Integrin αβ heterodimer cell surface receptors mediate adhesive interactions that provide traction for cell migration. Here, we test whether the integrin, when engaged to an extracellular ligand and the cytoskeleton, adopts a specific orientation dictated by the direction of actin flow on the surface of migrating cells. We insert GFP into the rigid, ligand-binding head of the integrin, model with Rosetta the orientation of GFP and its transition dipole relative to the integrin head, and measure orientation with fluorescence polarization microscopy. Cytoskeleton and ligand-bound integrins orient in the same direction as retrograde actin flow with their cytoskeleton-binding β-subunits tilted by applied force. The measurements demonstrate that intracellular forces can orient cell surface integrins and support a molecular model of integrin activation by cytoskeletal force. Our results place atomic, Å-scale structures of cell surface receptors in the context of functional and cellular, μm-scale measurements.
Why do integrins differ in basal activity, and how does affinity for soluble ligand correlate with cellular adhesiveness? We show that basal conformational equilibrium set points for integrin α4β1 are cell type specific and differ from integrin α5β1 when the two integrins are coexpressed on the same cell. Although α4β1 is easier to activate, its high-affinity state binds vascular cell adhesion molecule and fibronectin 100- to 1,000-fold more weakly than α5β1 binds fibronectin. Furthermore, the difference in affinity between the high- and low-affinity states is more compressed in α4β1 (600- to 800-fold) than in α5β1 (4,000- to 6,000-fold). α4β1 basal conformational equilibria differ among three cell types, define affinity for soluble ligand and readiness for priming, and may reflect differences in interactions with intracellular adaptors but do not predict cellular adhesiveness for immobilized ligand. The measurements here provide a necessary framework for understanding integrin activation in intact cells, including activation of integrin adhesiveness by application of tensile force by the cytoskeleton, across ligand-integrin-adaptor complexes.
Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization- or contractility-driven "retrograde flow" of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-αVβ3 integrins in fibroblasts shows that integrins are coaligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modeling, suggest that integrins in FA are coaligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues.
Transforming growth factor (TGF)-β is synthesized as a proprotein that dimerizes in the endoplasmic reticulum. After processing in the Golgi to cleave the N-terminal prodomain from the C-terminal growth factor (GF) domain in each monomer, pro-TGF-β is secreted and stored in latent complexes. It is unclear which prodomain and GF monomer are linked prior to proprotein convertase (PC) cleavage, and how much conformational change occurs following cleavage. We have determined a structure of pro-TGF-β1 with the PC cleavage site mutated, to mimic the structure of the TGF-β1 proprotein. Structure, mutation, and model building demonstrate that the prodomain arm domain in one monomer is linked to the GF that interacts with the arm domain in the other monomer in the dimeric structure, i.e., the prodomain arm domain and GF domain in each monomer are swapped. Swapping has important implications for the mechanism of biosynthesis in the TGF-β family and is relevant to the mechanism for preferential formation of heterodimers over homodimers for some members of the TGF-β family. Our structure, together with two previous ones, also provides insights into which regions of the prodomain-GF complex are highly structurally conserved, and which are perturbed by crystal lattice contacts.
Von Willebrand factor, an ultralarge concatemeric blood protein, must bind to platelet GPIbα during bleeding to mediate hemostasis, but not in the normal circulation to avoid thrombosis. Von Willebrand factor is proposed to be mechanically activated by flow, but the mechanism remains unclear. Using microfluidics with single-molecule imaging, we simultaneously monitored reversible Von Willebrand factor extension and binding to GPIbα under flow. We show that Von Willebrand factor is activated through a two-step conformational transition: first, elongation from compact to linear form, and subsequently, a tension-dependent local transition to a state with high affinity for GPIbα. High-affinity sites develop only in upstream regions of VWF where tension exceeds ~21 pN and depend upon electrostatic interactions. Re-compaction of Von Willebrand factor is accelerated by intramolecular interactions and increases GPIbα dissociation rate. This mechanism enables VWF to be locally activated by hydrodynamic force in hemorrhage and rapidly deactivated downstream, providing a paradigm for hierarchical mechano-regulation of receptor-ligand binding.Von Willebrand factor (VWF) is a blood protein involved in clotting and is proposed to be activated by flow, but the mechanism is unknown. Here the authors show that VWF is first converted from a compact to linear form by flow, and is subsequently activated to bind GPIbα in a tension-dependent manner.
Foot-and-mouth disease virus (FMDV) mediates cell entry by attachment to an integrin receptor, generally αvβ6, via a conserved arginine-glycine-aspartic acid (RGD) motif in the exposed, antigenic, GH loop of capsid protein VP1. Infection can also occur in tissue culture adapted virus in the absence of integrin via acquired basic mutations interacting with heparin sulphate (HS); this virus is attenuated in natural infections. HS interaction has been visualized at a conserved site in two serotypes suggesting a propensity for sulfated-sugar binding. Here we determined the interaction between αvβ6 and two tissue culture adapted FMDV strains by cryo-electron microscopy. In the preferred mode of engagement, the fully open form of the integrin, hitherto unseen at high resolution, attaches to an extended GH loop via interactions with the RGD motif plus downstream hydrophobic residues. In addition, an N-linked sugar of the integrin attaches to the previously identified HS binding site, suggesting a functional role.
Integrins αVβ6 and αVβ8 are specialized for recognizing pro-TGF-β and activating its growth factor by releasing it from the latency imposed by its surrounding prodomain. The integrin αVβ8 is atypical among integrins in lacking sites in its cytoplasmic domain for binding to actin cytoskeleton adaptors. Here, we examine αVβ8 for atypical binding to pro-TGF-β1. In contrast to αVβ6, αVβ8 has a constitutive extended-closed conformation, and binding to pro-TGF-β1 does not stabilize the open conformation of its headpiece. Although Mn(2+) potently activates other integrins and increases affinity of αVβ6 for pro-TGF-β1 25- to 55-fold, it increases αVβ8 affinity only 2- to 3-fold. This minimal effect correlates with the inability of Mn(2+) and pro-TGF-β1 to stabilize the open conformation of the αVβ8 headpiece. Moreover, αVβ8 was inhibited by high concentrations of Mn(2+) and was stimulated and inhibited at markedly different Ca(2+) concentrations than αVβ6 These unusual characteristics are likely to be important in the still incompletely understood physiologic mechanisms that regulate αVβ8 binding to and activation of pro-TGF-β.
Integrins undergo large-scale conformational changes upon activation. Signaling events driving integrin activation have previously been discussed conceptually, but not quantitatively. Here, recent measurements of the intrinsic ligand-binding affinity and free energy of each integrin conformational state on the cell surface, together with the length scales of conformational change, are used to quantitatively compare models of activation. We examine whether binding of cytoskeletal adaptors to integrin cytoplasmic domains is sufficient for activation or whether exertion of tensile force by the actin cytoskeleton across the integrin-ligand complex is also required. We find that only the combination of adaptor binding and cytoskeletal force provides ultrasensitive regulation. Moreover, switch-like activation by force depends on the large, >130 Å length-scale change in integrin extension, which is well tailored to match the free-energy difference between the inactive (bent-closed) and active (extended-open) conformations. The length scale and energy cost in integrin extension enable activation by force in the low pN range and appear to be the key specializations that enable cell adhesion through integrins to be coordinated with cytoskeletal dynamics.
Recognition by the leukocyte integrins αXβ2 and αMβ2 of complement iC3b-opsonized targets is essential for effector functions including phagocytosis. The integrin-binding sites on iC3b remain incompletely characterized. Here, we describe negative-stain electron microscopy and biochemical studies of αXβ2 and αMβ2 in complex with iC3b. Despite high homology, the two integrins bind iC3b at multiple distinct sites. αXβ2 uses the αX αI domain to bind iC3b on its C3c moiety at one of two sites: a major site at the interface between macroglobulin (MG) 3 and MG4 domains, and a less frequently used site near the C345C domain. In contrast, αMβ2 uses its αI domain to bind iC3b at the thioester domain and simultaneously interacts through a region near the αM β-propeller and β2 βI domain with a region of the C3c moiety near the C345C domain. Remarkably, there is no overlap between the primary binding site of αXβ2 and the binding site of αMβ2 on iC3b. Distinctive binding sites on iC3b by integrins αXβ2 and αMβ2 may be biologically beneficial for leukocytes to more efficiently capture opsonized pathogens and to avoid subversion by pathogen factors.
We show that the three conformational states of integrin α5β1 have discrete free energies and define activation by measuring intrinsic affinities for ligand of each state and the equilibria linking them. The 5,000-fold higher affinity of the extended-open state than the bent-closed and extended-closed states demonstrates profound regulation of affinity. Free energy requirements for activation are defined with protein fragments and intact α5β1 On the surface of K562 cells, α5β1 is 99.8% bent-closed. Stabilization of the bent conformation by integrin transmembrane and cytoplasmic domains must be overcome by cellular energy input to stabilize extension. Following extension, headpiece opening is energetically favored. N-glycans and leg domains in each subunit that connect the ligand-binding head to the membrane repel or crowd one another and regulate conformational equilibria in favor of headpiece opening. The results suggest new principles for regulating signaling in the large class of receptors built from extracellular domains in tandem with single-span transmembrane domains.
Integrins are adhesion receptors that transmit force across the plasma membrane between extracellular ligands and the actin cytoskeleton. In activation of the transforming growth factor-β1 precursor (pro-TGF-β1), integrins bind to the prodomain, apply force, and release the TGF-β growth factor. However, we know little about how integrins bind macromolecular ligands in the extracellular matrix or transmit force to them. Here we show how integrin αVβ6 binds pro-TGF-β1 in an orientation biologically relevant for force-dependent release of TGF-β from latency. The conformation of the prodomain integrin-binding motif differs in the presence and absence of integrin binding; differences extend well outside the interface and illustrate how integrins can remodel extracellular matrix. Remodelled residues outside the interface stabilize the integrin-bound conformation, adopt a conformation similar to earlier-evolving family members, and show how macromolecular components outside the binding motif contribute to integrin recognition. Regions in and outside the highly interdigitated interface stabilize a specific integrin/pro-TGF-β orientation that defines the pathway through these macromolecules which actin-cytoskeleton-generated tensile force takes when applied through the integrin β-subunit. Simulations of force-dependent activation of TGF-β demonstrate evolutionary specializations for force application through the TGF-β prodomain and through the β- and not α-subunit of the integrin.