contributed equally to this workThe membrane-distal headpiece of integrins has evolved to speci®cally bind large extracellular protein ligands, but the molecular architecture of the resulting complexes has not been determined. We used molecular electron microscopy to determine the threedimensional structure of the ligand-binding headpiece of integrin a 5 b 1 complexed with fragments of its physiological ligand ®bronectin. The density map for the unliganded a 5 b 1 headpiece shows a`closed' conformation similar to that seen in the a V b 3 crystal structure. By contrast, binding to ®bronectin induces an`open' conformation with a dramatic,~80°change in the angle of the hybrid domain of the b subunit relative to its I-like domain. The ®bronectin fragment binds to the interface between the b-propeller and Ilike domains in the integrin headpiece through the RGD-containing module 10, but direct contact of the synergy-region-containing module 9 to integrin is not evident. This ®nding is corroborated by kinetic analysis of real-time binding data, which shows that the synergy site greatly enhances k on but has little effect on the stability or k off of the complex.

Familial amyotrophic lateral sclerosis (FALS) is linked to over 90 point mutations in superoxide dismutase-1 (SOD1), a dimeric metalloenzyme. The postmortem FALS brain is characterized by SOD1 inclusions in the motor neurons of regions in which neuronal loss is most significant. These findings, together with animal modeling studies, suggest that aggregation of mutant SOD1 produces a pathogenic species. We demonstrate here that a mutant form of SOD1 (A4V) that is linked to a particularly aggressive form of FALS aggregates in vitro, while wild-type SOD1 (WT) is stable. Some A4V aggregates resemble amyloid pores formed by other disease-associated proteins. The WT dimer is significantly more stable than the A4V dimer, suggesting that dimer dissociation may be the required first step of aggregation. To test this hypothesis, an intersubunit disulfide bond between symmetry-related residues at the A4V dimer interface was introduced. The resultant disulfide bond (V148C-V148C') eliminated the concentration-dependent loss of enzymatic activity of A4V, stabilized the A4V dimer, and completely abolished aggregation. A drug-like molecule that could stabilize the A4V dimer could slow the onset and progression of FALS.

The ligand binding function of integrins can be modulated by various monoclonal antibodies by both direct and indirect mechanisms. We have characterized an anti-␤ 1 antibody, SG/19, that had been reported to inhibit the function of the ␤ 1 integrin on the cell surface. SG/19 recognized the wild type ␤ 1 subunit that exists in a conformational equilibrium between the high and low affinity states but bound poorly to a mutant ␤ 1 integrin that had been locked in a high affinity state. Epitope mapping of SG/19 revealed that Thr 82 in the ␤ 1 subunit, located at the outer face of the boundary between the I-like and hybrid domains, was the key binding determinant for this antibody. Direct visualization of the ␣ 5 ␤ 1 headpiece fragment in complex with SG/19 Fab with electron microscopy confirmed the location of the binding surface and showed that the ligand binding site is not occluded by the bound Fab. Surface plasmon resonance showed that ␣ 5 ␤ 1 integrin bound by SG/19 maintained a low affinity toward its physiological ligand fibronectin (Fn) whereas binding by function-blocking anti-␣ 5 antibodies resulted in a complete loss of fibronectin binding. Thus a class of the anti-␤ antibodies represented by SG/19 attenuate the ligand binding function by restricting the conformational shift to the high affinity state involving the swing-out of the hybrid domain without directly interfering with ligand docking.Integrins are cell adhesion molecules that bind to ligands in the extracellular matrix and on cell surfaces and regulate many biological functions including cell migration. The affinity of integrins for ligands is conformationally regulated (1-4). On physiological cell surfaces, integrins can assume multiple conformations with distinct affinity states, with the low affinity state being predominant. Physiological "inside-out" signals that impinge on integrin cytoplasmic domains and activating or inhibitory mAbs 1 that bind to the integrin extracellular domain are thought to alter the equilibrium between different conformational states. Electron microscopic (EM) studies of the extracellular domain, fluorescence resonance energy transfer and NMR studies on the cytoplasmic domains, and disulfide crosslinking of the transmembrane domains have indicated that close apposition of the membrane-proximal region of each subunit and the overall bent structure are hallmarks for the low affinity state of integrins, whereas the extended conformation with separated legs represents the high affinity state (5-8).Monoclonal antibodies have been instrumental to studies on the structure and function of integrins (9). Integrin mAbs can be divided into three classes: (i) "inhibitory mAbs" that perturb biological function i.e. ligand binding, (ii) "stimulatory mAbs" that augment ligand binding, and (iii) "neutral mAbs" that do not have any impact on activity. Although some inhibitory mAbs directly recognize the ligand binding site on the integrin molecule and act as ligand-mimetic competitive inhibitors, another class of inhibitory mAbs bind...