Conformational Changes in the Integrin औA Domain Provide a Mechanism for Signal Transduction via Hybrid Domain Movement

Abstract

The ligand-binding head region of integrin ␤ subunits contains a von Willebrand factor type A domain (␤A). Ligand binding activity is regulated through conformational changes in ␤A, and ligand recognition also causes conformational changes that are transduced from this domain. The molecular basis of signal transduction to and from ␤A is uncertain. The epitopes of mAbs 15/7 and HUTS-4 lie in the ␤ 1 subunit hybrid domain, which is connected to the lower face of ␤A. Changes in the expression of these epitopes are induced by conformational changes in ␤A caused by divalent cations, function perturbing mAbs, or ligand recognition. Recombinant truncated ␣ 5 ␤ 1 with a mutation L358A in the ␣7 helix of ␤A has constitutively high expression of the 15/7 and HUTS-4 epitopes, mimics the conformation of the ligand-occupied receptor, and has high constitutive ligand binding activity. The epitopes of 15/7 and HUTS-4 map to a region of the hybrid domain that lies close to an interface with the ␣ subunit. Taken together, these data suggest that the transduction of conformational changes through ␤A involves shape shifting in the ␣7 helix region, which is linked to a swing of the hybrid domain away from the ␣ subunit.Integrins mediate a wide variety of essential cell-matrix and cell-cell interactions and also participate in many common disease processes (1, 2). Integrins are heterodimers containing non-covalently associated ␣ and ␤ subunits; each subunit has a large extracellular domain linked to a transmembrane segment and a short cytoplasmic tail. Integrins participate in bi-directional signaling; ligand recognition is dynamically regulated by "inside-out" signaling, and ligand occupancy leads to "outsidein" signals that affect cell migration, growth, differentiation, and survival (3-5). Modulation of integrin activity is essential in such processes as leukocyte migration to sites of tissue injury and the aggregation of platelets to form a hemostatic plug. Integrin activation can be mimicked in vitro by divalent cations such as Mn 2ϩ or Mg 2ϩ (6). Three major conformational states of integrins can be distinguished using monoclonal antibodies (mAbs) 1 : an inactive (resting or low affinity) state, an active (or high affinity) state, and a ligand-occupied state (7). The conformations of the inactive and active states are discriminated by low and high expression, respectively, of activation epitopes (such as those recognized by 12G10, 15/7, and 9EG7 for the ␤ 1 subunit, see Refs. 8 -10). The ligand-occupied conformer expresses high levels of ligand-induced binding site (LIBS) epitopes (which are generally also activation epitopes) and shows decreased expression of ligand-attenuated binding site (LABS) epitopes (such as mAb 13 for the ␤ 1 subunit, see Ref. 11). The conformational states are in equilibrium; therefore, antibodies that recognize activation epitopes or LIBS tend to cause activation and stabilize the ligand-occupied state. Conversely, antibodies that recognize LABS appear to block ligand binding by preventing conformati...

Structural basis for allostery in integrins and binding to fibrinogen-mimetic therapeutics

“…The effects of mutations designed to induce hybrid domain swing-out 9,42 (Fig. 2d), allosteric activating or inhibitory mAb that bind to the hybrid domain 10,13 ( Supplementary Fig. 4), disulphide cross-links in the β6-α7 loop 43 and shortening of the α7-helix in the βI domain 44 , all support the conclusion that the closed and open conformations of the integrin headpiece have low and high affinity for ligand, respectively.…”

“…This marked change in tertiary structure is supported by mutational studies 3,6,[9][10][11] and solution X-ray scattering 12 . Ligand-mimetic compounds induce the extended, open headpiece conformation of integrins in solution and on the cell surface 3,6,[10][11][12][13] , and LIBS epitope exposure 14 . In contrast, when a ligand-mimetic is soaked into preformed crystals containing the bent integrin conformation with the closed headpiece, binding induces only localized structural changes near the ligand binding site 8 .…”

Allosteric β1 Integrin Antibodies That Stabilize the Low Affinity State by Preventing the Swing-out of the Hybrid Domain

“…2B and Ref. 32), supporting the indirect mode of inhibition by this class of mAbs. EM images of the integrin-SG/19 Fab complex clearly show that the mAb docks at the outer hinge connecting the I-like domain and the hybrid domain.…”

“…SG/7 also reacted much less well (ϳ70% reduction from wild type) to the wedge mutant (data not shown). A reduction of mAb 13 binding and augmentation of mAb HUTS-4 by an activating mutation in the ␤ 1 I-like domain ␣7-helix has been reported (32). These effects of the L358A mutation are much less pronounced (ϳ50% reduction and ϳ50% increase in binding of mAbs 13 and HUTS-4, respectively) than the G429N mutation.…”

“…This would prevent the outward swing of the hybrid domain and fix the closed conformation of the headpiece that represents the low affinity state (5). Recently, we (33) and others (32) have reported that shape shifting in the ␣7 helix region of the ␤ I-like domain upregulates ligand affinity. In the context of the whole molecule, this shape shifting causes an outward swing of the ␤ hybrid domain (3,5,26).…”