Crystal structure of the extracellular segment of integrin alpha Vbeta3

Abstract

Integrins are alphabeta heterodimeric receptors that mediate divalent cation-dependent cell-cell and cell-matrix adhesion through tightly regulated interactions with ligands. We have solved the crystal structure of the extracellular portion of integrin alphaVbeta3 at 3.1 A resolution. Its 12 domains assemble into an ovoid "head" and two "tails." In the crystal, alphaVbeta3 is severely bent at a defined region in its tails, reflecting an unusual flexibility that may be linked to integrin regulation. The main inter-subunit interface lies within the head, between a seven-bladed beta-propeller from alphaV and an A domain from beta3, and bears a striking resemblance to the Galpha/Gbeta interface in G proteins. A metal ion-dependent adhesion site (MIDAS) in the betaA domain is positioned to participate in a ligand-binding interface formed of loops from the propeller and betaA domains. MIDAS lies adjacent to a calcium-binding site with a potential regulatory function.

Fig. 1

Structure of the extracellular segment of αVβ3. (A) Stereoview of a simulated-annealing omit map at 1.5 σ, in the vicinity of Arg²⁶¹ (from β3) (magenta). Surrounding densities (cyan) (from α V) are from the same map. (B) Ribbon drawing (40) of crystallized αVβ3 [shown in blue (αV) and red (β3)]. (C) Model of the straightened extracellular segment of αVβ3. The two tails would extend into the plasma membrane in the native integrin. Translated and rotated EGF-3 and -4 show the approximate location of EGF-1 and -2 (gray). The PSI tracing (gray) is approximate. Connections of the untraced domains are in dotted lines. αV was straightened by extending the structure by 135° at the thigh–calf-1 interface (circled) and then rotating the calf module ~120° around its “long” axis to avoid clashes at the thigh–calf-1 interface. The same transformations were then applied to β3 (residue 445 onward). Arrows point to the position of the three longest inter-domain linkers 1, 2, and 3 in the structure. Amino acid domain boundaries are indicated in parenthesis. In this and other figures, “n” and “c” indicate NH₂- and COOH-terminus, respectively.

Fig. 2

Structure of the integrin β-propeller. (A) Bottom view of the seven-bladed (numbered) αV propeller. Disulfides (sticks) and glycans (spheres) are in red and Ca²⁺ ions are in green. (B) Super-imposition of the seven blades, with blade five shown in red. The cage residues (ϕ, aromatic; G, Gly; and P, Pro) are in red. The ϕϕGϕ sequence assumes the shape of a cup. Strands A to D are labeled. Top (C) and side (D) views of the central propeller–βA domain interface. The propeller's lower (red) and upper (blue) aromatic rings surrounding Arg²⁶¹ of β3 are shown. The 3₁₀-helix of β3 is in green. The lower ring residues are labeled in red. The prolines and glycines of the cage motif are shown as magenta and cyan spheres, respectively. F⁴²⁷ and D⁴³⁰ replace the proline and glycine residues in blade seven, respectively (41).

Fig. 3

Structural features of the βA domain. (A) Ribbon drawing of the βA domain. Disulfides and the glycan are in purple and red, respectively. (B) Residues [single letter (41)] forming MIDAS that could participate in metal ion (blue) coordination. (C) Calcium (yellow) coordination at ADMIDAS. (D) Superimposition, based on the central β sheet, of the βA domain (minus the insertion in the B-C loop) and the “open” and “closed” forms of αA of CD11b (all gray). Shown are the α7-helices of the βA domain (red) and the “open” (blue) and “closed” (green) forms of αA of CD11b. (E) Coordination of SILEN in the βA domain (red) and in “closed” αA of CD11b (green). The βA domain SILEN residues [single letter, (41)] I¹¹⁴, L¹³⁸, L²⁴⁵, and I³⁰⁷ (red) [equivalent to I¹³⁵, L¹⁶⁴, I²³⁶, and Y²⁶⁷, respectively, in αA of CD11b (green) (23)] coordinate I³⁴⁴ (equivalent in sequence to L³¹² in αA of CD11b). I³⁵¹ (corresponding to I³¹⁶ in αA of CD11b) is not coordinated in β3 SILEN but is partially exposed at the bottom of the structure.

Fig. 4

Architecture of integrin and G protein interfaces. The nucleotide-binding folds are in green, and the propellers are in gray. The integrin is shown in (A) and (B) and the G protein in (C) and (D). The helices at the core of each interface are indicated. Integrin's Arg²⁶¹ and G protein's Lys²¹⁰ project toward the respective propeller's central cavity. Gα (Ala³⁰–Leu³⁴⁸) and Gβ (Ser²–Asn³⁴⁰) are shown. G protein coordinates were from (33).

Fig. 5

Surface representation, done with GRASP (42), of the main αVβ3 interface in the head region. Contacting residues (distance cutoff 3.5 Å for hydrogen bonds and salt bridges, and 4.0 Å for van der Waals contacts) are shown. Arg²⁶¹ is in blue. Hydrophobic, ionic, and mixed contacts are in yellow, red, and orange, respectively. The β3 strands and helices contributing to the interface are indicated.

Fig. 6

The putative ligand-binding site at the integrin αβ interface. GRASP representation of the αV propeller (white) and the α3 A-domain (gray), viewed from the top of the head (the tails lying in the back). Arg¹⁴³–Phe¹⁵⁴ (red) and Gly¹⁷²–Gly¹⁸¹ (green) of αV correspond to the identified ligand-binding residues Arg¹⁴⁷–Tyr¹⁶⁶ and Gly¹⁸⁴–Gly¹⁹³, respectively, in αIIb. Ligand-binding residues in β3 (Asp¹⁷⁹–Thr¹⁸³, “ligand specificity” region (magenta); Asp¹¹⁹, Ser¹²¹, Ser¹²³, Glu²²⁰ (all MIDAS residues), and Arg²¹⁴ (yellow) are shown. Asp²¹⁷ in β3 (also in yellow) is involved in binding of a ligand-mimetic monoclonal antibody. The expected MIDAS metal ion is in blue.