Structural basis of transmembrane domain interactions in integrin signaling

Abstract

Cell surface receptors of the integrin family are pivotal to cell adhesion and migration. The activation state of heterodimeric alphabeta integrins is correlated to the association state of the single-pass alpha and beta transmembrane domains. The association of integrin alphaIIbbeta3 transmembrane domains, resulting in an inactive receptor, is characterized by the asymmetric arrangement of a straight (alphaIIb) and tilted (beta3) helix relative to the membrane in congruence to the dissociated structures. This allows for a continuous association interface centered on helix-helix glycine-packing and an unusual alphaIIb(GFF) structural motif that packs the conserved Phe-Phe residues against the beta3 transmembrane helix, enabling alphaIIb(D723)beta3(R995) electrostatic interactions. The transmembrane complex is further stabilized by the inactive ectodomain, thereby coupling its association state to the ectodomain conformation. In combination with recently determined structures of an inactive integrin ectodomain and an activating talin/beta complex that overlap with the alphabeta transmembrane complex, a comprehensive picture of integrin bi-directional transmembrane signaling has emerged.

Figure 1

Amino acid sequence of integrin αIIb and β3 transmembrane segments and flanking regions. Membrane-embedded residues,,,, are enclosed by a gray box. Residues 991–995 constitute the highly conserved GFFKR sequence motif of integrin α subunits.

Figure 2

NMR structures of the individual integrin αIIb and β3 transmembrane segments. (A) Structures of the monomeric αIIb and β3 TM segments (PDB entries 2k1a and 2rmz, respectively) and their estimated membrane embedding.,,, Selected side chains are shown in ball-and-stick representation. (B) An αIIb-homologous GFF structural motif is found in glycogen phosphorylase b (GPb; PDB entry 1a8i; residues 748–750). The αIIb TM segment and pertinent GPb helix are shown in blue and green, respectively. (C) For αIIb (blue), structural homology was found for a TM helix of cytochrome c oxidase (PDB entry 1xme, chain A), shown in green. The side chains of αIIb(F992–F993) and 1xme-A (A129-T130) are shown in ball-and-stick representation. F992 and A129 are structurally homologous. (D) The β peptide from the light-harvesting protein B-800/850 (homologous PDB entries 2fkw, chain S; 1nkz, chain F; and 1kzu, chain B also exhibits homology to αIIb. Shown is the structural alignment of integrin αIIb(P965–R995), in blue, with chain F of 1nkz, in green. The side chains of αIIb(F992–F993) and 1nkz-F(W39–L40) are shown in ball-and-stick representation. F993 and W39 are structurally homologous. The experimentally determined average positions of the lipid carbonyl, phosphate and choline groups (16, 20 and 22 Å from the center of the membrane) are depicted for 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine. The bulk water phase is assumed to be fully established at 28 Å.

Figure 3

NMR structure of the integrin αIIbβ3 transmembrane complex. The locations of the outer and inner membrane clasps, OMC and IMC, respectively, are indicated. The structure illustrates the intersubunit distance (NOE) restraints used to calculate the αIIbβ3 TM complex. For clarity, interproton NOE connectivities are denoted by dotted black lines between the carbon atoms that are covalently bonded to the hydrogen nuclei, giving rise to the NOEs. The structure shown is the average structure of an ensemble of 20 structures calculated without an αIIb(R995)-β3(D723) structural constraint. It exhibits a negligible backbone r.m.s.d. of 0.32 Å to the average structure calculated with such a restraint (PDB entry 2k9j). The depicted orientations are related by a rotation of ∼180° about the y axis.

Figure 4

Integrin bi-directional signaling. (A) Model of a resting integrin receptor. The αVβ3 ectodomain crystal structure was fused to an αVβ3 TM model based on the αIIbβ3 TM complex NMR structure., The α and β subunits are shown in blue and red, respectively. (B) Model of an active integrin conformation. The α(Calf-1/2) and β(TD/IE2-4) domains of the integrin ectodomain37 are shown dissociated with a schematic active headpiece. The dissociated β TM segment was overlaid with the activating talin2/β1D crystal structure (PDB entry 3g9w). Talin2 is shown in green and the direction of its effected β TM segment reorientation21 is indicated by green arrows. Two orientations, which are related by a rotation of ∼90° about the y axis, are shown for both models.