Integrin Activation Dynamics between the RGD-binding Site and the Headpiece Hinge

Abstract

Integrins form mechanical links between the extracellular matrix and the cytoskeleton. Although integrin activation is known to be regulated by an allosteric conformational change, which can be induced from the extracellular or intracellular end of the molecule, little is known regarding the sequence of structural events by which signals propagate between distant sites. Here, we reveal with molecular dynamics simulations of the FnIII 10 -bound ␣ V ␤ 3 integrin headpiece how the binding pocket and interdomain ␤A/hybrid domain hinge on the distal end of the ␤A domain are allosterically linked via a hydrophobic T-junction between the middle of the ␣1 helix and top of the ␣7 helix. The key results of this study are: 1) that this T-junction is induced by ligand binding and hinge opening, and thus displays bidirectionality; 2) that formation of this junction can be accelerated by ligand-mediated force; and 3) how formation of this junction is inhibited by Ca 2؉ in place of Mg 2؉ at the site adjacent to the metal ion-dependent adhesion site ("ADMIDAS"). Together with recent experimental evidence that integrin complexes can form catch bonds (i.e. become strengthened under force), as well as earlier evidence that Ca 2؉ at the ADMIDAS results in lower binding affinity, these simulations provide a common structural model for the dynamic process by which integrins become activated.Integrins anchor cells to the extracellular matrix. They are transmembrane heterodimers, composed of non-covalently bound ␣ and ␤ subunits that associate to form the extracellular, ligand-binding head, two multidomain "legs," two single-pass transmembrane helices, and two short cytoplasmic tails (Fig. 1A). All known integrin heterodimers contain the ␤A domain (also called I-like or ␤I domain), located at the extracellular end of the ␤-subunit. The top of the ␤A domain contains three metal ion binding sites, termed the "ligand-induced metal binding site" ("LIMBS"), 3 the "metal ion-dependent adhesion site" ("MIDAS"), and the "adjacent to the MIDAS" ("ADMIDAS").The LIMBS has been called the synergistic metal binding site instead, since it was found to contain a metal ion in a crystal structure of the unliganded ␣ IIb ␤ 3 integrin (44).Integrin-mediated adhesion often occurs under tensile forces such as fluid flow or myosin-mediated contractions that cells exert to sample the rigidity of their surroundings. Thus, to enable mechanosensing, integrins cannot be constitutively active. Rather, integrin activation is regulated by long-range conformational changes that can originate from the cytoplasmic or extracellular end of the integrin molecule (1). For example, ligand binding has been shown to induce the activating conformational change that leads to hinge opening between the ␤A and hybrid domains in the integrin headpiece (2-4). Vice versa, events originating from the cytoplasmic region of the molecule have been shown to switch the extracellular binding site to the high affinity state (5). This bidirectional reciprocity is typical for allosterically ...

Mechanotransduction Pathways Linking the Extracellular Matrix to the Nucleus

“…Integrin activation can be induced by binding molecules inside the cell (inside-out activation) (Puklin-Faucher and Sheetz, 2009), or forces exerted by the ECM and transmitted through ligand (outside-in activation) and a subsequent propagation of conformational changes from the tail to head and head to tail domains, respectively. Some suggest that during cell adhesion, conformational changes are induced simultaneously by inside-out and outside-in signals, and expedited by forces transmitted through ligands (Alon and Dustin, 2007;Puklin-Faucher and Sheetz, 2009;Puklin-Faucher et al, 2006). Simulations by Puklin-Faucher et al show acceleration in activation events from seconds to microseconds in the presence of mechanical forces (PuklinFaucher et al, 2006).…”

“…Additionally, there is evidence supporting the idea that it is heterodimerization of different integrin molecules that triggers the clustering phenomenon (Luo et al, 2005). Indeed, the challenge on the way of studying transmembrane proteins (e.g., integrin) embedded in the plasma membrane has led researchers to use alternative methods that neglect effects of the plasma membrane on integrin activation/clustering, whereas a major body of molecular level, computational studies of integrin are focused on simulating the ectodomain exclusively (Chen et al, 2011;Puklin-Faucher and Sheetz, 2009;Puklin-Faucher and Vogel, 2009). To reconcile these pieces of apparently contradictory evidence while taking into account effects of the plasma membrane, a series of MD simulation has been recently carried out that underlined the critical role of the plasma membrane in homooligomerization of integrin transmembrane-cytoplasmic domains upon activation .…”

Forcing Switch from Short- to Intermediate- and Long-lived States of the αA Domain Generates LFA-1/ICAM-1 Catch Bonds

“…Atomic force microscopy single-bond experiments have demonstrated that ␣ 5 ␤ 1 , an ␣A domain-lacking integrin, forms catch bonds with fibronectin (FN) in which force prolongs bond lifetimes in the 10 -30 pN range (19). Steered molecular dynamics simulations have suggested how force might activate integrin ␣A domains (20) and the headpiece of integrin ␣ V ␤ 3 (21)(22)(23)(24). However, many mechanistic details about the integrin mechanochemistry are still missing.…”

The human platelet antigen-1b (Pro33) variant of αIIbβ3 allosterically shifts the dynamic conformational equilibrium of this integrin toward the active state

“…3B) and variables characterizing the bending and splaying of the structure (Fig. 3C, D), which had been used successfully in related studies to characterize inactiveto-active transitions (27,37,64). The results display significant differences in the conformational states of both isoforms of αIIbβ3, with the ectodomain of the Pro33 variant showing a stronger tendency to move towards an open, extended conformation with more splayed legs than the Leu 33 isoform.…”