(A) Ribbon diagram of the superimposed crystal structures of low-affinity (1jlm.pdb) (cyan) and high-affinity (1ido.pdb) (gray) forms of CD11bA with the MIDAS metal ion shown in the respective color. The conformational switch from the low- to high-affinity state, which reshapes MIDAS, involves a 2Å 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 10Å axial downward displacement of the C-terminal α7 helix (in red)(direction of these movements is shown by arrows). Buried and exposed sidechains of Phe302 (F302) and Phe275 (F275) are in cyan and red, respectively. The three MIDAS loops (L1–L3) provide respectively, the motif Asp140xSer142xSer144 (D140xS142xS144 in single letter code) (where x is any amino acid), Thr209 (T209), and Asp242 (D242)(B, C). 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 representation with green carbons and red oxygens. The ligand glutamate is in gold. Hydrogen- and metal ion bonds are represented with dashed red lines. All molecular graphics images were generated using the UCSF Chimera package.

(A) Ribbon representation of the complex. CD11bA and the heavy (H) and light (L) chains of Fab 107 are colored cyan, pink and yellow, respectively. The ligand D107 side chain is shown, with oxygen atoms as red spheres, coordinating MIDAS Ca²⁺ (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) Sigma A-weighted 2Fo-Fc density map (in blue; contoured at 1.0 σ) of the MIDAS motif in the complex. Coordinating residues (single letter code) are shown as ball-and-stick representation with green carbons 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⁺ (cyan sphere). The average ion-ligating group distance in this structure (as well as in the high-affinity/107 complex (see below) is 2.42–2.51Å. The estimated coordinate error for the models (derived from the refinement program Buster) is 0.39–0.41Å, yielding a maximum distance of 2.8Å, in agreement with the symmetric bidentate carboxylate group-Ca²⁺ distance reported by Harding (56). Orientation of this figure is as in Fig. 1B, C.

(A) Main interacting residues (shown as stick models) from Fab 107 and low-affinity CD11bA, colored as in Fg. 1A. The interacting MIDAS loops L1 and L3 and 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 colored red and blue, respectively. The conformationally sensitive G243 Cα atom is in green. Hydrogen- and metal ion bonds are represented with dashed red lines. (B) Sensorgrams recording interaction of immobilized WT- (2,771 resonance units, RU), D107/G (5,182 RU) or E101/G (4,344 RU) forms of scFv 107 antibody with human low-affinity CD11bA (1.6μM) in Ca²⁺ and Mg²⁺ buffer (1mM each). (C) Sensorgrams of interaction of immobilized Fab 107 (in 1mM each of Ca²⁺ and Mg²⁺) with low-affinity human (hWT) and rat (rWT) CD11bA, and rat-to-human D178/E CD11bA (rD178/E) and D178, R203, N206, K210/E178, T203, L206, H210 (rDRNK/ETLH) chimera (0.5μM). 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.

(A) Ribbon representation of high-affinity CD11bA/Fab107 complex. D107 coordinates a MIDAS Ca²⁺ (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 I316/G activating mutation. (B) Superposed crystal structures of low-and high-affinity CD11bA complexed with Fab 107, colored in cyan and magenta, respectively. Orientation is similar to (A). (C) Major interacting residues on the MIDAS face (shown as stick models) from Fab107/low-affinity CD11bA (colored white), superposed on structure of closed CD11bA alone (1.jlm.pdb)(colored orange) and on that of open CD11bA alone (1ido.pdb)(colored gray). The MIDAS ion is colored in the respective color of each domain. MIDAS loops 1–3 (L1–3) are labeled. Orientation is the same as in Fig. 3A. Hydrogen- and metal ion bonds are represented with dashed red lines. See text for details.

(A) Histograms (mean ± SD, n=3 independent experiments, each in triplicate) showing effect of 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% drop in mAb 24 binding in presence of Fab 107 was significant at a p value of 0.018. B, C, Flow cytometry analysis of one of the experiments shown in (A) of K562 expressing WT CD11b/CD18 stained with mAb24 in the absence (B) and presence (C) of Fab107 or stained with mAb IB4 as positive control. The reduction in mAb24 binding in presence of Fab107 is reflected by the left shift relative to 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 absence or presence of Fab107 to K562 cells stably expressing the constitutively active high-affinity receptor. The 11 % drop is significant (p=0.025) and ranged from 11–16% in the three independent experiments. E, F, Flow cytometry analysis of the experiment shown in (D). Note the high baseline binding of mAb24 to the constitutively active Ile316/Gly integrin (compare with that to WT, Fig. 5B). Binding of mAb24 to K562 expressing Ile316/Gly CD11b/CD18 is reduced in presence of Fab107 (F) when compared in its absence (E). 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 FMLP-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, FMLP-treated vs. untreated cells). H, I, Flow cytometry analysis of the experiment shown in (G). Binding of mAb24 to FMLP-treated PMNs is markedly reduced in presence of Fab107 (I) when compared in its absence (H). IB4-stained PMNs are indicated. All binding reactions were carried out in buffer containing physiologic concentrations of Ca²⁺ and Mg²⁺ (1mM each).

Ribbon diagrams of complexes of CD11bA/Fab107 (in cyan); CD49bA-collagen peptide (in pink) (1dzi.pdb); CD11aA-ICAM1 (CD54) (in yellow)(1mq8.pdb); CD49aA-AQC2 Fab complex (in gray)(1mhp.pdb) and CD11aA-AL-57 Fab complex (in 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 sidechains of ligand Asp/Glu are shown in the respective colors, with oxygens in red. Structures were superposed on CD11bA structure using Matchmaker in Chimera.