Ligand binding activities of β1 and β4 integrins

Soluble extracellular fragments of integrins each containing a releasable C-terminal clasp (Fig. 1A) were purified as described in the Materials and Methods. The α- and β-subunits were linked by a disulfide-bonded clasp and migrated as a single band in the SDS-PAGE under nonreducing conditions (TEV− lanes in Fig. 1B). By contrast, they were separated into two bands after TEV protease treatment (TEV+ lanes in Fig. 1B). Thus, it was confirmed that all purified integrin heterodimers were linked via a disulfide-bonded clasp at the C-terminal, which can be released (or ‘unclasped’) by TEV protease treatment. These integrins were subjected to the following experiments (ligand binding assay and structural analysis by electron microscopy). As reported previously (Takagi et al., 2002), binding of αVβ3 integrin to its ligand was negligible in the presence of 5 mM Ca²⁺ but was upregulated 4- to 5-fold over the background (i.e. BSA control) level in the presence of 1 mM Mn²⁺ in both clasped and unclasped conditions (Fig. 1C, far left panel), indicating that the 5 mM Ca²⁺ and the 1 mM Mn²⁺ conditions correspond to the low- and high-affinity states, respectively. We deliberately employed a non-physiologically high concentration of Ca²⁺ (5 mM) to push the equilibrium toward the low-affinity state by saturating the βI domain ADMIDAS with Ca²⁺, which has been reported to have negative regulatory function (Mould et al., 2003). The ADMIDAS-bound Ca²⁺ is believed to stabilize the low-affinity conformation of the ligand-engaging MIDAS metal through its preference toward pentagonal bipyramidal over octahedral coordination geometry (Xia and Springer, 2014). When another RGD-dependent integrin α5β1 was subjected to the same assay using immobilized fibronectin, the results were essentially the same, although the overall binding signal was much higher (Fig. 1C, second panel). We then examined the ligand binding ability of all other non-RGD integrins toward laminin-10 (for integrins α3β1, α6β1 and α6β4) or type I collagen (for α2β1) using the same assay conditions. As shown in Fig. 1C, they all showed similar ligand binding to αVβ3 integrin; binding was negligible in 5 mM Ca²⁺ but high ligand binding activities were observed in the presence of Mn²⁺. Importantly, the extent of ligand binding under Mn²⁺ conditions for each integrin was almost the same irrespective of the state of the C-terminal clasp (clasped or unclasped), except for α6β4 integrin. In the case of the α6β4 integrin, unclasping resulted in diminished binding to laminin-10, although there was still clear binding above background levels and above the level seen in Ca²⁺ conditions. From these results, we conclude that 5 mM Ca²⁺ and 1 mM Mn²⁺ can be used as representative conditions to induce low- and high-affinity states, respectively, for all integrin ectodomain fragments used here.

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Fig. 1.

Recombinant integrin ectodomain proteins. (A) Domain organization of the integrin ectodomain constructs used in this study. Each domain is color-coded and schematically drawn, together with the disulfide-bonded coiled-coil ‘clasp’ fused at the C-terminus. The approximate position of the hinge during the global bending motion is indicated by a horizontal dashed line. (B) SDS-PAGE analysis of recombinant integrins. Purified recombinant integrin samples were either unclasped by TEV protease (+) or left untreated (−) and subjected to nonreducing SDS-PAGE using 10% gel and stained with Coomassie Brilliant Blue. Note that all intact integrin heterodimers migrate as a single band of 200-250 kDa but show two bands corresponding to α (∼120-150 kDa) and β (80-100 kDa) subunits after the removal of disulfide-bonded C-terminal clasp by the TEV protease treatment. Difference in the apparent size of the β1 band of the α2β1 sample compared with other β1 integrins is consistent with the different glycosylation capacities of the cell line used for protein production. (C) Ligand binding activities of various integrins. Binding of integrins to their respective primary ligand (laminin-10 for α3β1, α6β1 and α6β4; fibronectin for α5β1 and αVβ3; type I collagen for α2β1) was evaluated by a solid-phase binding assay under three different conditions. Open bars represent intact integrin in the presence of 5 mM Ca²⁺; gray bars show intact integrin in the presence of 1 mM Mn²⁺; black bars show unclasped integrin in the presence of 1 mM Mn²⁺. Binding is expressed as the ratio of absorbance values obtained with the ligand-coated wells relative to that with BSA control wells, where ratio=1 (dashed lines) means that there is no specific binding to the ligand. Data represent mean±s.d. from three independent experiments.

EM imaging of β1 and β4 integrins

Having established the experimental conditions to maintain various integrins in the low- and high-affinity states, we turned to negative-stain EM imaging to visualize the conformation of each integrin under both conditions. To this end, integrin samples were loaded onto a gel filtration column equilibrated with a buffer containing 5 mM Ca²⁺ (low-affinity state) or 1 mM Mn²⁺ (high-affinity state) and the monodisperse peak fraction containing the heterodimeric integrin ectodomain was used to make EM grids. We also included one more condition, where 1 mM Mg²⁺ was added to 5 mM Ca²⁺ buffer. Data were collected for all six integrins under four different conditions (clasped/5 mM Ca²⁺, clasped/5 mM Ca²⁺+1 mM Mg²⁺, clasped/1 mM Mn²⁺ and unclasped/1 mM Mn²⁺). As can be seen in the representative raw EM image (Fig. S1), all samples showed well-dispersed individual particles that allowed efficient image analysis. From the EM images obtained, ∼1000 particles in each condition were boxed out, and they were averaged after classification into 20 classes. We used αVβ3 integrin as a reference, because this is the most extensively studied integrin by multiple groups using EM analysis. As expected, nearly all αVβ3 particles exhibited a highly bent conformation in 5 mM Ca²⁺ regardless of the additional presence of Mg²⁺. In the Mn²⁺ condition, a large majority changed their shape and showed a completely extended and open conformation consistent with the ‘switchblade model’ (Fig. S2A). The behavior of α5β1 integrin was quite different from that of αVβ3, however, because it rarely assumed the acutely bent conformation in the low-affinity condition but rather exhibited varying shapes, with partly to fully extended conformations being predominant (Fig. 2A and Fig. S2B). In fact, this result is essentially the same as that reported by Springer and colleagues (Su et al., 2016). The authors showed that only a fraction of EM class averages of ligand-unbound and clasped α5β1 ectodomain fragment appeared as the acutely bent form in a buffer containing 1 mM Ca²⁺ and 1 mM Mg²⁺. More importantly, the shape distribution of α5β1 particles did not change when the high-affinity condition (i.e. 1 mM Mn²⁺) was employed (Fig. 2B and Fig. S2B), suggesting the lack of a strong correlation between the affinity state and the global conformation in α5β1. Despite high variability of the particle shapes of α5β1 in the EM images, most 2D class averages showed clear and distinctive features that helped us to assign each chain or domain present in the construct (Fig. 1A) into the densities, enabling us to interpret the 3D structure (Fig. 2C) and to classify particles according to their global conformation (see below).

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Fig. 2.

Projection averages of negatively stained α5β1 integrin. All 20 2D class averages obtained from the EM images of clasped α5β1 integrin in the presence of 5 mM Ca²⁺ (A) or 1 mM Mn²⁺ (B). (C) Three particularly well-resolved class averages with different bending angles from A (marked with asterisks) are enlarged and shown alongside the best-matching structural models (color-coded as in Fig. 1A). Bars: 25 nm.

We next performed similar EM imaging on laminin- and collagen-binding integrins that had escaped structural scrutiny until now, namely α3β1, α6β1, α6β4 and α2β1. In general, the results were almost the same as that of α5β1 integrin (Fig. 3 and Fig. S2C-F). No severely bent conformation was observed for any of these integrins, even in the low-affinity state, and the majority of the class averages exhibited an overall extended conformation. Furthermore, the conformational distribution seen in the Mn²⁺-activated condition for the three laminin-binding integrins was indistinguishable from that in the Ca²⁺ or Ca²⁺/Mg²⁺ condition, regardless of the presence of the C-terminal clasp (Fig. 3 and Fig. S2C-E). In particular, α6β4 integrin almost always assumed the completely extended structure regardless of the condition (Fig. S2E), suggesting that it is mostly refractory to conformational regulation. In the case of α2β1 integrin, there is an additional I (A) domain in the α-subunit, which was clearly visible at the top of the molecule in the class-averaged images (Fig. S2F). Although it shared the same trend with laminin-binding integrins of having the overall extended conformation in both low- and high-affinity conditions, there was a clear local conformational change upon addition of Mn²⁺, corresponding to head opening (i.e. the swing-out of the hybrid domain of the β-subunit). As the αI domain functions as an internal ligand to the β-subunit (Sen et al., 2013), Mn²⁺-treated α2β1 would represent the ‘active and liganded’ state rather than the ‘active and non-liganded’ state, as in the case of all other integrins lacking domain I. Therefore, these results are highly consistent with the notion that the β-hybrid swing is coupled to ligand binding rather than the affinity state of integrin (Su et al., 2016).

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Fig. 3.

The conformational distribution spectra for various integrins under low-affinity (5 mM Ca²⁺) and high-affinity (1 mM Mn²⁺) conditions. The colored bar graphs represent percentage distribution of five conformational groups within the 2D class averages obtained for each dataset. The five groups include: (1) compact integrin with a bending angle <90° (acutely bent, blue); (2) integrin with a bending angle between 90° and 120° (half bent, green); (3) integrin with a bending angle over 120° (slightly bent, yellow); (4) integrin showing fully extended conformation with closed headpiece (extended closed, orange); and (5) integrin showing fully extended conformation with open headpiece (extended open, magenta). Representative 2D averages are shown below the bar graph. For the original full class average gallery and particle number information, see Fig. S2.

Conformational spectra of β1 and β4 integrins

The individual class average was categorized into five groups based on their shape (Fig. S2), and the prevalence of each group was calculated from the numerical values obtained during single-particle analysis. As none of the integrins showed a significant difference in the conformational distribution between Ca²⁺ and Ca²⁺/Mg²⁺ conditions, as well as between clasped/Mn²⁺ and unclasped/Mn²⁺ samples, we will focus on the comparison between the clasped/Ca²⁺ and clasped/Mn²⁺ conditions. The population prevalence determined as above can be regarded as the approximate ‘conformational distribution spectra’ for each integrin or condition (Fig. 3). It is evident from this analysis that all examined β1 and β4 integrins prefer the extended conformation (colored in yellow, orange or magenta) over bent conformation (blue and green) under the Ca²⁺-induced low-affinity condition, and rarely assumed the severely bent conformation (blue) that is strongly favored by the well-studied β2 and β3 integrins. Furthermore, there seems to be no or very limited coupling between the affinity state and the global conformation of these integrins, because nearly identical conformational distributions were obtained for the Ca²⁺ and Mn²⁺ conditions. We are thus inclined to think that the switchblade model is not applicable to most if not all β1 and β4 integrins, at least as an affinity regulation mechanism. However, data must be interpreted in a careful manner to avoid overgeneralization, as the current study used only the soluble integrin ectodomain fragments. In addition, as the direct structural analysis is only possible with isolated integrin preparations, it is difficult to know the true conformational spectrum on the cell surface. For example, Springer and colleagues estimate that more than 98% of the α5β1 molecule on K562 cells are in the bent form (Li et al., 2017). It is possible that certain mechanisms exist on the cell surface to stabilize a bent conformation, at least for α5β1. Nevertheless, our data indicate that the ectodomain portions of β1 and β4 integrins lack the intrinsic property of rearrangement upon affinity manipulation by divalent cations.

Unlike the integrins present on circulating blood cells, including αIIbβ3, αVβ3, αXβ2 and αLβ2, which have to bind ligands during transient encounters, laminin-binding integrins (α3β1, α6β1, α7β1 and α6β4) and collagen-binding integrins (α1β1, α2β1, α10β1 and α11β1) on stationary cells generally have ample time to establish firm adhesion due to continuous contact with the extracellular components, and may not need mechanisms of rapid affinity upregulation. Thus the rapid ‘switchblade’ activation, in a strict sense, may only be applicable to those integrins on blood cells that emerged at a relatively late evolutionary point after the acquisition of the vascular system. Although there is strong coupling between ligand binding and global and local conformation of a wide variety of integrins (Takagi et al., 2002; Nishida et al., 2006; Chen et al., 2010, 2011; Rocco et al., 2008; Xie et al., 2004; Su et al., 2016), we do not have direct evidence that such coupling is applicable to laminin-binding integrins. This is mainly due to the limited structural information available for the laminin-integrin interaction (Takizawa et al., 2017). We predict that further structural analysis as well as development of biochemical tools, such as high-affinity laminin mimetic ligands to probe the interaction between laminin and integrin, will be essential to increase our understanding about this fundamental and ancient cell-matrix interaction event.