The structure described in this communication is that of αVβ3 in its active (ligand-competent) state. Activation in this case is artificially induced by truncating the transmembrane and cytoplasmic segments that normally restrain integrins into a default low-affinity state (29
). Naturally occurring β3 mutants (from some patients with thrombasthenia) and mutagenesis studies of the related integrin αIIbβ3 [reviewed in (1
)] identified three ligand-binding regions. Two of these are located in the αIIb propeller and the third involves the βA domain MIDAS. The βA domain also contains a fourth “ligand specificity” region. Replacing this region in β1 with that of β3 switched ligand-binding specificity of integrin αVβ1 into that of αVβ3 [reviewed in (1
)]. The two β3 regions and the αV residues that correspond to the two regions identified in αIIb cluster at the top of the integrin head (). We propose that ligand binding occurs in this area. Confirmatory evidence comes from rotary-shadowed preparations where fibrinogen is found associated with the top of αIIbβ3 (6
). The putative αVβ3 ligand-binding site is formed primarily by residues in loops D3-A3 (between blades 2 and 3) and B3-C3 (within blade 3) of the propeller and by residues in loop B-C and the MIDAS motif of the βA domain. The αA domain loops out from the COOH-terminal end of the D3-A3 loop in αA-integrins.
Major conformational changes throughout the β subunit and reorientation of the extracellular domains of the α and β subunits appear to coincide with integrin activation, whether induced physiologically or artificially (9
). Our crystal structure reveals a severely bent αVβ3 conformation. This arrangement differs dramatically from the extended conformations seen in cryoelectron microscopy images of integrins reconstituted in lipid bilayers (7
) or rotary shadowing of isolated integrins (4
). However, a minority of rotary shadowing images suggests that the tails may have collapsed on top of (or beneath) the head (5
). The dimensions of this oblong form (120 Å by 80 Å) agree nicely with our structure, suggesting that similar bending may occur in solution. The fact that extended and severely bent conformations of the same molecule are seen suggests that a highly flexible site, the genu, exists in the integrin. The extreme degree of bending found in the crystal structure is less likely to occur in a membrane-anchored integrin because such a conformation would likely constrain access of polymerized extracellular matrix to the ligand-binding site. Nevertheless, the high degree of flexibility at the genu provides a first glimpse of the spectrum of the quaternary changes possible during bi-directional signaling in integrins. The presence of a metal ion at the genu suggests one mechanism by which these changes could be regulated.
The structural similarities between the propeller–βA domains and the Gα-Gβ domains are intriguing (). In G proteins, GTP hydrolysis induces a major conformational shift in Gα, a component of which is the destabilization of the α2-helix in switch region II. This results in physical separation of the two subunits in the active state and unmasks ligand-binding sites on the propeller (33
). Does the structural homology between integrins and G proteins translate into analogous models of allosteric control? A large interface between the propeller and βA domains is seen in our structure, in contrast to G proteins where in the active state, the equivalent Gβ and Gα are completely dissociated. Other observations suggest, however, that the propeller–βA domain contact area may also be a dynamic interface that can be modulated. First, integrin αIIbβ3 can be reversibly dissociated into its individual subunits by brief treatment with EDTA (34
). Second, a conformational shift of the propeller relative to the βA domain has been reported (32
). Third, a split of the αIIbβ3 head into two distinct knobs was observed in the presence of RGD peptides (11
). In our structure, cation-π bonding between Arg261
and surrounding aromatic residues contributes to the αβ interface. Amide-aromatic interactions can be attractive or repulsive (27
), unlike ion pairs. Thus, it is conceivable that small changes in side chain orientation could modulate the stability of the αβ interface, perhaps allowing for dissociation under certain conditions.
The location of the metal ion– binding sites in αVβ3 helps clarify some of the complex and unresolved regulatory effects exerted by metal ions on integrin-ligand binding. Cells expressing a mutant α4β1 (lacking one or more metal-binding sites in the propeller) detach easily from substrate under shear flow (35
). The calcium ions in the propeller are not close to the proposed ligand-binding site shown in , but they may help to make more rigid the propeller-thigh interface and may thus regulate integrin-ligand interactions in an allosteric manner. The calcium ion at the thigh–calf-1 interface may play a similar role. Studies with β3 and α5β1 integrins have also shown that a high-affinity Ca2+
site is required for ligand-binding, and that a low-affinity Ca2+
site allosterically inhibits ligand binding (36
). The presence of two adjacent metal-binding sites (MIDAS and ADMIDAS) in the βA domain suggests underlying mechanisms for these effects.
The structure of αVβ3 reveals new domains, previously unpredicted domains, and creative use of the Ig scaffold. Integrin-ligand interactions are regulated not only by changes in affinity but also by altered avidity (receptor clustering) [reviewed in (38
)]. Integrins also bind in cis to several membrane receptors, an interaction that modulates their signaling functions [reviewed in (39
)]. The mosaic of domains revealed in the integrin structure can now serve as a foundation for future investigations into the structural basis of these interactions.