Neutrophil adhesion-dependent functions such as chemotaxis, spreading, and phagocytosis are inhibited by neutrophil inhibitory factor (NIF), a glycoprotein produced by the hookworm Ancylostoma caninum. The NIF binding site has been localized to the A-domain of integrin CR3 (CD11b/CD18) and shown to be metal-dependent. The recently solved crystal structure of the A-domain from CD11b revealed a putative metal ion-dependent adhesion site (MIDAS) on the top of the structure. To determine if NIF binds to the A-domain at its MIDAS face, amino acid substitutions involving 24 residues present in surface loops and adjacent helices in the structure were created. The expressed CD11b A-domain and CR3 heterodimers were then tested in a blinded manner for their ability to bind to biotinylated NIF. The solvent-exposed Gly143, Asp149, Glu178-Glu179, and Arg208, all located on the MIDAS face, in close proximity to the metal ion, were involved in CR3-NIF interaction. These data show that the natural integrin antagonist, NIF, binds to CR3 through the MIDAS region and identify putative contact residues in this region that could be targeted therapeutically.

The divalent cations Mg(2+) and Ca(2+) regulate the interaction of integrins with their cognate ligands, with Mg(2+) uniformly facilitating and Ca(2+) generally inhibiting such interactions in vitro. Because both cations are present in mm concentrations in vivo, the physiologic relevance of the in vitro observations is unclear. We measured the affinity of both cations to the inactive and active states of the ligand- and cation-binding A-domain (CD11bA) from integrin CD11b/CD18 in the absence and presence of the single-chain 107 antibody (scFv107), an activation-insensitive ligand-mimetic antibody. Using titration calorimetry, we found that Mg(2+) and Ca(2+) display equivalent (mm) affinities to inactive CD11bA. Activation induced a approximately 10-fold increase in the binding affinity of Mg(2+) to CD11bA with no change in that of Ca(2+) (106 microm +/- 16 and 2.1 mm +/- 0.19, respectively, n = 4). This increase is largely driven by favorable enthalpy. scFv107 induced a 50-80-fold increase in the binding affinity of Ca(2+) (but not Mg(2+) or Mn(2+)) to either form of CD11bA. Thus the affinity of metal ions to integrins is itself regulated by the activation state of these receptors and by certain ligands. These findings, which we expect will be applicable in vivo, elucidate a new level of regulation of the integrin-metal-ligand ternary complex and help explain some of the discrepant effects of Ca(2+) on integrin-ligand interactions.

Crystallographic and electron microscopy studies revealed genuflexed (bent) integrins in both unliganded (inactive) and physiologic ligandbound (active) states, suggesting that local conformational changes are sufficient for activation. Herein we have explored the role of local changes in the contact region between the membrane-proximal ␤-tail domain (␤TD) and the ligand-binding ␤A domain of the bent conformation in regulating interaction of integrin CD11b/CD18 (␣M␤2) with its physiologic ligand iC3b.We replaced the ␤TD CD loop residues D658GMD of the CD18 (␤2) subunit with the equivalent D672SSG of the ␤3 subunit, with AGAA or with NGTD, expressed the respective heterodimeric receptors either transiently in epithelial HEK293T cells or stably in leukocytes (K562), and measured their ability to bind iC3b and to conformation-sensitive mAbs. In the presence of the physiologic divalent cations Ca 2؉ plus Mg 2؉ (at 1 mM each), the modified integrins showed increased (in HEK293) or constitutive (in K562) binding to iC3b compared with wild-type receptors. K562 expressing the ␤TD-modified integrins bound in Ca 2؉ Mg 2؉ to the ␤A-directed high-affinity reporter mAb 24 but not to mAb KIM127, a reporter of the genu-straightened state. These data identify a role for the membrane proximal ␤TD IntroductionIntegrins are ␣␤ heterodimeric receptors normally expressed in an inactive state on the cell surface but can switch rapidly and reversibly to the active physiologic ligand-binding state in response to inside-out activation signals generated from within cells (reviewed by Hynes 1 ). The integrin ectodomain consists of a "head" segment on top of 2 "leg" segments 2,3 ( Figure 1). The head segment is formed of a 7-bladed ␤ propeller from the ␣ subunit that associates noncovalently with a von Willebrand factor type A (VWFA) domain (␤A or I-like) from the ␤ subunit. The ␣-subunit leg is formed of an Ig-like "thigh" domain followed by 2 large ␤-sandwich domains, calf-1 and calf-2. The ␤-subunit leg is formed of an Ig-like "hybrid" domain inserted into the N-terminal PSI domain, 4,5 followed by 4 EGF-like domains and a novel beta tail domain (␤TD) 2 (Figure 1). In native integrins, each leg terminates in a single membranespanning segment and a short cytoplasmic tail. The prototypical ligand Arg-Gly-Asp binds to the head segment such that the ligand aspartate engages ␤A through a metal ion coordinated at the metal ion-dependent adhesion site (MIDAS) and the ligand arginine fits an adjacent pocket in the propeller. 3 One half of the integrin ␣ subunits have an additional VWFA domain (␣A or I) inserted between blades 2 and 3 of the propeller. ␣A exists in inactive and active conformations, and the ␣1 helix, the ␤F-␣7 (F/␣7) loop, and ␣7 helix are known to be involved in this transition. [6][7][8] In the active conformation of ␣A-containing integrins, a C-terminal glutamate from active ␣A ligates the ␤A MIDAS, stabilizing the high-affinity state. 9 Integrins assume a compact conformation bent at their ␣␤ "knees" (located between the th...