Unique transmembrane domain interactions differentially modulate integrin αvβ3 and αIIbβ3 function

Abstract

Lateral transmembrane (TM) helix–helix interactions between single-span membrane proteins play an important role in the assembly and signaling of many cell-surface receptors. Often, these helices contain two highly conserved yet distinct interaction motifs, arranged such that the motifs cannot be engaged simultaneously. However, there is sparse experimental evidence that dual-engagement mechanisms play a role in biological signaling. Here, we investigate the function of the two conserved interaction motifs in the TM domain of the integrin β3-subunit. The first motif uses reciprocating “large-large-small” amino acid packing to mediate the interaction of the β3 and αIIb TM domains and maintain the inactive resting conformation of the platelet integrin αIIbβ3. The second motif, S-x3-A-x3-I, is a variant of the classical “G-x3-G” motif. Using site-directed mutagenesis, optical trap-based force spectroscopy, and molecular modeling, we show that S-x3-A-x3-I does not engage αIIb but rather mediates the interaction of the β3 TM domain with the TM domain of the αv-subunit of the integrin αvβ3. Like αIIbβ3, αvβ3 on circulating platelets is inactive, and in the absence of platelet stimulation is unable to interact with components of the subendothelial matrix. However, disrupting any residue in the β3 S-x3-A-x3-I motif by site-directed mutations is sufficient to induce αvβ3 binding to the αvβ3 ligand osteopontin and to the monoclonal antibody WOW-1. Thus, the β3-integrin TM domain is able to engage in two mutually exclusive interactions that produce alternate α-subunit pairing, creating two integrins with distinct biological functions.

Breaking the Backbone: Central Arginine Residues Induce Membrane Exit and Helix Distortions within a Dynamic Membrane Peptide

“…Single transmembrane model helices reveal significant biophysical features for larger membrane proteins and are especially important for understanding (a) single-span membrane proteins − and (b) polar or charged functional groups that are in direct contact with the lipids. ,,, Limitations arise when the interhelix protein–protein structural interactions in multihelix bundles , are not represented by the individual helices considered here. Nevertheless, the molecular features that underlie protein–lipid interactions, individual helix stability, and dynamics are well represented. , …”

“…Due to numerous experimental challenges with large membrane proteins, simplified model systems can be useful for understanding the physical chemistry of the lipid interactions of ionizable protein side chains. The membranes p a n n i n g p e p t i d e G W 5 , 1 9 A L P 2 3 ( a c e t y l -GGALW 5 (LA) 6 LW 19 LAGA-amide), 7 for example, has been useful for defining the titration properties of membraneimbedded ionizable Arg, Lys, His, and Glu residues. 8−10a GW 5,19 ALP23 is advantageous because its robust helix adopts a well-defined orientation within lipid bilayers, wherein the helix tilt is dependent on membrane acyl chain length.…”

“…The membranes p a n n i n g p e p t i d e G W 5 , 1 9 A L P 2 3 ( a c e t y l -GGALW 5 (LA) 6 LW 19 LAGA-amide), 7 for example, has been useful for defining the titration properties of membraneimbedded ionizable Arg, Lys, His, and Glu residues. 8−10a GW 5,19 ALP23 is advantageous because its robust helix adopts a well-defined orientation within lipid bilayers, wherein the helix tilt is dependent on membrane acyl chain length. 11 The chain length dependence is likely a result of helix adaptation to the lipid-peptide hydrophobic mismatch.…”

“…Molecular dynamics simulations additionally predict that the helix movement is accompanied by local membrane thinning. 14 By contrast, Arg placed at the deadcenter of the GW 5,19 ALP23 helix, at position 12, causes the helix to adopt multiple states. In DOPC membranes, both coarse-grained molecular simulations and solid-state NMR experiments predict the presence of three major states.…”

“…The third state is entirely different, with the complete helix exiting the lipid bilayer to adopt an interfacial orientation, perpendicular to the membrane normal. Modest amounts of cholesterol, about 10 mol %, in the DOPC membrane force essentially the entire R 12 GW 5,19 ALP23 population into this interfacial state. 15 The multistate features and membrane-exit property of R 12 GW 5,19 ALP23 are likely dictated by a Trp "cage" surrounding the R12 guanidium group, which effectively is trapped between the two aromatic side chains and restricted from favorable interactions with the bilayer head groups.…”