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Cryo-EM structure of the αvβ8 integrin reveals a mechanism for stabilizing integrin extension

Abstract

Integrins are conformationally flexible cell surface receptors that survey the extracellular environment for their cognate ligands. Interactions with ligands are thought to be linked to global structural rearrangements involving transitions between bent, extended-closed and extended-open forms. Thus far, structural details are lacking for integrins in the extended conformations due to extensive flexibility between the headpiece and legs in this conformation. Here we present single-particle electron cryomicroscopy structures of human αvβ8 integrin in the extended-closed conformation, which has been considered to be a low-affinity intermediate. Our structures show the headpiece rotating about a flexible αv knee, suggesting a ligand surveillance mechanism for integrins in their extended-closed form. Our model predicts that the extended conformation is mainly stabilized by an interface formed between flexible loops in the upper and lower domains of the αv leg. Confirming these findings with the αvβ3 integrin suggests that our model of stabilizing the extended-closed conformation is generalizable to other integrins.

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Fig. 1: Structure of the αvβ8 integrin in extended-closed conformation.
Fig. 2: The αvβ8 headpiece rotates about a flexible αv knee to survey the environment.
Fig. 3: The αv leg stabilizes integrin extension of the αvβ3 integrin.
Fig. 4: The stabilizing mechanism of the extended αv integrin.
Fig. 5: The extended αvβ3 integrin shows increased ability to mediate adhesion.

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Acknowledgements

We thank M. Braunfeld for supporting the cryo-EM facility at UCSF. LIBS antibodies were a gift from M. Ginsberg (University of California San Diego). This work was supported in part by grants the NIH (U54HL119893 and R01HL113032 to S.L.N.; R01HL134183 to S.L.N. and Y.C.; R01GM098672, S10OD020054 and S10OD021741 to Y.C.; and P41CA196276 to J.M.) and from the University of California Office of the President Tobacco-Related Disease Research Program to S.L.N. Y.C. is an Investigator of the Howard Hughes Medical Institute.

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Authors

Contributions

A.C., M.G.C. and S.W. performed cryo-EM and structural biology. A.C. and M.G.C. performed ns-EM. A.C. and S.I. performed biochemical experiments. S.I., A.C. and S.L.N. designed, generated and characterized mutant integrins. A.C., M.G.C., S.I., S.W., S.L.N. and Y.C. conceived experiments and wrote the manuscript. J.M., J.L., J.B. and S.L.N. produced, characterized, cloned and engineered monoclonal antibodies.

Corresponding authors

Correspondence to Stephen L. Nishimura or Yifan Cheng.

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Integrated supplementary information

Supplementary Figure 1 Models of integrin conformational changes and activation mechanisms.

a, The individual leg, head and sub-domains are shown using common nomenclature. b, In the switchblade model, the bent conformation moves through an extended-closed conformation to an extended-open conformation. In this model, the bent conformation is low-affinity, the extend-closed is a low-affinity intermediate, and the extended-open is the high-affinity fully active conformation. c, In the bent model, affinity regulation occurs solely in the bent conformation. d, In the case of αvβ8, affinity regulation must occur in the extended-closed conformation. In all models, affinity changes corresponding to movements in the headpiece are indicated by pink arrows.

Supplementary Figure 2 Cryo-EM micrograph, local resolution estimates and orientation distribution of the αvβ8-8B8-68 complex.

a, Representative electron micrograph and b, 2D class averages of frozen hydrated αvβ8-8B8-68 complex. The scale bar is 500 Å in the micrograph, and 200 Å in 2D class averages. c, Fourier Shell Correlation (FSC) curves of whole αvβ8-8B8-68 complex (blue solid line) and headpiece alone (blue dashed line). d, FSC curve between the density map and the fitted atomic model of αvβ8-8B8-68 complex. e, Local resolution of αvβ8-8B8-68 complex (left) and angular distributions of all particles used for calculating 3D reconstruction (right). The figures are color coded as indicated. f, Local resolution of αvβ8-8B8-68 complex with focused alignment in its headpiece (left) and angular distributions of all particles used for calculating 3D reconstruction (right), which is almost identical to the angular distribution of whole molecule reconstruction.

Supplementary Figure 3 Processing schematic for αvβ8-8B8-68 complex.

A schematic flowchart showing the classification scheme of αvβ8-8B8-68 complex. Particle numbers at each step and for each class are indicated. 3D reconstructions of six out of eight subclasses reached sub-nanometer resolution, despite small particle numbers for each class. Local resolution for each subclass are color coded as indicated. Two classes that did not yield comparable reconstructions were excluded from further analysis.

Supplementary Figure 4 Contacts between the αv thigh and calf-1 domains stabilize the extended conformation.

a and b, Same views of density maps for all subclasses show the progressive loss of contact between αv-leg and β8 upper leg (a), and disappearance of the β8 lower leg (b). The color code is: αv-green; β8-blue.

Supplementary Figure 5 Extension of αvβ3 does not change its intrinsic ligand affinity; integrin β leg sequence alignment.

a, Receptor binding of αvβ3 ectodomains (WT and αvc-cβ3) to vitronectin-N in basal (Ca2+) or activating (Mn2+) cation conditions (n = 3, ± s.e.m.). b, Receptor binding of αvβ8 ectodomains (WT and αvc-cβ8) to L-TGF-β (n = 3, ± s.e.m.). c, Sequence alignment for the leg of all β integrins pairing with αv. Yellow highlights conserved cysteines in the β legs, notable exceptions in the β8 leg are highlighted in blue.

Supplementary information

Supplementary Figures 1–5 and Supplementary Tables 1–3

Reporting Summary

Supplementary Video 1

Molecular animation of the headpiece movement. Animated movie of ribbon models of the six αvβ8-8B8-68 subclasses aligned to the αv-calf-1,2 region of subclass (iv). The movie depicts αvβ8-8B8-68 in the membrane and moves sequentially through subclasses (i), (ii), (iii), (v) and (vi), each time moving back through subclass (iv).

Source Data, Figure 5

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Cormier, A., Campbell, M.G., Ito, S. et al. Cryo-EM structure of the αvβ8 integrin reveals a mechanism for stabilizing integrin extension. Nat Struct Mol Biol 25, 698–704 (2018). https://doi.org/10.1038/s41594-018-0093-x

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