Electron tomography reveals diverse conformations of integrin αIIbβ3 in the active state

Abstract

We used electron tomography to determine the three-dimensional (3D) structure of integrin αIIbβ3 in the active state. We found that we obtained better density maps when we reconstructed a 3D volume for each individual particle in the tilt series rather than to extract the particle-containing subvolumes from a 3D reconstruction of the entire specimen area. The 3D tomographic reconstructions of 100 particles revealed that activated αIIbβ3 adopts many different conformations. An average of all the individual 3D reconstructions nicely accommodated the crystal structure of the αVβ3 headpiece, confirming the locations assigned to the α- and β-subunit in the density map. The most striking finding of our study is the structural flexibility of the lower leg of the β-subunit as opposed to the conformational stability of the leg of the α-subunit. The good fit of the atomic structure of the βI domain and the hybrid domain in the active state showed that the hybrid domain swings out, and most particles used for tomography are in the active state. Multivariate statistical analysis and classification applied to the set of 3D reconstructions revealed that more than 90% reconstructions are grouped into the classes that show the active state. Our results demonstrate that electron tomography can be used to classify complexes with a flexible structure such as integrins.

Introduction

Integrins are a large family of cell surface glycoproteins that mediate cell–cell and cell–extracellular matrix (ECM)⁵ interactions (Hemler et al., 1990, Hynes, 1992). In vertebrates, integrins are heterodimers composed of one of the 19 known α-subunits and one of the eight known β-subunits (Humphries, 2000, Hynes, 1992). The combination of an α- and a β-subunit defines an individual receptor, and since not all subunit combinations are realized, only approximately 25 distinct integrins have been identified to date. αIIbβ3, an integrin restricted to platelets and cells of the megakaryocytic lineage, has been one of the most intensively studied integrins. The stimulation of platelets by antagonists such as thrombin, ADP, collagen, or immobilized von Willebrand factor (vWF) increases the affinity of αIIbβ3 for fibrinogen and vWF. Cross-linking of platelets through these ligands causes platelet aggregation that leads to an outside-in signal that stabilizes the platelet aggregation.

Since αIIbβ3 on the platelet requires fast activation in response to antagonists, for example when vessels are injured, a central issue concerning integrin αIIbβ3 is the molecular mechanism that underlies its quick activation process. During the last 4 years, significant advances have been made in our understanding of the activation mechanism of integrins. These advances are based on the recently determined structures of integrins and their domains by X-ray crystallography, nuclear magnetic resonance studies, and electron microscopy (EM) (Adair and Yeager, 2002, Beglova et al., 2002, Takagi et al., 2002, Takagi et al., 2003, Xiong et al., 2001, Xiong et al., 2002). The crystal structures of the extracellular portion of integrin αVβ3 revealed an ovoid “head” and two tails comprising 12 domains (Xiong et al., 2001). A noteworthy feature of the crystal structure is the bent conformation, in which the headpiece and tailpiece form an acute angle, contrary to previous electron microscopic studies of negatively stained or metal-shadowed integrins, which showed extended molecules (Carrell et al., 1985, Weisel et al., 1992). Based on single-particle EM projection averages of recombinant αVβ3 as well as biochemical and cell biological experiments, Takagi et al. (2002) have shown that the bent conformation corresponds to an inactive conformation of the integrin, which upon activation extends upward in a switchblade-like opening. A three-dimensional (3D) reconstruction of unliganded αIIbβ3 derived by single-particle EM of vitrified specimens (Adair and Yeager, 2002) is different from the expectations based on previous EM studies as well as the crystal structure of αVβ3. To date, the 3D structure of an intact integrin in the active state has not been reported. Since the flexibility of the leg of the β-subunit (Takagi et al., 2002) makes it difficult to apply image averaging such as single-particle analysis to integrins, we used electron tomography to visualize integrins in the high-affinity state. Electron tomography of single macromolecules, originally developed by Hoppe et al., 1968, Hoppe et al., 1976a, Hoppe et al., 1976b, Hoppe et al., 1976c. They also carried out the first ever averaging of tomographically reconstructed macromolecules on a ribosome (Knauer et al., 1983, Oettl et al., 1983). Walz et al. (1997) first attempted volume extraction alignment and classification after the reconstruction of a full field of macromolecules prepared in vitreous ice. This method can avoid two-dimensional (2D) image averaging that leads to faulty averaging of heterogeneous structures. Multivariate statistical analysis of the 3D reconstructions and docking of the crystal structure of the integrin headpiece into the averaged 3D map showed that our tomographic approach enabled us to obtain the correct 3D structure of the flexible protein and the hybrid domain of intact αIIbβ3 swings out when it forms a complex with an RGD peptide.