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    Single-molecule characterization of subtype-specific β1 integrin mechanics
    Jo, M.H., et al. Single-molecule characterization of subtype-specific β1 integrin mechanics. Nat Commun 13, 1, 7471 (2022).Abstract
    jo_2022_37809.pdf
    Although integrins are known to be mechanosensitive and to possess many subtypes that have distinct physiological roles, single molecule studies of force exertion have thus far been limited to RGD-binding integrins. Here, we show that integrin α4β1 and RGD-binding integrins (αVβ1 and α5β1) require markedly different tension thresholds to support cell spreading. Furthermore, actin assembled downstream of α4β1 forms cross-linked networks in circularly spread cells, is in rapid retrograde flow, and exerts low forces from actin polymerization. In contrast, actin assembled downstream of αVβ1 forms stress fibers linking focal adhesions in elongated cells, is in slow retrograde flow, and matures to exert high forces (>54-pN) via myosin II. Conformational activation of both integrins occurs below 12-pN, suggesting that post-activation subtype-specific cytoskeletal remodeling imposes the higher threshold for spreading on RGD substrates. Multiple layers of single integrin mechanics for activation, mechanotransduction and cytoskeleton remodeling revealed here may underlie subtype-dependence of diverse processes such as somite formation and durotaxis.
    A general chemical principle for creating closure-stabilizing integrin inhibitors
    Lin, F.-Y., et al. A general chemical principle for creating closure-stabilizing integrin inhibitors. Cell 185, 19, 3533-3550.e27 (2022).Abstract
    lin_2022_38059.pdf
    Integrins are validated drug targets with six approved therapeutics. However, small-molecule inhibitors to three integrins failed in late-stage clinical trials for chronic indications. Such unfavorable outcomes may in part be caused by partial agonism, i.e., the stabilization of the high-affinity, extended-open integrin conformation. Here, we show that the failed, small-molecule inhibitors of integrins αIIbβ3 and α4β1 stabilize the high-affinity conformation. Furthermore, we discovered a simple chemical feature present in multiple αIIbβ3 antagonists that stabilizes integrins in their bent-closed conformation. Closing inhibitors contain a polar nitrogen atom that stabilizes, via hydrogen bonds, a water molecule that intervenes between a serine residue and the metal in the metal-ion-dependent adhesion site (MIDAS). Expulsion of this water is a requisite for transition to the open conformation. This change in metal coordination is general to integrins, suggesting broad applicability of the drug-design principle to the integrin family, as validated with a distantly related integrin, α4β1.
    Dynamics of integrin α5β1, fibronectin, and their complex reveal sites of interaction and conformational change
    Su, Y., Iacob, R.E., Li, J., Engen, J.R. & Springer, T.A. Dynamics of integrin α5β1, fibronectin, and their complex reveal sites of interaction and conformational change. J Biol Chem 102323 (2022).Abstract
    su_2022_38109.pdf
    Integrin α5β1 mediates cell adhesion to the extracellular matrix (ECM) by binding fibronectin (Fn). Selectivity for Fn by α5β1 is achieved through recognition of an RGD motif in the 10th type-III Fn domain (Fn10) and the synergy site in the 9th type-III Fn domain (Fn9). However, details of the interaction dynamics are unknown. Here, we compared synergy-site and Fn-truncation mutations for their α5β1-binding affinities and stabilities. We also interrogated binding of the α5β1 ectodomain headpiece fragment to Fn using hydrogen deuterium exchange mass spectrometry (HDX MS) to probe binding sites and sites of integrin conformational change. Our results suggest the synergistic effect of Fn9 requires both specific residues and a folded domain. We found some residues considered important for synergy are required for stability. Additionally, we show decreases in fibronectin HDX are localized to a synergy peptide containing contacting residues in two β-strands, an intervening loop in Fn9, and the RGD-containing loop in Fn10, indicative of binding sites. We also identified binding sites in the α5-subunit β-propeller domain for the Fn9 synergy site and in the β1-subunit βI domain for Fn10 based on decreases in α5β1 HDX. Interestingly, the dominant effect of Fn binding was an increase in α5β1 deuterium exchange distributed over multiple sites that undergo changes in conformation or solvent accessibility and appear to be sites where energy is stored in the higher-energy, open-integrin conformation. Together, our results highlight regions important for α5β1 binding to Fn and dynamics associated with this interaction.
    Regulation by metal ions and the ADMIDAS of integrin α5β1 conformational states and intrinsic affinities
    Anderson, J.M., Li, J. & Springer, T.A. Regulation by metal ions and the ADMIDAS of integrin α5β1 conformational states and intrinsic affinities. Mol Biol Cell mbcE21110536 (2022).Abstract
    anderson_2022_37926.pdf
    Activation of integrins by Mn2+ is a benchmark in the integrin field, but how it works and whether it reproduces physiologic activation is unknown. We show that Mn2+ and high Mg2+ concentrations compete with Ca2+ at the ADMIDAS and shift the conformational equilibrium toward the open state, but the shift is far from complete. Additionally, replacement of Mg2+ by Mn2+ at the MIDAS increases the intrinsic affinities of both the high affinity open and low affinity closed states of integrins, in agreement with stronger binding of Mn2+ than Mg2+ to oxygen atoms. Mutation of the ADMIDAS increases the affinity of closed states and decreases the affinity of the open state and thus reduces the difference in affinity between the open and closed states. An important biological function of the ADMIDAS may be to stabilize integrins in highly discrete states, so that when integrins support cell adhesion and migration, their high and low affinity correspond to discrete on- and off-states, respectively.
    Low affinity integrin states have faster ligand binding kinetics than the high affinity state
    Li, J., Yan, J. & Springer, T. Low affinity integrin states have faster ligand binding kinetics than the high affinity state. eLife 10, e73359, (2021). Publisher's VersionAbstract
    li-2021-37916.pdf
    Integrin conformational ensembles contain two low-affinity states, bent-closed and extended-closed, and an active, high-affinity, extended-open state. It is widely thought that integrins must be activated before they bind ligand; however, one model holds that activation follows ligand binding. As ligand-binding kinetics are not only rate limiting for cell adhesion but also have important implications for the mechanism of activation, we measure them here for integrins α4β1 and α5β1 and show that the low-affinity states bind substantially faster than the high-affinity state. On and off-rates are similar for integrins on cell surfaces and as ectodomain fragments. Although the extended-open conformation's on-rate is ~20-fold slower, its off-rate is ~25,000-fold slower, resulting in a large affinity increase. The tighter ligand-binding pocket in the open state may slow its on-rate. Low affinity integrin states not only bind ligand more rapidly, but are also more populous on the cell surface than high affinity states. Thus, our results suggest that integrin binding to ligand may precede, rather than follow, activation by "inside-out signaling".
    W, X. & TA, S. Metal ion and ligand binding of integrin α5β1. Proc Natl Acad Sci USA 111, 50, 17863-8 (2014).Abstract
    17863full.pdf

    Integrin α5β1 binds to an Arg-Gly-Asp (RGD) motif in its ligand fibronectin. We report high-resolution crystal structures of a four-domain α5β1 headpiece fragment, alone or with RGD peptides soaked into crystals, and RGD peptide affinity measurements. The headpiece crystallizes in a closed conformation essentially identical to that seen previously for α5β1 complexed with a Fab that allosterically inhibits ligand binding by stabilizing the closed conformation. Soaking experiments show that binding of cyclic RGD peptide with 20-fold higher affinity than a linear RGD peptide induces conformational change in the β1-subunit βI domain to a state that is intermediate between closed (low affinity) and open (high affinity). In contrast, binding of a linear RGD peptide induces no shape shifting. However, linear peptide binding induces shape shifting when Ca(2+) is depleted during soaking. Ca(2+) bound to the adjacent to metal ion-dependent adhesion site (ADMIDAS), at the locus of shape shifting, moves and decreases in occupancy, correlating with an increase in affinity for RGD measured when Ca(2+) is depleted. The results directly demonstrate that Ca(2+)binding to the ADMIDAS stabilizes integrins in the low-affinity, closed conformation. Comparisons in affinity between four-domain and six-domain headpiece constructs suggest that flexible integrin leg domains contribute to conformational equilibria. High-resolution views of the hybrid domain interface with the plexin-semaphorin-integrin (PSI) domain in different orientations show a ball-and-socket joint with a hybrid domain Arg side chain that rocks in a PSI domain socket lined with carbonyl oxygens.

    Dong, X., Hudson, N.E., Lu, C. & Springer, T.A. Structural determinants of integrin β-subunit specificity for latent TGF-β. Nat Struct Mol Biol. 21, 12, 1091-6 (2014).Abstract
    Nat_Struc-2014.pdf

    Eight integrin α-β heterodimers recognize ligands with an Arg-Gly-Asp (RGD) motif. However, the structural mechanism by which integrins differentiate among extracellular proteins with RGD motifs is not understood. Here, crystal structures, mutations and peptide-affinity measurements show that αVβ6 binds with high affinity to a RGDLXXL/I motif within the prodomains of TGF-β1 and TGF-β3. The LXXL/I motif forms an amphipathic α-helix that binds in a hydrophobic pocket in the β6 subunit. Elucidation of the basis for ligand binding specificity by the integrin β subunit reveals contributions by three different βI-domain loops, which we designatespecificity-determining loops (SDLs) 1, 2 and 3. Variation in a pair of single key residues in SDL1 and SDL3 correlates with the variation of the entire β subunit in integrin evolution, thus suggesting a paradigmatic role in overall β-subunit function.