ARTICLE

https://doi.org/10.1038/s41467-021-24184-8 OPEN Structural mechanism of laminin recognition by integrin

Takao Arimori 1, Naoyuki Miyazaki 1,2, Emiko Mihara 1, Mamoru Takizawa3, Yukimasa Taniguchi3, ✉ Carlos Cabañas 4,5,6, Kiyotoshi Sekiguchi3 & Junichi Takagi 1

Recognition of laminin by integrin receptors is central to the epithelial cell adhesion to basement membrane, but the structural background of this molecular interaction remained

1234567890():,; elusive. Here, we report the structures of the prototypic laminin receptor α6β1 integrin alone and in complex with three-chain laminin-511 fragment determined via crystallography and cryo-electron microscopy, respectively. The laminin-integrin interface is made up of several binding sites located on all five subunits, with the laminin γ1 chain C-terminal portion pro- viding focal interaction using two carboxylate anchor points to bridge metal-ion dependent adhesion site of integrin β1 subunit and Asn189 of integrin α6 subunit. Laminin α5 chain also contributes to the affinity and specificity by making electrostatic interactions with large surface on the β-propeller domain of α6, part of which comprises an alternatively spliced X1 region. The propeller sheet corresponding to this region shows unusually high mobility, suggesting its unique role in ligand capture.

1 Laboratory for Protein Synthesis and Expression, Institute for Protein Research, Osaka University, Suita, Osaka, Japan. 2 Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance, University of Tsukuba, Ibaraki, Japan. 3 Division of Matrixome Research and Application, Institute for Protein Research, Osaka University, Suita, Osaka, Japan. 4 Cell-cell Communication & Inflammation Unit, Centro de Biología Molecular Severo Ochoa (CSIC- UAM), Madrid, Spain. 5 Department of Immunology, Ophthalmology and Otorhinolaryngology (IOO), Faculty of Medicine, Universidad Complutense de ✉ Madrid, Madrid, Spain. 6 Instituto de Investigación Sanitaria Hospital 12 Octubre (i+12), Madrid, Spain. email: [email protected]

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ntegrins are one of the most important classes of receptors that organs6–8. Laminin-511 (LM511, α5, β1, and γ1 chain composi- Imediate adhesion of cells to extracellular matrix (ECM) com- tion) is one of the most abundant laminin isoforms present in ponents, which is the hallmark of multicellular organisms. adult epithelial tissues, but also involved in development and is Integrins exist as a heterodimeric molecule comprising two type I implicated in the selective differentiation of stem cells9,10. Intact transmembrane subunits α and β, yielding various α/β combina- laminin heterotrimer is a very large (>800 kDa) molecule with a tions with distinct ligand specificity1. For example, Caenorhabditis cross-shaped appearance, and its integrin binding site has been elegans has only one β subunit (pat-3), shared by two α subunits narrowed down to the C-terminal one-tenth of the molecule (pat-2 and ina-1)2, while humans have eight β and eighteen α called “E8” fragment11.We12 and others13 have succeeded in subunits to assemble more than twenty-four heterodimeric recombinantly producing E8 fragment of human LM511 and integrins, which can be divided into four classes (Fig. 1a). Phy- mouse LM111 (mLM111) and solved their crystal structures at logenetic analysis has suggested that pat-2 and ina-1 have evolved 1.80 and 2.13 Å resolutions, respectively. These structures into two distinct classes, the Arg-Gly-Asp (RGD)-binding class revealed the architecture of the E8 fragment, where the coiled-coil and the laminin-binding class, respectively3. Therefore, the domain that bundles the α/β/γ chains and the closely-arranged laminin-binding class represents one of the most ancient integrin three laminin globular (LG) domains (LG1-3) of the α chain form classes conserved throughout the metazoan evolution, playing a ladle-like shape. Although we have provided evidence that the fundamental roles in the cell attachment to the basement mem- C-terminal region of the LMγ1 chain (referred to as LMγ1-tail or brane during development4,5. simply γ1-tail hereafter) and the bottom face of the LG domains Laminins are heterotrimeric molecules consisting of α, β, and γ conspire to form integrin binding interface, we have been unable chains and are major component of basement membranes. There to show that LMγ1-tail peptides can directly bind to integrins as are five α chains (α1–5), three β chains (β1–3), and three γ chains many RGD-based peptides and mimetic compounds do, making (γ1–3) in mammals, which constitute at least 16 laminin isoforms it difficult to reach a consensus about the identity of the binding each showing some preferential expression in certain tissues/ site(s).

abLaminin receptors β4 α6β1 integrin genu TM α6 N β-propeller Thigh Calf-1 Calf-2 C α6

αIIb Leukocyte-specific α3 α7 αE* PSI

β7 receptors Hybrid I-EGF β3 TM α5 α4 αD* β1 NCβI β-tail β1 β5 αV β2 αX* α8 α9 β6 αM* RGD receptors αL* β8 α6β1 headpiece α1* α11* (velcro) α2* α10* 1618A-zip α6(1-618)-A-zip β-propeller Thigh

1445 Collagen receptors β1(1-445)-B-zip-PA βI PA Hybrid B-zip PSI c TS2/16

βI d β-propeller Y214 D211 G217 (10aa) (12aa) IIId Hybrid Q198 IIIc C442 Thigh IIIb Q61 P228 PSI IIIa

Fig. 1 Crystal structure of the α6β1 integrin headpiece in complex with TS2/16 Fv-clasp. a Integrin α/β pairs and classification. 18 α subunits (red circles, with the αI domain-containing ones indicated by asterisks) and 8 β subunits (blue circles) found in human forming 24 heterodimeric pairs are represented by the α-β connecting lines. Integrin subunits or heterodimers with at least partial ectodomain structures determined are denoted by thick circles. Each heterodimer falls into one of the four distinct classes. Modified from the Fig. 1 of ref. 1. b Domain organization of α6β1 integrin and design of the α6β1 headpiece construct used for the structural analysis. c Ribbon presentation of the overall structure. Integrin α6 and β1 subunits are colored in hot pink and sky blue, respectively, and TS2/16 VH-SARAH and TS2/16 VL-SARAH composing TS2/16 Fv-clasp are in green and cyan, respectively. The location of the PSI domain (not included in the model) is denoted by gray circle, with the boundary residues (Q61 and C442) labeled. d An expanded view of the blade III β-sheet of the α6 subunit-β-propeller. The region indicated by a rectangle in (c) containing the X1 region (yellow) is shown with an Fo-Fc electron density map corresponding to the outermost strand (IIId) contoured at 3.0 σ. The modeled heptapeptide 211DGPYEVG217 fitted in the map is also shown as stick models.

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Our understanding about the mechanism of ECM ligand format “Fv-clasp” described previously35. In the 2.89 Å resolution recognition by integrins has been advanced enormously by the structure, one α6β1-TS2/16 Fv-clasp complex was contained in structural analyses of numerous integrin heterodimers by X-ray an asymmetric unit (Supplementary Table 1). Four out of the crystallography as well as cryoelectron microscopy (cryo-EM). five domains contained in the headpiece construct were resolved, The prevailing concept is that the ligand provides carboxylate while the PSI domain of the β1 subunit showed poor electron moiety to make direct coordination bond with the metal ion density and omitted from the final model (Fig. 1c). The β1chain present in a site called Metal-Ion-Dependent Adhesion Site structure was essentially the same as that in the published (MIDAS) conserved in all β subunits14–17. This in turn changes α5β1 structures14,36, and the resolved α6domains(β-propeller and the coordination geometry of MIDAS and results in a large thigh) were highly similar to α5andotherα subunits. The α6 β- downward shift of the α7 helix in the βI domain, leading to the propeller domain assumes canonical 7-bladed propeller fold with >60° swing-out of the β hybrid domain18. This local conforma- the seven four-strand anti-parallel β sheets forming the domain tional change of integrin β facilitates global shape-shifting or core (Supplementary Fig. 1a). However, the two loops preceding “extension” of integrin heterodimer, and eventually transform the the outermost strand in the blade III (strand IIId) were disordered. integrin into the force-transmission and signaling device that links In the sequence spanning K199 to V227, only β-strand IIId, ECM ligands and the cytoskeleton19,20. The global conformational 211DGPYEVG217, showed traceable electron density (Fig. 1d). change has also been observed with non-RGD integrins such as Density for the large Y214 residue allowed us to confidently assign α4β7, αXβ2, and αLβ2 using low-resolution negative-stain electron – the sequence to the structure register of this segment. No other microscopy (EM)21 23. Furthermore, the ligand-induced local published integrin β-propeller domains show such long disordered conformational change at the MIDAS leading to the hybrid loops. Furthermore, this region coincides with the alternatively domain swing-out (or head-opening) has been visualized for many spliced “X1 region” (residues I193-L235) found only in laminin- RGD-integrins at atomic resolution16,18,24. In contrast to those binding α subunits37, suggesting its possible role in ligand capture integrins, however, no structural information about the laminin- and/or specificity. This point will be discussed later in more detail. binding integrins is available to date (Fig. 1a, structurally deter- The TS2/16 used to facilitate crystallization is an activating mined integrins are circled with thick lines), except for the antibody that increases ligand binding affinity of all β1 integrins. low-resolution negative-stain EM images12,25. Therefore, structure Its binding epitope had been mapped to N207/K208/V211 located determination of laminin-binding integrin class before and after in the lower half of the α2 helix of the βI domain38, which is the binding of laminin is immensely needed to gain mechanistic confirmed by the current structure (Fig. 2a). In the crystal, the understanding of this interaction fundamental to multicellular TS2/16-bound α6β1 adopts a closed conformation with the β1 organisms. hybrid domain tucked inside39. Furthermore, the structure of the We aimed at solving the 3D structure of laminin-integrin TS2/16-bound βI domain was indistinguishable from that in α5β1 complex. Among the four laminin-binding integrin heterodimers, integrin structure crystallized without TS2/16 (PDB: 4wjk), we focused on α6β1 because it is known to exhibit the highest showing overall RMSD of 0.37 Å for 216 Cα atoms when binding affinity toward LM51126. The α6 subunit is widely dis- superposed (Fig. 2a). This clearly indicates that TS2/16 binding tributed in various tissues, but when both β1 and β4 are present, does not automatically induce global or local conformational α6 is preferentially paired with β427. α6β1 integrin is expressed on changes of β1 subunit, consistent with the previous negative-stain cells of hematopoietic origin, as well as some neural cells. Inter- EM results showing that it can bind to α5β1 integrin regardless of estingly, it is the major integrin isoform expressed on the surface its conformational state39. of human stem cells, and adhesion to ECM via integrin α6 plays All integrin β1 subunits contain three metal-binding sites important roles in proliferation, migration, differentiation, and – called Synergistic Metal ion Binding Site (SyMBS), MIDAS, and self-renewal of stem cells28 31. In fact, the E8 fragment from ADjacent to MIDAS (ADMIDAS), each known to bind Ca2+, LM511 is currently used as an important substrate for the culture Mg2+, and Ca2+ under physiological buffer conditions. However, of embryonic stem cells and induced pluripotent stem cells32.As we could not see clear electron density for ADMIDAS metal in α6 integrins are considered as useful markers for cancer stem cells our structure, while electron densities were clearly visible for and also fundamentally involved in the pathogenesis and reg- metals in SyMBS and MIDAS using a simulated-annealing omit ulation of cancer33,34, elucidation of their binding mechanism map (Fig. 2b). Comparison of the current structure with the toward LM511 is of utmost medical importance. Ca2+-containing ADMIDAS structure of α5β1 (4wjk) indicates Here, we report the crystal structure of the laminin receptor its incompatibility with Ca2+ coordination at this site due to a integrin α6β1 as well as the cryo-EM structure of the α6β1- sidechain flip of the D137 and a ~1.8 Å shift of the A342 carbonyl LM511 complex. We succeed in visualizing the direct coordina- (Fig. 2c), making us conclude that the ADMIDAS is unoccupied tion bond between the E1607 in the LMγ1-tail and the MIDAS in the current α6β1 structure. Although ~0.5 mM Ca2+ was metal in β1 integrin, as well as many more amino acid residues present in the crystal, which had been directly flash frozen before involved in the interaction, suggesting that the ability of integrin the diffraction experiments, the presence of Ca2+-chelating to recognize basement membrane laminin is mediated by a phosphate ions in the crystallization buffer may have lowered cluster of small and potentially weak interactions that come from its effective concentration, causing the loss of ADMIDAS Ca2+. residues distributed across all five subunits. In addition, we find a unique feature of a loop segment in α6 β-propeller that may play important role in ligand capture. Structure determination of the α6β1-LM511-TS2/16-HUTS-4 quaternary complex by single-particle cryo-EM. We next determined the structure of α6β1 headpiece in complex with its Results ligand LM511 by a cryo-EM approach. As for the LM511, we Crystal structure of the integrin α6β1 headpiece in complex utilized the E8-like truncated LM511 (tLM511) design reported with TS2/16 Fv-clasp. To gain structural insights into the laminin- previously12. In order to maximize the stability of the complex as integrin interaction, we first determined the crystal structure of well as its conformational homogeneity, we utilized two anti- ligand binding fragment of human α6β1(α6β1 headpiece, Fig. 1b). bodies in the Fv-clasp format; the affinity-enhancing TS2/16 and Crystallization was facilitated by complex formation with an anti- the conformation-stabilizing HUTS-4. The latter was included β1 antibody TS2/16, in a hyper-crystallizable antibody fragment because it is known to bind inside the β1 hybrid domain in the

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a b (SyMBS) (MIDAS) (ADMIDAS) α2

α1 N224 D130 Q169 S132

V211 D226 D137 D138 E229 S134

K208 S227 D259

N207 P228 A342 TS2/16 βI domain c NAG(α6β1)

N343(α6β1) D137(α6β1)

A342(α6β1)

D137(α5β1) A342(α5β1)

Ca2+: ADMIDAS (α5β1)

Fig. 2 Structure of the βI domain. a Structural comparison of β1 integrins before and after the TS2/16 binding. The β1 subunit portions from the TS2/16- bound α6β1 headpiece (sky blue) and the unbound α5β1 headpiece (PDB ID:4wjk, orange) are superposed at the βI domain and shown as Cα-tracing. TS2/ 16 Fv-clasp bound to α6β1 is shown as a translucent ribbon diagram. The α2 helix of βI domain harboring TS2/16 epitope residues (N207/K208/V211) and the adjacent α1 helix are labeled. b Metal ion coordination sites in the α6β1 headpiece structure. Simulated-annealing Fo-Fc omit map for metal ions contoured at 2.5 σ is shown in magenta. Ca2+ and Mg2+ ions assigned in the SyMBS and the MIDAS are shown as green and yellow spheres, respectively.

No electron density was observed at the ADMIDAS (dotted circle). c 2Fo-Fc electron density map contoured at 1.2 σ around the ADMIDAS region in the crystal structure of the α6β1-TS2/16 Fv-clasp complex (sky blue), superposed onto the α5β1 headpiece structure (orange) as in a.Ca2+ ion bound to the α5β1 headpiece is shown as an orange sphere. open conformation and expected to prevent closure by acting like condition (i.e., in the presence of 1 mM Mn2+)15,18. Despite the a wedge40. A quaternary complex comprising α6β1 headpiece, lack of ADMIDAS metal in the crystal structure of the ligand- tLM511, TS2/16 Fv-clasp, and HUTS-4 Fv-clasp (a total mass of unbound state (Fig. 2b), we assume that it is occupied in this ~290 kDa) was purified by SEC (Supplementary Notes and Sup- complex structure based on the buffer condition (i.e., inclusion of plementary Fig. 2), and cryo-EM images were collected using 1mM Mn2+ and absence of chelating species) as well as the Titan Krios equipped with a Falcon 3EC direct electron detector. volume of the map at the corresponding region (Supplementary Star-shaped particles representing the quaternary complex were Fig. 4a). After multiple rounds of refinements, a final atomic visible in both raw images and after 2D averaging (Supplementary model of the quaternary complex composed of α6β1 headpiece, Fig. 3a, b). From a total of >2 million particle images, a single- tLM511, TS2/16 Fv-clasp, and HUTS-4 Fv-clasp (simply called particle analysis derived a cryo-EM density map at 3.9 Å reso- α6β1-LM511 complex hereafter) was built (Fig. 3b, Supplemental lution (Fig. 3a and Supplementary Fig. 3). As the first step toward Movie 1). the reliable atomic model building, we fitted the crystal structures Overall, the LM511 and the α6 subunit parts showed no of each unit contained in the complex individually to the map. significant structural differences from their solitary crystal The structural units used for the fitting included followings; α6-β structures, indicating the lack of conformational adjustments propeller (this work), α6-Thigh (this work), β1-βI (this work), β1- upon the interaction (Fig. 4a, c). In contrast, the β1subunit Hybrid (PDB: 4wk0), β1-PSI (PDB: 4wk0), LM511-coiled-coil underwent both global and local conformational changes. First, (PDB: 5xau), LMα5-LG1 (PDB: 5xau), LMα5-LG2 (PDB: 5xau), the hybrid domain of the β1 subunit is swung out, and α6β1 LMα5-LG3 (PDB: 5xau), TS2/16 Fv-clasp (PDB: 5xcx), and adopts an open-head conformation similar to that observed in HUTS-4 Fv-clasp (this work, Supplementary Fig. 1b). Models of other ligand-bound β subunits (Fig. 4a)19,41–44. In addition, the the N-terminal half of the coiled-coil domain of tLM511 and a α1 helix in the βI domain moved ~3 Å toward the MIDAS, and long helix located at the bottom of HUTS-4 Fv-clasp were the α7 helix shifted downward to allow the swing-out of the omitted from the model due to the low quality of the corre- hybrid domain (Fig. 4a, expanded panel). The down-shift of the sponding cryo-EM map. All the crystal structures could be fitted α7 helix was accompanied by a large positional change of an N- very well in the map except for the βI domain of the integrin acetyl glucosamine linked to N343 at the beginning of the α7, β1 subunit, where apparent discrepancies between the crystal which was evident in the EM map (Supplementary Fig. 4b). structure and the map were observed for the α1 and α7 helices, The above configurations of the α1andα7 helices as well as the forcing us to manually build the model. The positions of the swung-out hybrid domain seen in the α6β1-LM511 complex are cations and the conformation of the coordinating residues were very similar to those in the “open-head” conformation of ligand- modeled using the crystal structure of the αIIbβ3 in open con- bound αIIbβ3 (Supplementary Fig. 5a), indicating that the same formation (PDB: 2vdo) as a guide. We assigned three Mn2+ ions ligand-induced conformational change mechanism found in in the SyMBS, MIDAS, and ADMIDAS, following the previously many RGD-binding integrins also applies to laminin-binding reported integrin crystal structures determined under a similar integrins.

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a LM511

TS2/16 90º α6 (integrin)

β1 (integrin)

HUTS-4

b β1 LM γ1

LM α5 βI 90º β-propeller

Hybrid Thigh PSI

Fig. 3 Cryo-EM structure of a quaternary complex of the α6β1 headpiece, tLM511, TS2/16 Fv-clasp, and HUTS-4 Fv-clasp. a Cryo-EM density map. The map is colored as follows; integrin α6 subunit in pale pink, integrin β1 subunit in light blue, LM511 in pale green, TS2/16 Fv-clasp in pale yellow, and HUTS-4 Fv-clasp in pale orange. The map is drawn at the contour level of 0.05 in Chimera. b Ribbon diagram of the atomic model of the quaternary complex. Integrin and TS2/16 are shown in the same color scheme as Fig. 1c. Laminin α5, β1, and γ1 chains are shown in light green, blue, and yellow, respectively.

VH-SARAH and VL-SARAH of HUTS-4 Fv-clasp are shown in orange and purple, respectively.

Integrin-antibody interfaces. HUTS-4 is a conformation- using fluorescent TS2/16 monomer. In fact, the affinity of TS2/16 reporting antibody against β1 that preferentially binds to in the presence of Mn2+ and RGD was ~70% higher than that ligand-occupied β1 integrins. Consistent with previous epitope in the resting condition (Supplementary Fig. 5d), confirming mapping result, HUTS-4 recognized species-specific residues the theoretical prediction. More precise understanding of the E371 and K417 located at the inner face of the lower region of the mechanism of the allosteric integrin activation by TS2/16 should β1 hybrid domain, explaining why it cannot bind to closed β1 await further structural and functional analyses. integrin with tucked hybrid domain (Fig. 4b and Supplementary Fig. 5b)45. Unexpectedly, HUTS-4 also lightly touches the PSI domain, which may have contributed to the resolution of this Recognition of laminin β1 and γ1 chains by α6β1 integrin. The domain in the cryo-EM map by fixing the PSI-hybrid inter- cryo-EM structure of the quaternary complex enabled the detailed domain angle. analysis of the laminin-integrin interface. tLM511 bound to the In the laminin-bound α6β1 cryo-EM structure, TS2/16 bound top of the α6β1 headpiece using its bottom portion where coiled- to βI domain using the same interface seen in the crystal structure coil of three chains converge near the C-terminus of the LMβ1 without laminin. To our surprise, superposition of ligand chain, burying 1372.7 Å2 and 1330.6 Å2 of solvent accessible unbound structure (X-ray) with the ligand bound form (EM) at surfaces of LM511 and α6β1 integrin, respectively (Fig. 5a). This the Fv portion of TS2/16 revealed that there was virtually no large interface is supported by numerous contacts on both sides difference between them on either antibody or integrin side (Fig. 5a). We have previously shown, via mutational studies, that (Supplementary Fig. 5c), indicating that the TS2/16 binding does a conserved acidic residue located in the C-terminal region of the not cause conformational changes in the βI domain, at least with laminin γ chain plays a central role in the integrin binding by an extent noticeable at the resolution of the current cryo-EM directly coordinating to the MIDAS cation12,46, although we have structure (3.9 Å). As TS2/16 is an activating antibody, theoretical been unable to observe direct binding between LMγ1-tail peptides consideration of binding energetics tells us that it must bind with and integrins. In the cryo-EM map of the α6β1-LM511 complex, higher affinity to the ligand-bound (open) integrin than the density extending from P1604 (the last LMγ1 residue that was unbound (closed) integrin. Therefore, we compared the binding visible in the LM511 crystal structure12) to penetrate into the affinity of TS2/16 toward ligand unbound and ligand-bound α5β1 integrin β1 subunit was observed (Fig. 5b), allowing us to suc- integrins on K562 cell surface, in a FACS-based binding assay cessfully construct a model of the LMγ1-tail all the way to the C-

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a b N343-NAG

βI ADMIDAS MIDAS α2 α1

Hybrid α7 Hybrid

HUTS-4 c

Coiled-coil domain LG3

90º LG2 LG1

Fig. 4 Structural changes upon ligand binding. a Comparison of α6β1 headpiece conformations before and after ligand binding. The crystal structure of unliganded α6β1 headpiece (gray) was superposed at the βI domain onto the cryo-EM structure of the LM511 complex (color-coded as in Fig. 3b). An expanded view near the βI MIDAS (boxed) is shown in the right. The global conformational rearrangement (i.e., a swing-out of the β1 hybrid domain) and the local conformational rearrangement (i.e., shifts in α1 and α7 helices) are denoted by red and orange arrows, respectively. The N-acetylglucosamine (NAG) residue attached to N343 is shown in stick model. b. HUTS-4 cannot bind to the tucked hybrid domain in the closed headpiece conformation. The hybrid domain and HUTS-4 in the cryo-EM structure (color-coded as in Fig. 3b) was superposed at the hybrid domain onto the crystal structure of closed α6β1 headpiece (gray). c LM511 does not undergo conformational change. The crystal structure of tLM511 (PDB ID: 5xau, gray) was superposed onto the cryo-EM structure (color-coded as in Fig. 3b) at the LG1-3 domains. terminal P1609. Thus our structure shows that integrin binding electrostatic interactions. To verify the specificity of these interac- causes γ1E1607 to become ordered, which is well positioned in tions, we conducted structure-guided mutagenesis analysis. The the laminin sequence to form a direct coordination to the MIDAS soluble full-length ectodomain of α6β1 in which two subunits metal (Fig. 5b). In addition to the γ1E1607-MIDAS engagement, are tied by a coiled-coil motif called velcro (α6β1ec-velcro) and the we also found a potential hydrogen bonding interaction between N-terminally PA-tagged tLM511 (PA-tLM511) were used for the carboxyl group of the C-terminal P1609 of LMγ1 and the side immunoprecipitations (Fig. 6a). First, charge reversal mutations chain of N189 of integrin α6, which is strictly conserved among were introduced in the II-III loop residues to make a series of all laminin-binding integrin α chains (Fig. 5b, e). Thus, the γ1 tail integrin α6 mutants, D153K, R155D, R157D, and K161D, and their region seems to use two anchor points (E1607 and P1609) to binding toward the wild-type PA-tLM511 was evaluated. When bridge β1 and α6, in a way analogous to the subunit-bridging “R” mixtures of integrin- and laminin-expressing conditioned media and “D” portions of RGD peptide ligands14–17. Furthermore, we were immunoprecipitated from the laminin side using anti-PA tag noted that the C-terminal residue of LMβ1, L1786, is accom- antibody (NZ-1)-immobilized Sepharose, only the wild-type and modated in a small dead-end space formed by integrin α6, the K161D mutant integrins were co-precipitated (Fig. 6b, upper integrin β1, and LMγ1, likely contributing to the binding (Sup- panel). When pulled-down from the integrin side using anti-velcro plementary Fig. 6a). Since the position of this LMβ1 terminal antibody (2H11)-immobilized Sepharose, binding of tLM511 again residue relative to the γ1 chain is fixed by the γ1Cys1600- was seen only with wild-type and the K161D mutant integrins, β1Cys1785 disulfide bond conserved across all the integrin- although all mutant integrins were expressed at comparable levels binding laminin trimers, this single-residue contact by LMβ chain (Fig. 6b, lower panel). These results suggest that D153, R155, and may also constitute an important determinant for binding R157 in the II-III loop all contribute critically to the LM511 specificity. binding, while K161 contribution is marginal. We then performed a reciprocal experiment by introducing mutations in LMα5chain α β Recognition of laminin α5 chain by α6β1 integrin. Compared to to see their effect on the binding to the wild-type 6 1. As shown the focal mode of integrin binding by β1andγ1 chains using their in Fig. 6c, mutants K3099D and D3102R showed severely C-terminal ends, LMα5 uses wider surface at the bottom of LG2 decreased binding activity compared to wild-type LM511, while domain, which is recognized exclusively by integrin α6 subunit that of D2942R was only slightly diminished. (Fig. 5a). At the heart of this interface, a cluster of charged residues The same experimental setup was used to investigate the α from the loop connecting blade II and III of α6 (referred to as II-III potential contribution of the N189 of integrin 6 in the γ loop hereafter) point toward laminin, each of which is accom- recognition of the C-terminal carboxylate of LM 1 suggested in α β panied by residues with complementary charges on the laminin the previous section. Thus, N189 in the context of 6 1ec-velcro side (Fig. 5a, c), suggesting a formation of inter-molecular was mutated to Asp residue, expecting that the negative charge

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LG1 a LG2 R3045(α5) P1609(γ1) K3092(α5)

R3042(α5) D2942(α5) E1607(γ1)

LM511 D3102(α5) K3099(α5)

D153

R157 SyMBS R155 MIDAS

E215 K161 α6β1 integrin N189 D211 ADMIDAS

b cd

LG2 LG2 P1604 D2942 LMγ1 tail K3092 R3042 R3045 D3102 D211 N189 E1607 R155 K161 E215 β1 K3099 R157 G217 D153 Q198 ADMIDAS P228 α6 P1609 MIDAS SyMBS II-III loop X1 region

Fig. 5 Interaction between α6β1 integrin and LM511. a Open book view of the binding interface. (Left) Surface representations. Atoms in contact with the partner molecule (within 4 Å) are shown in orange. (Right) Cartoon representations. The key residues in this paper are shown as stick models and labeled. The surface and the cartoon figures are viewed from the same orientation. b Cryo-EM map near the laminin γ1-tail, drawn at the contour level of 0.05 in Chimera. The atomic model of the γ1-tail region (T1603-P1609) is shown in yellow stick model. Note that E1607 (red stick model) in the γ1-tail is in a suitable position to coordinate MIDAS cation and that the terminal carboxyl group of P1609 is pointing toward N189 of integrin α6 subunit. Close-up views for the contacts between the laminin LG2 domain and the II-III loop region c or the X1 region d of the α6 β-propeller domain. e Multiple sequence alignment of integrin α subunits for blade III and its flanking loop regions. Positions for the four β-strands constituting the blade III (strands IIIa-IIId) are denoted by blue arrows above the alignment, and the actual β-strand segments in each structurally determined α subunit are underlined. The Asn189 conserved in all laminin-binding integrins and the segments that are truncated in α6 mutants used in Fig. 6f are boxed in red and magenta, respectively. The α6 residues mutated for the functional study shown in Fig. 6 are labeled on top of the alignment, color-coded according to their fold reduction in the laminin affinity when mutated to Ala by red (>10-fold), orange (5–10 fold), and green (<5-fold). See also Fig. 6g. may repel the γ1 C-terminal carboxylate without altering the mobile LMγ1 terminus to help LMγ1-E1607 to coordinate the geometrical shape of the α6 surface. As shown in Fig. 6b, lane 7, MIDAS cation. the N189D mutant completely lost the binding ability toward As the pull-down assays described above are qualitative and LM511. We have previously reported that shortening of the C- have a very limited dynamic range, we next sought to establish a terminal end of LMγ1 by one or two residues led to a decreased more quantitative assay to evaluate the contribution of each α6 affinity to α6β146. It is thus likely that the interaction between α6 residue to the overall binding, using more conservative Ala N189 and the terminal carboxyl group of LMγ1 is required for mutations. To this end, we inserted superfolder green fluorescent optimum binding, probably by capturing the otherwise highly protein (sfGFP) between the PA tag and the truncated laminin

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ab c integrin α6 laminin α5 PA tag α-PA (NZ-1) mutants mutants

PA-tLM511E8 Wild-type D2942R K3099D D153K R155D R157D K161D N189D mock mock D3102R Wild-type

250k I 250k I IP IP 150k 150k NZ-1 NZ-1 100k L 100k L α6β1ec-velcro

250k 250k IP I IP I A-zip B-zip 150k 150k 2H11 2H11 velcro 100k L 100k L 1234567 12345 α-velcro (2H11) d Control g WT

F D153A Δ R155A

Mean R157A N189A (arbitrary units/cell) N189D

sfGFP-tLM511 (nM) e Control WT D205A

F E210A Δ D211A

Mean E215A E219A (arbitrary units/cell) E221A

f sfGFP-tLM511 (nM)

Control WT F

Δ X1-ΔN X1-ΔC

Mean X1-ΔNC (arbitrary units/cell)

sfGFP-tLM511 (nM)

Fig. 6 Mutagenesis of the interface residues. a Schematics of the α6β1 integrin ectodomain (α6β1ec-velcro) and tLM511 (PA-tLM511) constructs used for the pull-down binding assay. Binding of integrin mutants to wild-type laminin b and laminin mutants to wild-type integrin c. Each construct was transfected into Expi293F cells, and culture media of each pair of integrin/laminin were mixed and subjected to immunoprecipitation using anti-PA tag antibody (NZ-1, upper panel) or anti-velcro tag antibody (2H11, lower panel), followed by SDS-PAGE analysis under non-reducing condition. Band positions of α6β1ec- velcro and PA-tLM511 are indicated by I and L, respectively. Immunoprecipitation experiments were repeated twice. Binding analysis of sfGFP-fused tLM511 to the integrin mutants expressed on the cell surface. Expi293F cells transiently expressing α6β1 integrins with the II-III loop mutations d, X1-region mutations e, and loop truncation mutations f were incubated with increasing concentrations of sfGFP-tLM511 to measure its binding as described in the “Method” section. Each panel shows data from a representative experiment out of three independent ones. g Laminin binding affinity of mutant α6β1 integrins. Equilibrium dissociation constants (KD) derived from the binding isotherms like the ones shown in d–f by non-linear regression analysis are shown. When the binding was below the level of control non-transfected cells (black circles in d–f), that mutant integrin is judged as no binding. Data are mean ± SD of three independent experiments. Relative impact of each mutation on the binding is expressed as the fold-increase in the KD value from the wild-type. Uncropped gel images and source data are provided in the Source Data file.

α5 subunit in the construct described above, and the binding of the integrated fluorescence values were plotted against the ligand resultant sfGFP-tLM511 to cells transiently expressing human concentration to derive equilibrium KD values (Fig. 6d, g). As the α6β1 was evaluated by FACS. Concentration-dependent shift parent Expi293 cells express endogenous α6 subunit at low level in the fluorescence histogram was observed in the presence of (Supplementary Fig. 7b), binding of sfGFP-tLM511 to Expi293 0.5 mM Mn2+ (Supplementary Fig. 7a), and the net increase of (black lines in Fig. 6d–f) was regarded as a baseline. This

8 NATURE COMMUNICATIONS | (2021) 12:4012 | https://doi.org/10.1038/s41467-021-24184-8 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24184-8 ARTICLE experiment revealed that binding to R155A mutant (purple line to multiple reasons including the lack of convenient ligand- in Fig. 6d) was below the level of control, indicating that this mimetic compounds like RGD, difficulty in producing well- residue was indeed indispensable for the laminin binding activity. behaved and biologically active laminin fragment, and intrinsic In contrast, the D153A and R157A mutants retained reasonable flexibility of integrin ectodomain. We have applied several unique binding ability with 3.6- and 2.3-fold increased KD values, methodologies to overcome these hurdles and solved the structure respectively (Fig. 6g), suggesting that their contribution to the of the integrin α6β1 headpiece in complex with the E8 fragment of binding were partial. We also extended this assay format to LM511 by cryo-EM, which is rapidly growing as a critical method evaluate the role of N189 by measuring the ligand affinity of the for integrin structural determination47,48. The complex exhibits charge-manipulated N189D as well as more conservative N189A so-called open head conformation with the β1 hybrid domain mutants, and found that both mutants completely lost the wide open, in sharp contrast to the tucked hybrid domain found binding ability, confirming the importance of the N189(α6)- in the crystal structure of the same integrin in the absence of E1607(LMγ1) hydrogen bond. laminin. We have observed that full-length α6β1 integrin assumes primarily extended overall conformation before the ligand binding and postulated that the linkage between the large global con- Potential role of the X1 region of α6 in the laminin binding.In formational change and the ligand affinity upregulation found addition to the II-III loop residues described above, the alter- in many integrins may not be applicable to laminin-binding natively spliced X1 region of α6 is also buried upon the binding of integrins25. However, the current structure confirmed that the laminin LG2 (Fig. 5a, d). This region was highly mobile in the conformational rearrangement (head opening) is tightly linked to absence of laminin as described earlier (Fig. 1d), but upon the ligand binding in α6β1 integrin as well, indicating that it is a the LM511 docking the cryo-EM map showed visible density for general feature shared among major integrin classes, except for the the two segments flanking the IIId strand (Supplementary Fig. 4c) αvβ8 integrin that is known to maintain closed conformation after in contrast to the total lack of electron densities in the crystal the binding of protein ligand48,49. structure. Guided by the EM map, we modeled these loop seg- The structure of the complex provided the final proof for the ments as shown in Supplementary Fig. 4c (cyan), although they much-debated central role of the MIDAS metal in engaging the were omitted from the final model deposited in the PDB or from LMγ1 E1607 carboxylate12. In addition to this key laminin- the Fig. 5 because the map was not clear enough at the side chain integrin interaction, the structure unraveled several new interface level to allow confident residue assignment. When the electro- features that had not been appreciated before. First, the LMγ1-tail static potential of the top face of the integrin α6 was calculated provides two anchor points (the MIDAS-coordinating E1607 and after incorporating the putative loops, it showed highly negative the α6-abutting P1609) to bridge two integrin subunits, much like surface near the X1 region (Supplementary Fig. 6b, bottom; the α-β bridging RGD peptide ligand. In many published struc- modeled regions were bounded by dotted lines). On the laminin tures of RGD-binding integrins in complex with RGD-like side, a cluster of basic residues including R3042, R3045, and ligands, the “D” in the ligand invariably serves as the primary K3092 reach out toward the X1 region, making highly electro- anchor point to engage MIDAS-bound cation, while the “R” or positive surface (Supplementary Fig. 6b, top). Thus, the con- equivalent basic residue at the -2 position is often bound by tribution of the X1 region to the interface is evident, although specific residue(s) in the α subunit (e.g., α5-Q221/D227, αV- precise residue-wise contacts could not be visualized in the 3.9 Å D218, and αIIb-D224, denoted by red in Fig. 5e), although there cryo-EM structure. In order to investigate the role of the X1 are few exceptions to this rule50.Inα6β1-LM511 complex, the region in more detail, we systematically mutated acidic residues primary anchor point is the MIDAS-coordinating E1607 in the in the X1 region and evaluated their ability to bind laminin using LMγ1-tail, and the second anchor is formed between the terminal the flow cytometry quantitative assay. Although substituting the carboxylate of P1609 at +2 position and the conserved N189 of acidic residues with Ala generally lowered the affinity toward α6. Interestingly, we note that RGD-binding α subunits all have sfGFP-tLM511, the effect was partial for D205A, E210A, and Tyr/Phe at the position equivalent to α6 N189 (Fig. 5e), and they E221A mutants, while D211A, E215A, and E219A mutants invariably contribute to the ligand binding by contacting with the completely lost the activity (Fig. 6e, g). There was a trend that the aliphatic portion of the Arg. Also, the corresponding Tyr187 impact of the Ala mutation was severer when the residue was (Fig. 5e) in α4 plays important role in the recognition of α4- located closer to the IIId strand (Figs. 5e and 6g). Of note, specific compound RO0505376 by making ring stacking with the mutation of E215 in the middle of the IIId strand abolished the third aromatic moiety of the compound21. In contrast, the cor- binding, although this residue was not even in contact with responding residue in I-domain integrins are usually small amino laminin in the complex structure (Fig. 5d). We then removed acids (Fig. 5e), which may be explained by the lack of the second mobile segments flanking the IIId strand that do not bear func- anchor point in their ligand, the internal peptide connecting the tionally essential residues from either side (X1-ΔN or X1-ΔC) or I-domain to the β-propeller51. Thus, the subunit-bridging, two- both (X1-ΔNC), in expectation that the modifications may reduce point anchoring mode seems to be conserved among ligands of the flexibility of the X1 region and increase the binding by sta- non-I domain integrins, although the direction of the tripeptide bilizing the binding-compatible conformation of the IIId strand. portion is reversed in LM compared to the RGD ligands. As the truncation was designed so as to keep the length of the Another important interface concerns the blade II-III loop in loops the same as in the α3 subunit (Fig. 5e), these rather long the integrin α subunits’ β-propeller domain. In the complex, the (7–8 residues) truncations did not affect the expression level of bottom face of the LMα5 LG2 domain docks onto this loop with a the resultant mutant α6 (Supplementary Fig. 7b). When subjected high electrostatic complementarity. The conformation of this to the tLM511 binding assay, however, none of these truncation loop in the apo form is nearly identical to that of the complex, mutants supported meaningful laminin binding (Fig. 6f), indi- suggesting its significant contribution to the binding energy cating that the mobile X1 region in its entirety is important for by presenting a preformed LM docking platform at the top of the overall binding activity. the α6 subunit. On the laminin side, a set of charged residues at the edge of the LG2 domain are responsible for accepting these Discussion residues (Fig. 5c). Interestingly, these residues are well conserved For many years, it has been difficult to obtain structural infor- in LMα1 chain in LM111 (Supplementary Fig. 8), which is a good mation about laminin-integrin interactions at atomic detail, owing adhesion substrate for α6β126. Therefore, we predict that the

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II-III loop plays similar role in recognizing LM111. We also note region will bring the bottom face of the LG1-3 into close proxi- that the importance of the II-III loop in the recognition of protein mity to integrin, followed by the II-III loop engagement to fix the ligands may be generalized to most if not all integrin α subunits. relative orientation between the integrin and the LG trimer. Then In I-domain integrins, for example, it is the loop harboring the the LMγ1-tail penetrates into the groove between the α6 and ligand-binding I-domain, which can be considered as an internal β1 subunits and needs to sample only small space to find its protein ligand52.InαIIb integrin, the II-III loop forms so-called target, the MIDAS metal. The twin engagement via LMγ1 P1609- cap subdomain unique to this subunit, which has been not only α6 N189 and LMγ1 E1607-MIDAS completes the local con- structurally shown to make direct contact with a fibrinogen γ formational rearrangement of α1 and α7 helices in the βI domain, peptide16,24, but also predicted to mediate more extensive inter- which makes the hybrid domain to swing out. Eventually, α6β1is action with the fibrinogen protein53. Furthermore, this loop converted into the extended-open conformation, and establishes contains D154 in integrin α5 (highlighted purple in Fig. 5e), the firm linkage between the basement membrane and the which has been shown to play a key role during the interaction cytoskeleton of epithelial cells. with the fibronectin ligand by accepting R1379 on the synergy site Structures of αVβ6orαVβ8 integrin headpiece in complex with located in the 9th FnIII repeats14. Although there is no experi- a protein ligand L-TGF-β have been solved by X-ray crystal- mental structure available for the α4orα9 integrins bound to lography and cryo-EM, respectively, where the ligand recognitions protein ligands (e.g., VCAM-1), this region may be a good place were mediated primarily via β subunit43,48. The current study to start a mutational analysis experiment to map the key residues presents another structural framework for the interaction between responsible for the high affinity binding of protein ligands. integrin and its large ECM ligand, and shows how and to what The alternatively spliced X1/X2 region has long been postu- extent the integrin α subunit contributes to the laminin recogni- lated to be important for ligand binding in laminin binding tion. It also makes it clear that structural analysis of integrin integrins, since in α7 the alternatively spliced versions exhibit bound by a large protein ligand is critical in delineating the full distinct specificity against different laminins54. The X1 sequence picture of ECM recognition by integrins. Integrin research has of α6 is highly conserved with that of the X1 variant of α7 subunit been enormously propelled by the discovery of the RGD tripeptide including the acidic residues important for the binding, but dif- in 198457, which marked an epoch in the history of cell adhesion fers substantially with the alternatively spliced α7 X2 variant research. However, the unusual success of the RGD peptide may including the overall length (Fig. 5e). It is known that α6 does not have caused a misconception in the field that protein-protein have X2 variant, although a longer variant containing extra interfaces can often be narrowed down to a linear peptide insertion of X2 after the X1 (α6X1X2) is expressed as a minor sequence. The ECM component laminin uses multiple stretches of species55. Interestingly, corresponding region of α3 subunit is amino acid sequences distributed widely in the structure to for- much shorter. Our structure directly confirmed the critical mulate complicated 3D pharmacophore, in order to secure the involvement of this region in the ligand binding of α6X1β1 specificity and high affinity toward specific integrin receptors. This integrin by showing that the X1 loop is in fact located in the feature, at least in the case of laminin, has prevented us from interface. However, the way α6 X1 region contributes to the devising a small molecule capable of mimicking or inhibiting the laminin binding may not follow the classical lock-and-key type of interaction. However, we hope that the structural information protein recognition mechanism, because the sidechain con- provided here, particularly that of the core binding interface near formation of the critical interface residues identified by Ala the LMγ1-tail, may help designing useful chemical tools to further mutagenesis experiments (e.g., E210 and E219) remained our understanding of the cell-basement membrane interactions. ambiguous even after the ligand docking. Considering the fact that these residues can be crosslinked to basic residues in the LM5 Methods 56 LG2 domain when they were mutated to Cys , they may make Residue numbering scheme. Residue numbering for integrin subunits are based direct but transient contact with laminin. Based on these con- on the mature protein sequence following the convention in integrin research siderations, it is tempting to speculate that the long and mobile literatures, while laminin subunits are numbered starting with the initiation Met as X1 region is specialized in facilitating the long-range electrostatic residue 1. capture of ligands, like in a fly fishing. We propose a hypothetical α β model of the laminin recognition mechanism by α6β1 in Fig. 7. Sample preparation for the structural analysis. The integrin 6 1 headpiece and tLM511 used for the structural analysis were prepared as previously described12,35. First, the long-range ligand search via the mobile and charged X1 For production of the α6β1 headpiece fragment, coiled-coil motifs called A-zip and

LM511 LM511

LG3 LG2 LG1 LG2 E1607

N189 α1 II-III loop X1 LG2 βI α7 α1 β-propeller α7

Hybrid α6β1 (closed) α6β1 (open)

Fig. 7 Hypothetical model of the LM511 recognition by α6β1 integrin. In the first step, a long-range electrostatic search via the mobile and charged X1 region attracts the LG domain of laminin to integrin (the first panel). The II-III loop fixes the relative orientation between the integrin and laminin (the second panel). The flexible γ1-tail is fixed via two anchor points (i.e., LMγ1 P1609-α6 N189 and LMγ1 E1607-MIDAS), which induces local conformational rearrangement of α1 and α7 helices in the integrin βI domain, making the hybrid domain swing out (the third panel). Eventually, α6β1 is converted into the extended-open conformation (the last panel).

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B-zip were appended to the C-termini of a truncated α6 (residues 1-618) and a plunged into liquid ethane and then transferred to a cryo-electron microscope truncated β1 (residues 1-445), respectively, to stabilize the heterodimer. A 12- (Titan Krios, Thermo Fisher Scientific) incorporating a field emission gun, a Cs residue PA tag was also appended to the C-terminus of the β1 construct to facilitate corrector (CEOS, GmbH), a Volta phase plate, and a direct electron detection purification. After the anti-PA-tag antibody (NZ-1) immunoaffinity purification, camera (Falcon 3EC, Thermo Fisher Scientific). The microscope was operated at the tag regions were removed by TEV protease digestion (the cleavage positions are 300 kV with a nominal magnification of ×59,000, which resulted in a calibrated indicated by red arrowheads in Fig. 1b) prior to the final purification on a Superdex pixel size of 1.113 Å. Volta phase contrast movies were recorded on the Falcon 3EC 200 Increase 10/300 GL column (Cytiva) equilibrated with 20 mM Tris, 150 mM camera in linear mode as described previously63. Each exposure of 2 s was frac- NaCl, pH 7.5 (TBS). For the tLM511 production, hexahistidine-tagged tLMα5 tionated into 26 movie frames, leading to a total electron dose of 20 e-/Å2, with a (residues E2655–A3327), HA-tagged tLMβ1 (residues D1714–L1786), and FLAG- nominal defocus ranging from -0.6 to -0.8 μm (Supplementary Table 2). Two data tagged tLMγ1 (residues D1528–P1609) were co-transfected into FreeStyleTM 293 sets, data set #1 (4,155 movies) and #2 (3,613 movies), were collected with EPU cells (Thermo Fisher Scientific) according to manufacturer’s instruction, and the (Thermo Scientific) in the same experimental condition. resultant tLM511 protein was purified from the conditioned media through two The workflow of cryo-EM reconstruction was summarized in Supplementary successive affinity chromatographies using cOmplete His-Tag Purification Resin Fig. 3. Movie frames were aligned and summed using MotionCor2 software64 to (Roche) and DDDDK-tagged Protein PURIFICATION GEL (MBL, Nagoya, obtain a dose-weighted and motion-corrected image. Estimation of the contrast Japan), followed by a final purification on a Superdex 200 Increase 10/300 GL transfer function (CTF) was performed using the Gctf software65. The following column (Cytiva). Anti-β1 integrin antibodies were produced in the Fv-clasp format image analysis was performed using the RELION 3.0 software package66. A total of fl as follows. Brie y, VH (residues 1-113) and VL (residues 1-108) genes of HUTS-4 1,540,870 particles (data set #1) and 1,119,413 particles (data set #2) were 40 were obtained by RT-PCR cloning from the hybridoma cells , while the VH and automatically picked from 4155 and 3613 movies, respectively, and then they were 35 fi VL sequences for TS2/16 have been reported . The bacterial expression plasmids separately used for reference-free 2D classi cation. Particles in the good 2D classes for VH-SARAH and VL-SARAH were constructed by using pET11c. Cys87 in the (827,006 particles in data set #1 and 693,594 particles in data set #2) were subjected fi fi VL domain of HUTS-4, a free Cys residue, was substituted with Tyr by a Quick- to rst round of 3D classi cation. The best class with 2 Fv-clasps bound in two data Change mutagenesis. Method for expression, refolding, and purification of the TS2/ sets was selected (class ii in data set #1 and class I in data set #2), and then a total of 16 and HUTS-4 Fv-clasps were essentially the same as that described previously35. 537,875 particles were combined and subjected to second round of 3D classification focused on a region at the molecular interface between the laminin and the integrin (Supplementary Fig. 3). Because of the flexibility of the thigh domain in the α Size exclusion chromatography (SEC) analysis. tLM511, TS2/16 Fv-clasp, and/ β α β subunit and the hybrid domain in the subunit and the incomplete binding of or HUTS-4 Fv-clasp were mixed with the integrin 6 1 headpiece at 1.5-fold molar HUTS-4 Fv-clasp, these regions were omitted from the 3D classification and excess. After 1-hour incubation at room temperature, the samples were subjected to refinement mask. The best class (class 3: 429,521 particles) was selected and used SEC on a Superdex 200 Increase 10/300 GL column (Cytiva, 28990944) equili- for 3D refinement and post-processing, which yielded a map at a 3.9 Å resolution brated with TBS containing 1 mM MgCl2 and 1 mM CaCl2 or TBS containing 1 (map A). To improve the density map at the laminin-integrin interface, we mM MnCl2 and 0.1 mM CaCl2. The peak top fraction sample obtained from the fi fi α β attempted further 3D focused classi cation and re nement. After density SEC analysis of the 6 1 headpiece, tLM511, TS2/16 Fv-clasp, and HUTS-4 Fv- subtraction of the flexible parts of the thigh domain in the α6 subunit and the clasp mixture eluted in the 1 mM MnCl2/0.1 mM CaCl2 condition (indicated by a hybrid domain in the β1 subunit of the integrin together with the HUTS-4 Fv- red asterisk in Supplementary Fig. 2b) was analyzed by 5-20% gradient SDS-PAGE, clasp, images were subjected to further two rounds (3rd and 4th) of 3D followed by staining with Coomassie Brilliant Blue (CBB). classification on the laminin-integrin interface. Finally, the best class (class C: 157,515 particles) was selected and used for 3D refinement and post-processing. Crystallization. For the crystallization of the α6β1 headpiece-TS2/16 Fv-clasp However, this process did not improve the resolution but rather worsened it complex, α6β1 headpiece was mixed with TS2/16 Fv-clasp at 1.5-fold molar excess (Supplementary Fig. 3c, map B, 4.3-Å resolution). Thus, we used the 3.9-Å and subjected to SEC on a Superdex 200 Increase 10/300 GL column equilibrated resolution map (map A) for the atomic model building and structure fi with TBS containing 1 mM MgCl2 and 1 mM CaCl2. The puri ed sample was interpretation. concentrated to 8.3 mg/ml by ultrafiltration using Amicon ultra (Millipore, 50 kDa cutoff, UFC505024). For the crystallization of HUTS-4 Fv-clasp, the purified Modeling of the α6β1 headpiece-tLM511 complex. Initial atomic model of the HUTS-4 Fv-clasp sample was also concentrated to 10 mg/ml using Spin-X UF α β fi (Corning, 30 kDa cutoff, 431484). Crystallization screening was carried out by the 6 1 headpiece-tLM511 complex was built by tting the crystal structures sitting-drop vapor diffusion method at 20 °C using The Classics Suite (Qiagen, described in the Results section as rigid bodies to the cryo-EM map using UCSF Chimera67. The atomic model of the LMγ1-tail region was built de novo in 130701), Wizard Classic 1&2 (Rigaku, 1009530 and 1009531), and ProPlex 62 fi (Molecular Dimensions, MD1-38) crystallization kits. Crystals of the α6β1 head- COOT . The models were manually adjusted using COOT to t to the cryo-EM fi fi 61 piece-TS2/16 Fv-clasp complex appeared in a few drops, and the crystallization map, and re ned using the real-space re nement in Phenix . As for the positions of the metals and the conformation of the coordinating residues in the integrin βI conditions were optimized using hanging drop vapor diffusion method at 20 °C. α β The diffraction quality crystals were obtained under the condition of 23% domain, the model was built by referring to the structure of IIb 3 in the open- head conformation (PDB: 2vdo) because of the indecisive map. The final model PEG1000, 0.2 M NaCl, 0.1 M Na/K phosphate, pH 6.5. Crystals of the HUTS-4 Fv- 68 fi clasp were appeared under ~20 conditions in the initial screening. A crystal was validated using MolProbity , and re nement and validation statistics are summarized in Supplementary Table 2. All structural figures were prepared by obtained under the condition of 1.26 M ammonium sulfate, 0.1 M MES, pH 6.0 fi (Wizard 2-No. 45) in the screening plate was used for data collection. PyMOL software (Schrödinger, LLC) unless otherwise speci ed.

X-ray diffraction and structure determination. Crystals of the α6β1 headpiece- Immunoprecipitation. Expression constructs for the soluble integrin α6β1 ecto- TS2/16 Fv-clasp complex were harvested and flash-frozen directly in liquid domain were prepared as previously described69. Briefly, the A-zip and the B-zip nitrogen, while crystals of the HUTS-4 Fv-clasp were transferred to well solution peptides that form a disulfide-linked coiled-coil called velcro were appended at the supplemented with 20% (v/v) glycerol before freezing. Diffraction data for the α6β1 C-termini of α6 ectodomain (authentic signal sequence followed by residues 1-988) headpiece-TS2/16 Fv-clasp complex and HUTS-4 Fv-clasp were collected at 100 K and β1 ectodomain (authentic signal sequence followed by residues 1-708), at beamlines TPS BL05A of National Synchrotron Radiation Research Center respectively, and cloned into a pcDNA3.1-based vector. For the PA-tagged tLM511 (Hsinchu, Taiwan) and BL-17A of Photon Factory (Tsukuba, Japan), respectively. construction, synthetic DNAs coding for tLMα5 (residues E2655–A3327), tLMβ1 Diffraction data were processed and scaled using XDS58. Initial phases were (residues D1714–L1786), and tLMγ1 (residues D1528–P1609) were used. A PA tag determined by molecular replacement method with PHASER59 from the CCP4 was appended at the N-terminus of the tLMα5. These three DNA segments were package60. PDB IDs of search models used for the molecular replacement were individually cloned into a pcDNA3.1-based vector containing a prolactin signal 4wk0, 3vi3, and 5xcx for the α6β1 headpiece-TS2/16 Fv-clasp complex and 3qq9, sequence. All single-residue mutations were introduced by QuickChange strategy. 4kaq, and 5xct for HUTS-4 Fv-clasp. Structure refinements were carried out using Each of the integrin and laminin samples were transiently expressed using the Phenix61, and manual model modifications were performed periodically using Expi293 expression system (Thermo Fisher Scientific) according to the method COOT62. Data collection statistics and refinement parameters are given in Sup- provided by the manufacturer. Culture supernatants were harvested 4 days after the plementary Table 1. transfection, and appropriate combinations of integrin and laminin culture media were mixed and subjected to co-immunoprecipitation using anti-PA tag antibody (NZ-1)-immobilized Sepharose or anti-velcro tag antibody (2H11)-immobilized Cryo-EM data collection and processing. For the cryo-EM experiments, α6β1 Sepharose. After washing with TBS containing 1 mM MgCl2 and 1 mM CaCl2, the headpiece, tLM511, TS2/16 Fv-clasp, and HUTS-4 Fv-clasp were mixed in a molar bound samples were eluted with SDS-PAGE sample buffer and subjected to 10% fi ratio of 1:1.5:1.5:1.5 and puri ed on a Superdex 200 Increase 10/300 GL column SDS-PAGE analysis under non-reducing condition, followed by CBB staining. μ equilibrated with TBS containing 1 mM MnCl2 and 0.1 mM CaCl2. A 2.5 l of the sample solution of the quaternary complex (70 μg/ml) was applied to glow- discharged Quantifoil holey carbon grids (Quantifoil R2/1, Mo 300 mesh) covered Preparations of fluorescently labeled TS2/16 Fv-clasp and tLM511. To be used with a thin, amorphous carbon film of thickness 5–10 nm. The grids were incu- in the quantitative binding analysis to cell surface integrins, TS2/16 Fv-clasp and bated in the Vitrobot Mark IV (Thermo Fisher Scientific) at 4 °C and 100% tLM511 were fluorescently labeled by fusing with superfolder green fluorescent fi humidity for 30 s. After blotted with lter papers for 3 s, the grids were immediately protein (sfGFP). For making TS2/16 Fv-clasp-sfGFP, both VH-SARAH and VL-

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SARAH coding regions were cloned in-frame into a mammalian expression vector 8. Hohenester, E. Structural biology of laminins. Essays Biochem 63, 285–295 containing mouse nidogen-1 signal sequence, and sfGFP gene (GenBank accession (2019). ASL68970.1) and hexahistidine tag were appended at the C-terminal of VH- 9. Shibata, S. et al. Selective laminin-directed differentiation of human induced SARAH and VL-SARAH, respectively. These plasmids were co-transfected into pluripotent stem cells into distinct ocular lineages. Cell Rep. 25, 1668–1679 Expi293F cells and the secreted fusion protein was purified from the culture e1665 (2018). supernatants using Ni-NTA-agaraose chromatography. For making fluorescent 10. Miner, J. H. & Yurchenco, P. D. Laminin functions in tissue morphogenesis. tLM511 fragment, sfGFP gene was inserted after the PA tag in the PA-tLMα5 Annu. Rev. Cell. Dev. Biol. 20, 255–284 (2004). construct described in the previous section, and the resultant PA-sfGFP-tLMα5 11. Edgar, D., Timpl, R. & Thoenen, H. The heparin-binding domain of laminin is β γ plasmid was co-expressed with 1 and 1 constructs using the Expi293F cells. The responsible for its effects on neurite outgrowth and neuronal survival. EMBO fi trimeric fusion protein PA-sfGFP-tLM511 was puri ed from the culture super- J. 3, 1463–1468 (1984). natants using NZ-1-immobilized Sepharose using the protocol described 12. Takizawa, M. et al. Mechanistic basis for the recognition of laminin-511 by 70 previously . For both sfGFP fusions, protein concentrations were determined alpha6beta1 integrin. Sci. Adv. 3, e1701497 (2017). fluorometrically with the NanoDrop 3300 fluorospectrometer (Thermo Fisher fi fi 13. Pulido, D., Hussain, S. A. & Hohenester, E. Crystal Structure of the Scienti c), using the puri ed sfGFP as a standard. Heterotrimeric Integrin-Binding Region of Laminin-111. Structure 25, 530–535 (2017). Flow cytometry-based binding assays. For the quantitative binding analysis of 14. Nagae, M. et al. Crystal structure of alpha 5 beta 1 integrin ectodomain: TS2/16 to cell surface β1 integrin, K562 cells were suspended in 20 mM Hepes, Atomic details of the fibronectin receptor. J. Cell. Biol. 197, 131–140 (2012). 150 mM NaCl, pH 7.5, containing 0.1% BSA (HBS-BSA) at 2 ×105 cells/ml and 15. Xiong, J. P. et al. Crystal structure of the extracellular segment of integrin incubated with varying concentrations of TS2/16 Fv-clasp-sfGFP in the presence of alpha Vbeta3 in complex with an Arg-Gly-Asp ligand. Science 296, 151–155 1 mM CaCl2 and 1 mM MgCl2 (for ligand unbound state) or 0.5 mM MnCl2, and (2002). 1 mM GRGDSP peptide (for ligand-bound state). After the incubation at room 16. Springer, T. A., Zhu, J. & Xiao, T. Structural basis for distinctive recognition of temperature for 2 h, the cells were directly subjected to flow cytometry on an fibrinogen gammaC peptide by the platelet integrin alphaIIbbeta3. J. Cell Biol. EC800 system (Sony) without any washing steps. The acquired histogram data 182, 791–800 (2008). fl were used to extract uorescence signal associated with each cell in a linear scale, 17. Dong, X., Hudson, N. E., Lu, C. & Springer, T. A. Structural determinants of and values from all gated cells (>5000 cells/histogram) were used to obtain mean integrin beta-subunit specificity for latent tgf-beta. Nat. Struct. Biol. 21, fl fl cellular uorescence in an arbitrary unit. Background uorescence obtained in the 1091–1096 (2014). fl absence of uorescent protein (generally ~0.5 arbitrary units /cell) were subtracted 18. Zhu, J. Q., Zhu, J. H. & Springer, T. A. Complete integrin headpiece opening from these values and plotted against the concentration of TS2/16, and the data in eight steps. J. Cell Biol. 201, 1053–1068 (2013). were analyzed using PRISM software (ver 9.1.0, GraphPad Software, LLC.) to α 19. Takagi, J., Petre, B. M., Walz, T. & Springer, T. A. Global conformational calculate KD values. For the quantitative analysis of tLM511 binding to various 6 rearrangements in integrin extracellular domains in outside-in and inside-out mutant integrins, Expi293F cells in suspension culture were transiently transfected signaling. Cell 110, 599–611 (2002). with various mutant versions of full-length human α6 together with wild-type β1 20. Campbell, I.D. & Humphries, M.J. Integrin structure, activation, and integrin subunits using the protocol recommended by the manufacturer. After interactions. Cold Spring Harb. Perspect. Biol. 3 (2011). 2 days post transfection, the cells were suspended in HBS-BSA containing 0.5 mM 21. Yu, Y. M. et al. Structural specializations of alpha(4)beta(7), an integrin that MnCl and incubated with varying concentrations of sfGFP-tLM511 at room 2 mediates rolling adhesion. J. Cell Biol. 196, 131–146 (2012). temperature for 2 h, and directly subjected to flow cytometry without washing 22. Nishida, N. et al. Activation of leukocyte beta2 integrins by conversion from steps. The analysis of the binding data and calculation of the KD values were – performed as in the case of TS2/16 binding, although the background fluorescence bent to extended conformations. Immunity 25, 583 594 (2006). of the Expi293F cells were higher than K562 cells (actual values shown in Sup- 23. Chen, X. et al. Requirement of open headpiece conformation for activation of – plementary Fig. 7a). leukocyte integrin alphaXbeta2. Proc. Natl Acad. Sci. USA 107, 14727 14732 (2010). 24. Xiao, T., Takagi, J., Coller, B. S., Wang, J. H. & Springer, T. A. Structural basis Reporting summary. Further information on research design is available in the Nature for allostery in integrins and binding to fibrinogen-mimetic therapeutics. Research Reporting Summary linked to this article. Nature 432,59–67 (2004). 25. Miyazaki, N., Iwasaki, K. & Takagi, J. A systematic survey of conformational Data availability states in beta1 and beta4 integrins using negative-stain electron microscopy. J. Atomic coordinates of HUTS4 Fv-clasp, integrin α6β1-TS2/16 Fv-clasp complex, and Cell Sci. 131 (2018). fi integrin α6β1-LM511-TS2/16 Fv-clasp-HUTS4 Fv-clasp complex have been deposited in 26. Nishiuchi, R. et al. Ligand-binding speci cities of laminin-binding integrins: a the Protein Data Bank (PDB) with accession codes 7CEA, 7CEB, and 7CEC, respectively. comprehensive survey of laminin-integrin interactions using recombinant Cryo-EM data of integrin α6β1-LM511-TS2/16 Fv-clasp-HUTS4 Fv-clasp complex have alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4 integrins. Matrix Biol. – been deposited in the EM Data Bank (EMDB) with accession code EMD-30342 . PDB 25, 189 197 (2006). IDs of search models used for the molecular replacement are 4wk0, 3vi3, and 5xcx for the 27. Hemler, M. E., Crouse, C. & Sonnenberg, A. Association of the VLA alpha α6β1 headpiece-TS2/16 Fv-clasp complex and 3qq9, 4kaq, and 5xct for HUTS-4 Fv-clasp. 6 subunit with a novel protein. A possible alternative to the common VLA – GenBank accession code for sfGFP gene is ASL68970.1. All the data supporting the beta 1 subunit on certain cell lines. J. Biol. Chem. 264, 6529 6235 (1989). findings of this study are available within the article and its Supplementary Information 28. Shen, Q. et al. Adult SVZ stem cells lie in a vascular niche: a quantitative – files, and from the corresponding author upon reasonable request. Source data are analysis of niche cell-cell interactions. Cell Stem Cell 3, 289 300 (2008). fl provided with this paper. 29. Yang, Z. et al. CD49f acts as an in ammation sensor to regulate differentiation, adhesion, and migration of human mesenchymal. Stem Cells Stem Cells 33, 2798–2810 (2015). Received: 19 September 2020; Accepted: 4 June 2021; 30. Villa-Diaz, L. G., Kim, J. K., Laperle, A., Palecek, S. P. & Krebsbach, P. H. Inhibition of focal adhesion kinase signaling by integrin alpha6beta1 supports human pluripotent stem cell self-renewal. Stem Cells 34, 1753–1764 (2016). 31. Toya, S. P. et al. Integrin alpha6beta1 Expressed in ESCs Instructs the Differentiation to Endothelial Cells. Stem Cells 33, 1719–1729 (2015). References 32. Miyazaki, T. et al. Laminin e8 fragments support efficient adhesion and expansion 1. Hynes, R. O. Integrins: bidirectional, allosteric signaling machines. Cell 110, of dissociated human pluripotent stem cells. Nat. Commun. 3 (2012). 673–687 (2002). 33. Krebsbach, P. H. & Villa-Diaz, L. G. The role of integrin alpha6 (CD49f) in stem 2. Brown, N. H. Cell-cell adhesion via the ECM: integrin genetics in fly and cells: more than a conserved biomarker. Stem Cells Dev. 26, 1090–1099 (2017). worm. Matrix Biol. 19, 191–201 (2000). 34. Zhou, Z. et al. alpha6-Integrin alternative splicing: distinct cytoplasmic 3. Hynes, R. O. & Zhao, Q. 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38. Luo, B. H., Strokovich, K., Walz, T., Springer, T. A. & Takagi, J. Allosteric 66. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure beta1 integrin antibodies that stabilize the low affinity state by preventing the determination in RELION-3. Elife 7 (2018). swing-out of the hybrid domain. J. Biol. Chem. 279, 27466–27471 (2004). 67. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory 39. Su, Y. et al. Relating conformation to function in integrin alpha5beta1. Proc. research and analysis. J. Comput. Chem. 25, 1605–1612 (2004). Natl Acad. Sci. USA. 113, E3872–E3881 (2016). 68. Chen, V. B. et al. MolProbity: all-atom structure validation for 40. Luque, A. et al. Activated conformations of very late activation integrins macromolecular crystallography. Acta Crystallogr. D. Biol. Crystallogr. 66, detected by a group of antibodies (HUTS) specific for a novel regulatory 12–21 (2010). region (355-425) of the common beta 1 chain. J. Biol. Chem. 271, 69. Takagi, J., Erickson, H. P. & Springer, T. A. C-terminal opening mimics 11067–11075 (1996). “inside-out” activation of integrin a5b1. Nat. Struct. Biol. 8, 412–416 41. Takagi, J., Strokovich, K., Springer, T. A. & Walz, T. Structure of integrin (2001). alpha5beta1 in complex with fibronectin. EMBO J. 22, 4607–4615 (2003). 70. Fujii, Y. et al. PA tag: a versatile protein tagging system using a super high 42. Eng, E. T., Smagghe, B. J., Walz, T. & Springer, T. A. Intact alpha(IIb)beta(3) affinity antibody against a dodecapeptide derived from human podoplanin. integrin is extended after activation as measured by solution x-ray scattering Protein Expres Purif. 95, 240–247 (2014). and electron microscopy. J. Biol. Chem. 286, 35218–35226 (2011). 43. Dong, X. et al. Force interacts with macromolecular structure in activation of TGF-beta. Nature 542,55–59 (2017). Acknowledgements 44. Wang, J. et al. Atypical interactions of integrin alphaVbeta8 with pro-TGF- We would like to thank Yukinari Kato and Mika Kato-Kaneko for performing molecular beta1. Proc. Natl Acad. Sci. USA 114, E4168–E4174 (2017). cloning of HUTS-4 antibody, Kenji Iwasaki for the support of the cryo-EM data 45. Mould, A. P. et al. Conformational changes in the integrin beta A domain acquisition and analyses, and the staff of the beamlines at Photon Factory and National provide a mechanism for signal transduction via hybrid domain movement. J. Synchrotron Radiation Research Center for their help with X-ray data collection. This Biol. Chem. 278, 17028–17035 (2003). work was supported in part by JSPS KAKENHI grant number JP18H02389 from Japan 46. Ido, H. et al. The requirement of the glutamic acid residue at the third position Society for the Promotion of Science to T.A., and by the Platform Project for Supporting from the carboxyl termini of the laminin gamma chains in integrin binding by Drug Discovery and Life Science Research (Basis for Innovative Drug Discovery and Life laminins. J. Biol. Chem. 282, 11144–11154 (2007). Science Research (BINDS)) funded by Japan Agency for Medical Research and Devel- 47. Nesic, D. et al. Cryo-electron microscopy structure of the alphaiibbeta3- opment (AMED) under Grant Number JP19am0101075 to J.T. The data collection and abciximab complex. Arterioscler. Thromb. Vasc. Biol. 40, 624–637 (2020). analyses were performed using the Molecular Cryo-Electron Microscope facility of 48. Campbell, M. G. et al. Cryo-EM reveals integrin-mediated TGF-beta activation Institute for Protein Research, Osaka University. without release from latent TGF-beta. Cell 180, 490–501 e416 (2020). 49. Wang, J., Su, Y., Iacob, R. E., Engen, J. R. & Springer, T. A. General structural Author contributions features that regulate integrin affinity revealed by atypical alphaVbeta8. Nat. T.A. designed and performed experiments, analyzed the data and wrote the manuscript. Commun. 10, 5481 (2019). N.M. performed experiments, analyzed the data, and wrote the manuscript. E.M., M.T., 50. Lin, F. Y., Zhu, J., Eng, E. T., Hudson, N. E. & Springer, T. A. beta-Subunit Y.T., and C.C. performed experiments, and analyzed the data. K.S. analyzed the data and Binding Is Sufficient for Ligands to Open the Integrin alphaIIbbeta3 wrote the manuscript. J.T. conceived the experimental design, analyzed the data and Headpiece. J. Biol. Chem. 291, 4537–4546 (2016). wrote the manuscript. All authors contributed to the preparation of the manuscript. 51. Sen, M., Yuki, K. & Springer, T. A. An internal ligand-bound, metastable state of a leukocyte integrin, alpha(X)beta(2). J. Cell Biol. 203, 629–642 (2013). 52. Takagi, J. & Springer, T. A. Integrin activation and structural rearrangement. Competing interests Immunological Rev. 186, 141–163 (2002). The authors declare no competing interests. 53. Kamata, T., Tieu, K. K., Irie, A., Springer, T. A. & Takada, Y. Amino acid residues in the alpha IIb subunit that are critical for ligand binding to integrin Additional information alpha IIbbeta 3 are clustered in the beta-propeller model. J. Biol. Chem. 276, Supplementary information 44275–44283 (2001). The online version contains supplementary material 54. von der Mark, H. et al. Alternative splice variants of alpha 7 beta 1 integrin available at https://doi.org/10.1038/s41467-021-24184-8. selectively recognize different laminin isoforms. J. Biol. Chem. 277, 6012–6016 Correspondence (2002). and requests for materials should be addressed to J.T. 55. Delwel, G. O., Kuikman, I. & Sonnenberg, A. An alternatively spliced exon in the Peer review information Nature Communications thanks Timothy Springer, Roy Zent, — extracellular domain of the human alpha 6 integrin subunit functional analysis of and other, anonymous, reviewers for their contributions to the peer review of this work. – the alpha 6 integrin variants. Cell Adhes. Commun. 3, 143 161 (1995). Peer review reports are available. 56. Taniguchi, Y., Takizawa, M., Li, S. & Sekiguchi, K. Bipartite mechanism for laminin-integrin interactions: Identification of the integrin-binding site in lg Reprints and permission information is available at http://www.nature.com/reprints domains of the laminin alpha chain. Matrix Biol. 87,66–76 (2020). 57. Pierschbacher, M. D. & Ruoslahti, E. Cell attachment activity of fibronectin Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in can be duplicated by small synthetic fragments of the molecule. Nature 309, published maps and institutional affiliations. 30–33 (1984). 58. Kabsch, W.Xds. Acta Crystallogr. D. Biol. Crystallogr. 66, 125–132 (2010). 59. Mccoy, A. J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007). Open Access This article is licensed under a Creative Commons 60. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Attribution 4.0 International License, which permits use, sharing, Acta Crystallogr. D. Biol. Crystallogr. 67, 235–242 (2011). adaptation, distribution and reproduction in any medium or format, as long as you give 61. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for appropriate credit to the original author(s) and the source, provide a link to the Creative macromolecular structure solution. Acta Crystallogr. D. Biol. Crystallogr. 66, Commons license, and indicate if changes were made. The images or other third party 213–221 (2010). material in this article are included in the article’s Creative Commons license, unless 62. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development indicated otherwise in a credit line to the material. If material is not included in the of Coot. Acta Crystallogr. D. Biol. Crystallogr. 66, 486–501 (2010). article’s Creative Commons license and your intended use is not permitted by statutory 63. Kato, K. et al. Structure of a cyanobacterial photosystem I tetramer revealed by regulation or exceeds the permitted use, you will need to obtain permission directly from cryo-electron microscopy. Nat. Commun. 10, 4929 (2019). the copyright holder. To view a copy of this license, visit http://creativecommons.org/ 64. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion licenses/by/4.0/. for improved cryo-electron microscopy. Nat. Methods 14,331–332 (2017). 65. Zhang, K. Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193,1–12 (2016). © The Author(s) 2021

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