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Homo-and hetero-oligomeric interactions between the transmembrane (TM) helices of integrin ␣ and ␤ subunits may play an important role in integrin activation and clustering. As a first step to understanding these interactions, we used the TOXCAT assay to measure oligomerization of the wild-type ␣ IIb TM helix and single-site TM domain mutants. TOXCAT measures the oligomerization of a chimeric protein containing a TM helix in the Escherichia coli inner membrane via the transcriptional activation of the gene for chloramphenicol acetyltransferase. We found the amount of chloramphenicol acetyltransferase induced by the wild-type ␣ IIb TM helix was approximately half that induced by the strongly dimerizing TM helix of glycophorin A, confirming that the ␣ IIb TM domain oligomerizes in biological membranes. Mutating each of the ␣ IIb TM domain residues to either Ala, Leu, Ile, or Val revealed that a GXXXG motif mediates oligomerization. Further, we found that the residue preceding each glycine contributed to the oligomerization interface, as did the residue at position i ؉ 4 after the second Gly of GXXXG. Thus, the sequence XXVGXXGGXXXLXX is critical for oligomerization of ␣ IIb TM helix. These data were used to generate an atomic model of the ␣ IIb homodimer, revealing a family of structures with right-handed crossing angles of 40°to 60°, consistent with a 4.0-residue periodicity, and with an interface rotated by 50°relative to glycophorin A. Thus, although the ␣ IIb TM helix makes use of the GXXXG framework, neighboring residues have evolved to engineer its dimerization interface, enabling it to subserve specific and specialized functions.By interacting with macromolecular extracellular ligands, integrins mediate essential cell-cell and cell-matrix interactions (1). Further, integrin occupancy transduces information into the cell interior that regulates processes as diverse as cytoskeletal organization, cell migration, cell proliferation, and cellular differentiation, whereas signals initiated within the cell regulate the ability of integrins to interact with ligands. The structural basis for integrin regulation is an area of intense study. For several integrins, there is a correlation between their activation state and the relative positions of the cytoplasmic (CYTO) 1 domains of their ␣ and ␤ subunits (2-5). Thus, these integrins are inactive when their CYTO domains are in proximity and are active when the domains are far apart (6). Binding of cytoplasmic proteins such as talin to the cytoplasmic domain of one subunit or the other may influence this equilibrium, providing additional opportunities for regulating the activation state of the integrin (7).The role of transmembrane (TM) domains in the integrin activation process is currently not well understood. On the basis of in vacuo molecular modeling, Gottschalk et al. (8) proposed a model in which the ␣ and ␤ subunit TM domains interact extensively in both the active and inactive states, with the interaction pattern changing during activation. Other models based...

Homomeric and heteromeric interactions between the ␣IIb and ␤3 transmembrane domains are involved in the regulation of integrin ␣IIb␤3 function. These domains appear to interact in the inactivated state but separate upon integrin activation. Moreover, homomeric interactions may increase the level of ␣IIb␤3 activity by competing for the heteromeric interaction that specifies the resting state. To test this model, a series of mutants were examined that had been shown previously to either enhance or disrupt the homomeric association of the ␣IIb transmembrane domain. One mutation that enhanced the dimerization of the ␣IIb transmembrane domain indeed induced constitutive ␣IIb␤3 activation. However, a series of mutations that disrupted homodimerization also led to ␣IIb␤3 activation. These results suggest that the homo-and heterodimerization motifs overlap in the ␣IIb transmembrane domain, and that mutations that disrupt the ␣IIb͞␤3 transmembrane domain heterodimer are sufficient to activate the integrin. The data also imply a mechanism for ␣IIb␤3 regulation in which the integrin can be shifted from its inactive to its active state by destabilizing an ␣IIb͞␤3 transmembrane domain heterodimer and by stabilizing the resulting ␣IIb and ␤3 transmembrane domain homodimers.␣IIb␤3 ͉ integrin regulation ͉ transmembrane domains I ntegrins reside on cell surfaces in an equilibrium between inactive and active conformations that can be shifted in either direction by altering the distance between the stalks that anchor integrins in cell membranes (1). At the cellular level, integrin activation is regulated by cellular agonists, but how this occurs is uncertain. In the case of the platelet integrin ␣IIb␤3, membrane-proximal segments of the ␣IIb and ␤3 cytoplasmic (CYT) domains are thought to directly interact to constrain the integrin in an inactive state (2). Agonist-stimulated talin binding to the ␤3 CYT domain may relieve this constraint, inducing ␣IIb␤3 activation (3).The ␣IIb and ␤3 transmembrane domains are also in proximity when the integrin is inactive and separate upon integrin activation (4). Moreover, these domains readily undergo homomeric interactions in micelles (5), and both homomeric and heteromeric interactions have been detected in biological membranes (6, 7). Thus, in the platelet membrane where the concentration of these domains is high, the ␣IIb and ␤3 helices might be expected to form homooligomers in the activated state, crosslinking individual molecules and stabilizing focal adhesions (8). Indeed, we tested this possibility previously by placing Asn, a residue known to strengthen homomeric transmembrane (TM) interactions (9, 10), at successive positions across a 10-residue segment of the ␤3 TM domain and found that mutations along one face of the helix led to constitutive ␣IIb␤3 activation and integrin clustering (11).However, there are two distinct mechanisms by which TM domain mutations might activate integrins. Besides increasing the tendency of a highly expressed integrin to form homooligomers, TM domain ...

Previous studies suggest that the toxic soluble-oligomeric form of different amyloid proteins share a common backbone conformation, but the amorphous nature of this oligomer prevents its structural characterization by experiment. Based on molecular dynamics simulations we proposed that toxic intermediates of different amyloid proteins adopt a common, nonstandard secondary structure, called α-sheet. Here we report the experimental characterization of peptides designed to be complementary to the α-sheet conformation observed in the simulations. We demonstrate inhibition of aggregation in two different amyloid systems, β-amyloid peptide (Aβ) and transthyretin, by these designed α-sheet peptides. When immobilized the α-sheet designs preferentially bind species from solutions enriched in the toxic conformer compared with non-aggregated, nontoxic species or mature fibrils. The designs display characteristic spectroscopic signatures distinguishing them from conventional secondary structures, supporting α-sheet as a structure involved in the toxic oligomer stage of amyloid formation and paving the way for novel therapeutics and diagnostics.DOI: http://dx.doi.org/10.7554/eLife.01681.001