapes MIDAS, involves a 2 Å inward movement of the α1 helix and the metal ion and a 180° flip of the Cα atom of Gly243 (in red) of MIDAS loop 3, forcing the solvent exposure of buried residues Phe275 and Phe302 and a 10 Å axial downward displacement of the C-terminal α7 helix (in red) (direction of these movements is shown by arrows). Buried and exposed side chains of F302 and F275 are in cyan and red, respectively. B and C, The three MIDAS loops (L1–L3) provide, respectively, the motif D140xS142xS144 (where x is any amino acid), T209, and D242. In the low-affinity CD11bA conformation (B), the two serines and D242 make direct bonds to the metal ion, while T209 makes indirect contact via a water molecule (ω1) (in the T209-ω1-metal-D242 closed MIDAS configuration); two other water molecules (ω2 and ω3) complete the octahedral coordination sphere of the MIDAS Mg2+ (or Mn2+) ion. In the high-affinity conformation (C), D242 moves from the primary to the secondary coordination sphere, increasing the net positive charge near the MIDAS ion, thus enhancing its electrostatic monodentate interaction with the ligand carboxylate, which replaces water molecule ω3. The MIDAS metal coordination thus switches to the T209-metal-ω1-D242 open MIDAS configuration. Coordinating residues and water molecules are shown as ball-and-stick representations with green carbon and red oxygen atoms. The ligand Glu is in gold. Hydrogen and metal ion bonds are represented with dashed red lines. All of the molecular graphics images were generated using the UCSF Chimera package.

FIGURE 2.
FIGURE 2.

Crystal structure of Fab 107 in complex with low-affinity CD11bA. A, Ribbon representation of the complex. CD11bA and the H and L chains of Fab 107 are cyan, pink, and yellow, respectively. The ligand D107 side chain is shown, with oxygen atoms as red spheres, coordinating the MIDAS Ca2+ ion (cyan sphere). Ligated CD11bA assumes the low-affinity conformation (featured by burial of F302 and F275 and packing of the C-terminal α7 helix against the body of the domain). The three MIDAS loops (L1–L3) are labeled. Molecular graphics images were generated using the UCSF Chimera package. B, A σ-weighted 2Fo − Fc density map (blue, contoured at 1.0σ) of the MIDAS motif in the complex. Coordinating residues are shown as ball-and-stick representations with green carbon and red oxygen atoms. Water molecules (ω) are depicted as red spheres. Hydrogen and metal ion bonds are represented with dashed red lines. The ligand D107 (in gold) bidentately coordinates the MIDAS Ca2+ ion (cyan sphere). The average ion-ligating group distance in this structure (as well as in the high-affinity CD11bA/Fab 107 complex; see below) is 2.42–2.51 Å. The estimated coordinate error for the models (derived from the refinement program Buster) is 0.39–0.41 Å, yielding a maximum distance of 2.8 Å, in agreement with the symmetric bidentate carboxylate group–Ca2+ distance reported by Harding (56). The orientation of this figure is as in Fig. 1B and 1C.

In addition to the Asp107–MIDAS Ca2+ ion contact, the mAb epitope comprises residues in two MIDAS ion-coordinating loops and the short BC loop, with important contacts involving Arg208, Phe246, and Glu178-Glu179 in CD11bA (Fig. 3A). Arg208 from MIDAS loop L2 inserts into a cleft between the Fab variable H and L chains and makes a salt bridge with Glu101 of the VH3 loop and side chain and main chain contacts with Tyr109 (from VH3 loop) and Tyr97 (from VL3 loop). Phe246 from MIDAS loop L3 (which also contains the metal-coordinating residue Asp242) inserts into a hydrophobic pocket walled off by three tyrosines from Fab 107 (Fig. 3A). Glu178-Glu179 in the BC loop of CD11bA contact Arg52 of the VH2 loop. Binding experiments have shown that the ligand Asp107 and Glu101 of the mAb (Fig. 3B) and Arg208, Phe246, and Glu178-Glu179 of CD11bA (30) are essential for the mAb 107–CD11bA interaction, in agreement with the structure and underscoring the role of these contacts in stabilizing the ligand Asp–Ca2+ interaction. Further validation of the structure comes from the examination of the sequence of rat CD11bA (which does not bind mAb 107): with the exception of the human-to-rat substitutions Lys279Gln, Thr203Arg, and Glu178Asp, all of the other contact residues are conserved. Kinetic analysis of rat-to-human chimeras showed that swapping the rat residues Gln279, Arg282, Arg203, Asn206, and Lys210 with the respective human residues Lys279, Gln282, Thr203, Leu206, and His210 did not restore the binding of mAb 107 to the resulting rat-to-human chimeric protein (data not shown). However, replacing rat Asp178 with human Glu178 in rat CD11bA largely reestablished binding to mAb 107 (Fig. 3C). This binding became almost identical to that with human WT CD11bA when Asp178Glu was combined with Arg203, Asn206, Lys210/Thr203, Leu206, and His210 substitution (Fig. 3C). These studies demonstrate that Glu178 is mainly responsible for the primate specificity of the mAb.

FIGURE 3.
FIGURE 3.

The integrin/Fab 107 binding interface and its validation. A, Main interacting residues (shown as stick models) from Fab 107 and low-affinity CD11bA are colored as in Fig. 2A. The interacting MIDAS loops L2 and L3, the BC loop from CD11bA, and VL1, VL3, VH2, and VH3 loops from Fab 107 are labeled. Main chain oxygen and nitrogen atoms of E244 [a key physiologic ligand binding residue in the αA domain of integrin CD11a (14)] are red and blue, respectively. The conformationally sensitive G243 Cα atom is green. Hydrogen and metal ion bonds are represented with dashed red lines. B, Sensorgrams recording the interaction of immobilized WT (2771 resonance units, RU), D107G (5182 RU), or E101G (4344 RU) forms of scFv 107 with human low-affinity CD11bA (1.6 μM) in Ca2+ and Mg2+ buffer (1 mM each). C, Sensorgrams of interaction of immobilized Fab 107 (in 1 mM each of Ca2+ and Mg2+) with low-affinity human (hWT) and rat (rWT) CD11bA and rat-to-human D178E CD11bA (rD178/E) and D178, R203, N206, K210/E178, T203, L206, and H210 (rDRNK/ETLH) chimeras (0.5 μM). Measurements from two independent experiments yielded Kd values (in nM) of 4.97 ± 0.24, 56.46 ± 17.2, and 7.57 ± 0.22 for hWT, rD178/E, and rDRNK/ETLH, respectively. Fab 107 did not bind to low-affinity rWT.

Crystal structure of Fab 107 in complex with high-affinity CD11bA

Structures of the monomeric high-affinity form of the αA domain from several integrins in complex with ligands yielded an open conformation (12–14), where a ligand Asp/Glu side chain binds the MIDAS Mg2+ (or Mn2+) ion monodentately, completing the octahedral coordination sphere around the metal and allowing the large conformational switch in the domain. The crystal structure of Ile316Gly CD11bA in complex with Fab 107 (Fig. 4A, Table I) differs from the previously determined open structure of this domain (12) in two key features: each of the four CD11bA molecules in the asymmetric unit adopts the low-affinity conformation (Fig. 4B) and a Ca2+ ion is again found at MIDAS, bound bidentately by the ligand Asp107. The conformationally active α1 and α7 helices (Fig. 1A) in both low- and high-affinity CD11bA/Fab 107 complexes make discontinuous crystal contacts with symmetry-related molecules (combined respective interface areas of 444 and 468 Å2). It is unlikely that such contacts prevent the switch into the open conformation upon ligation of MIDAS by the ligand Asp107, because the CD11bA/Fab 107 complexes have been obtained by cocrystallization.

FIGURE 4.
FIGURE 4.

Crystal structure of Fab 107 in complex with high-affinity CD11bA and comparison with structures of closed and open CD11bA forms. A, Ribbon representation of the high-affinity CD11bA/Fab 107 complex. D107 coordinates a MIDAS Ca2+ ion (cyan sphere) bidentately, but the high-affinity CD11bA assumes the low-affinity (closed) conformation. Orientation is as shown in Fig. 2A. The observed unraveling of the lower segment of the α7 helix (arrow) is caused by the helix-breaking I316G activating mutation. B, Superimposed crystal structures of low- and high-affinity CD11bA complexed with Fab 107, cyan and magenta, respectively. Orientation is similar to that in A. C, Major interacting residues on the MIDAS face (shown as stick models) from low-affinity CD11bA/Fab 107 (white), superimposed on the structure of closed CD11bA alone (orange) (1.jlm.pdb) and on that of open CD11bA alone (gray) (1ido.pdb). The MIDAS ion is colored in the respective color of each domain. MIDAS loops L1–L3 are labeled. The orientation is the same as that in Fig. 3A. Hydrogen and metal ion bonds are represented with dashed red lines. See text for details.

Our published kinetic studies revealed ∼7-fold lower affinity of mAb 107 to the open versus closed CD11bA conformation (30). This difference may be explained now by the present structures: Should Fab 107 have approached open CD11bA, Ser144 from MIDAS loop 1 likely would clash with the ligand Asp107, and the side chain of Glu244 (from MIDAS loop 3) would have to be sandwiched between Ser33 and Tyr31 of the Fab 107 VL1 loop (Fig. 4C), which is energetically unfavorable.

Effects of mAb 107 binding on conformational changes in the holoreceptor

The effect of mAb107 binding on conformational switching of the domain in the context of the full-length cellular integrin was examined next. We used the binding of mAb 24 as the reporter of the conformational switch from the low- to high-affinity state in the A domain (36, 37). Binding of Fab 107 to recombinant WT (low-affinity) CD11b/CD18, stably expressed on K562 cells, in buffer containing physiologic concentrations of Ca2+ and Mg2+ (1 mM each), failed to switch the integrin to the high-affinity conformation; instead, baseline binding of mAb 24 to the integrin was reduced significantly (Fig. 5A–C). Thus, binding of mAb 107 to WT CD11bA MIDAS in the full-length integrin does not elicit the agonist-like activities observed upon the binding of peptide or nonpeptide small molecule ligand mimetics to integrins (4).

FIGURE 5.
FIGURE 5.

Effects of Fab 107 on binding of the activation reporter mAb 24 to full-length CD11b/CD18. A, Histograms (mean ± SD, n = 3 independent experiments, each in triplicate) showing the effects of the absence and presence of unlabeled Fab 107 on binding of mAb 24 to the recombinant WT (low-affinity) integrin stably expressed on K562 cells. mAb 24 binding was expressed as a percentage of binding of the heterodimer-specific mAb IB4. The 45% decrease in mAb 24 binding in the presence of Fab 107 was significant at a p value of 0.018. B and C, Flow cytometry analysis of one of the experiments shown in A of K562 cells expressing WT CD11b/CD18 stained with mAb 24 in the absence (B) and presence (C) of Fab 107 or stained with mAb IB4 as a positive control. The reduction in mAb24 binding in the presence of Fab 107 is reflected by the left shift relative to mAb IB4-stained cells. D, Histograms (mean ± SD of triplicate determinations) of a representative experiment, one of three independent experiments carried out, showing binding of mAb 24 in the absence or presence of Fab 107 to K562 cells stably expressing the constitutively active high-affinity receptor. The 11% decrease is significant (p = 0.025) and ranged from 11 to 16% in the three independent experiments. E and F, Flow cytometry analysis of the experiment shown in D. Note the high baseline binding of mAb 24 to the constitutively active Ile316Gly integrin (compare with that to WT, Fig. 5B). Binding of mAb 24 to K562 cells expressing Ile316Gly CD11b/CD18 is reduced in the presence of Fab 107 (F) when compared to its absence (E). mAb IB4-stained cells represent the internal positive control. G, Histograms (mean ± SD of triplicate determinations) of a representative experiment, one of four carried out, showing FMLF-induced increased expression of mAb 24 epitope on agonist-activated native integrin on human neutrophils and its inhibition by Fab 107 binding to levels significantly lower than those found even in untreated neutrophils (p = 0.001, FMLF-treated versus untreated cells). H and I, Flow cytometry analysis of the experiment shown in G. Binding of mAb 24 to FMLF-treated polymorphonuclear neutrophils is reduced markedly in the presence of Fab 107 (I) when compared to its absence (H). mAb IB4-stained polymorphonuclear neutrophils are indicated. All of the binding reactions were carried out in buffer containing physiologic concentrations of Ca2+ and Mg2+ (1 mM each).

We have shown previously that replacing the invariant Ile316 in the C-terminal α7 helix with Gly in CD11bA generates a constitutively active integrin (31) by destabilizing the hydrophobic packing of the α7 helix against the body of the domain. Binding of Fab 107 to this Ile316Gly high-affinity receptor stably expressed on K562 cells in similar buffer conditions produced a small but significant reduction in mAb 24 binding to the integrin (Fig. 5D–F). To assess the effect of bound Fab 107 on the activation of the native receptor, binding of mAb 24 to native CD11b/CD18 was examined in the absence or presence of unlabeled Fab 107 after the preactivation of human neutrophils with FMLF in buffer containing physiologic concentrations of Ca2+ and Mg2+. As shown in Fig. 5G–I, unlabeled Fab 107 almost abolished the binding of mAb 24 to the native agonist-preactivated integrin.

Discussion

The key finding in this report is that the function-blocking mAb 107 binds the MIDAS face of CD11bA in a manner similar to that of physiologic ligands and current ligand-mimetic antagonists but with two notable differences: 1) the mAb-derived ligand Asp107 stabilizes Ca2+ instead of Mg2+ at MIDAS by binding to the metal ion bidentately instead of the usual monodentate binding mode seen with ligand–Mg2+ binding, and 2) ligation of MIDAS by the mAb did not elicit the conformational changes in CD11bA or in the holoreceptor that normally lead to cell adhesion.

Of the two divalent cations Mg2+ and Ca2+ that are abundant in peripheral blood, the octahedral environment at MIDAS is suited ideally for binding Mg2+ (43). This selectivity is perhaps reflective of the evolutionary origin of the extracellular A domains from an ancient intracellular von Willebrand factor type A domain, where Mg2+ is more abundant (44). Fitting the larger Ca2+ (ionic radius 1.0 Å) (45) over Mg2+ (ionic radius 0.72 Å) in the octahedral environment of MIDAS has been shown to be thermodynamically unfavorable and would result in significant structural rearrangement of the surroundings and in a decrease in the affinity for the natural ligand (25).

The bidentate coordination mode of the MIDAS metal explains the higher affinity of Ca2+ over Mg2+ at MIDAS in low- or high affinity CD11bA/Fab 107 complexes when measured by titration calorimetry [40 μM versus 1.0 mM in the low-affinity CD11bA/mAb107 complex and 25 versus 100 μM in the high-affinity CD11bA/scFv107 complex (31)]. The derived energy difference of 2.0 kcal/mol in metal ion binding to CD11bA in solution (31) also is in the range of the energy difference found between monodentate and bidentate coordination modes in other metalloproteins (46). Further, formation of the extra carboxylate bond–Ca2+ may explain the measured negative binding enthalpy of Ca2+ versus Mg2+ to closed and open MIDAS (31).

Binding studies carried out on K562 cells stably expressing WT full-length CD11b/CD18, which is expressed normally in the default low-affinity state on these cells, showed that binding of Fab 107 inhibited rather than induced the activating tertiary changes in the receptor, as reported by mAb 24 (Fig. 5A–C). This finding is consistent with the crystal structure of the low-affinity CD11bA/Fab 107 complex (Fig. 2A, 2B). Fab 107 also significantly inhibited binding of mAb 24 to the constitutive high-affinity receptor stably expressed on K562 cells, but the effect was rather small (Fig. 5D–F). This may be explained by the constitutive contacts that the mutationally stabilized high-affinity CD11bA is expected to make with βA in the context of the heterodimer (22), which are detected by mAb 24. These contacts are absent when high-affinity CD11bA is present in isolation in solution, where it can exist in an equilibrium that allows some of the molecules to populate the low-affinity conformation (47, 48). In the more physiologic situation where the native integrin is activated by agonists such as FMLF, subsequent binding of Fab 107 prevented the conformational switch of CD11bA to the high-affinity state (Fig. 5G–I). This blockade may result from the conversion of the physiologically activated native receptor into the low-affinity conformation or from skewing of the conformational equilibrium known to exist in the surface receptor between low- and high-affinity conformations (23) in favor of the former. In support of the latter scenario is the preferential binding of mAb 107 to the low-affinity state (30).

Although the importance of heptacoordination in stabilizing Ca2+ at MIDAS is established from the structural and functional data presented, important interactions mainly involving residues Phe246, Arg208, and Glu178 are required for the ligand Asp107 to bidentately ligate the MIDAS metal ion (Fig. 3B, 3C) (30). It could be argued that these interactions account for locking CD11bA in the closed state. Alternatively, bidentate ligation by the ligand Asp to the MIDAS Ca2+ ion, once established, could be the primary factor preventing the activation of the conformational switch usually seen in the structures of monodentately ligated MIDAS metal cations. We favor the latter possibility for a couple of reasons. First, the heptacoordination of the MIDAS Ca2+ ion mandates that Asp242 remains in the primary coordination sphere of the metal ion, preventing the critical activating 180° flip of conserved Gly243, which is necessary to switch the domain into the high-affinity state (Fig. 4C). In contrast, no notable change in the main chains of Phe246, Arg208, and Glu178 between inactive and active conformations is observed (Fig. 4C) to affect the conformational switch directly. Second, in studies of other metal ion binding sites that similarly contain an inner shell Asp/Glu, the carboxylate binding mode of the Asp/Glu has been shown to be critical not only in the binding affinity and selectivity of the metal cation but also in regulating the function of metalloproteins (43, 49). For example, the switch from the monodentate to bidentate Asp/Glu binding mode, which shifts the metal cation from Mg2+ to Ca2+, plays an important role in catalysis by several metalloenzymes (reviewed in Ref. 49).

The bidentate binding mode of the ligand Asp107, which stably coordinates Ca2+ at MIDAS, favoring the closed conformation of CD11bA, contrasts with the invariant monodentate coordination of the MIDAS Mg2+/Mn2+ ion in complexes of αA-containing integrins with physiologic ligands (13, 14, 16, 18). In these structures where αA assumes the open conformation, the ligand Asp/Glu originates from or at the end of a β strand or a rigid helix (Fig. 6), structural features that may favor the monodentate coordination mode in the octahedral coordination preferred by Mg2+, with Asp242 forced to move to the secondary metal coordination sphere, facilitated by the 180° flip in the adjacent Gly243 main chain (Fig. 4C). This integrin ligand adaptation thus may be necessary for triggering outside-in signaling. Interestingly, examination of the available crystal structures of the ligand-mimetic mAbs (20, 21) that ligate the MIDAS ion monodentately and result in an open MIDAS configuration reveals that the ligand Asp/Glu originates from shorter VH3 loops (Fig. 6), which also may favor the monodentate coordination mode, in comparison with the longer VH3 loop in Fab 107 that favors bidentate ligation of the MIDAS metal. The hookworm-derived natural integrin antagonist neutrophil inhibitory factor binds the MIDAS face of CD11bA with nanomolar affinity in the presence of Ca2+, and its binding is blocked by mAb 107 but not by other anti-CD11bA mAbs that do not ligate MIDAS (50–52). The Ca2+ dependency of this interaction suggests that the bidentate ligation mode may extend to some natural neutrophil inhibitory factor-like ligand-mimetic integrin antagonists as well. Structural studies will be needed to test this prediction.

FIGURE 6.
FIGURE 6.

Mode of interaction of ligand carboxylates in solved structures of αA domains in complex with physiologic ligands or ligand-mimetic mAbs. Ribbon diagrams of complexes of CD11bA/Fab 107 (cyan), CD49bA/collagen peptide (pink) (1dzi.pdb), CD11aA/ICAM1 (CD54) (yellow) (1mq8.pdb), CD49aA/AQC2 Fab complex (gray) (1mhp.pdb), and CD11aA/AL-57 Fab complex (magenta) (3hi6.pdb). Structures of CD11aA complexes with ICAM3 (1top.pdb) and ICAM5 (3bn3.pdb) are superposable on those of CD11aA/ICAM1 and have not been included to reduce complexity of the figure. The MIDAS metal ions (spheres), the Cα’s (spheres), and side chains of ligand Asp/Glu are shown in their respective colors, with oxygen atoms in red. Structures were superimposed on the CD11bA structure using Matchmaker in Chimera.

A major problem with existing ligand-mimetic integrin antagonists has been their potential to activate the integrin, thus inducing the harmful cell adhesion response that these drugs were designed to prevent (4, 8, 53). For example, patients treated with an oral ligand mimetic targeting platelet αIIbβ3 had a paradoxically worse outcome owing to increased cardiovascular thrombotic events (54). Such serious side effects underscore the need for new approaches in designing integrin antagonists that do not elicit the activating conformational shifts upon MIDAS ligation. One approach used a small molecule that engages the ligand Arg binding site in the propeller in the αA-lacking integrin αIIbβ3, thus impairing the ligation of the MIDAS metal by the ligand Asp in the Arg-Gly-Asp motif (55). The present data suggest that stabilizing the inhibitory Ca2+ ion at MIDAS by inducing a ligand carboxylate monodentate to bidentate switch may provide a promising novel solution for designing integrin antagonists devoid of partial agonism.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. Frank J. Rotella and Dr. Rongguang Zhang for assistance with data collection and Dr. Vineet Gupta for providing K562 stably expressing high-affinity CD11b/CD18. The crystal structures shown in this report are derived from work performed at Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source using the beam line 19ID.

Footnotes

  • 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 deposited 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.

  • 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 fragment
    WT
    wild-type.

  • Received August 18, 2011.
  • Accepted October 19, 2011.

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