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BackgroundProteins of the tetraspanin family contain four transmembrane domains (TM1-4) linked by two extracellular loops and a short intracellular loop, and have short intracellular N- and C-termini. While structure and function analysis of the larger extracellular loop has been performed, the organization and role of transmembrane domains have not been systematically assessed.ResultsAmong 28 human tetraspanin proteins, the TM1-3 sequences display a distinct heptad repeat motif (abcdefg)n. In TM1, position a is occupied by structurally conserved bulky residues and position d contains highly conserved Asn and Gly residues. In TM2, position a is occupied by conserved small residues (Gly/Ala/Thr), and position d has a conserved Gly and two bulky aliphatic residues. In TM3, three a positions of the heptad repeat are filled by two leucines and a glutamate/glutamine residue, and two d positions are occupied by either Phe/Tyr or Val/Ile/Leu residues. No heptad motif is apparent in TM4 sequences. Mutations of conserved glycines in human CD9 (Gly25 and Gly32 in TM1; Gly67 and Gly74 in TM2) caused aggregation of mutant proteins inside the cell. Modeling of the TM1-TM2 interface in CD9, using a novel algorithm, predicts tight packing of conserved bulky residues against conserved Gly residues along the two helices. The homodimeric interface of CD9 was mapped, by disulfide cross-linking of single-cysteine mutants, to the vicinity of residues Leu14 and Phe17 in TM1 (positions g and c) and Gly77, Gly80 and Ala81 in TM2 (positions d, g and a, respectively). Mutations of a and d residues in both TM1 and TM2 (Gly25, Gly32, Gly67 and Gly74), involved in intramolecular TM1-TM2 interaction, also strongly diminished intermolecular interaction, as assessed by cross-linking of Cys80.ConclusionOur results suggest that tetraspanin intra- and intermolecular interactions are mediated by conserved residues in adjacent, but distinct regions of TM1 and TM2. A key structural element that defines TM1-TM2 interaction in tetraspanins is the specific packing of bulky residues against small residues.

Lymphocytic choriomeningitis virus (LCMV), a rodentborne arenavirus, is a rare, zoonotic cause of aseptic meningitis in Europe and North America. It is carried by the common house mouse (Mus musculus), [...]

The integrin [alpha]IIb[beta]3 is a transmembrane (TM) heterodimeric adhesion receptor that exists in equilibrium between resting and active ligand binding conformations. In resting [alpha]IIb[beta]3, the TM and cytoplasmic domains of [alpha]IIb and [beta]3 form a heterodimer that constrains [alpha]IIb[beta]3 in its resting conformation. To study the structure and dynamics of the cytoplasmic domain heterodimer, we prepared a disulfide-stabilized complex consisting of portions of the TM domains and the full cytoplasmic domains. NMR and hydrogendeuterium exchange of this complex in micelles showed that the [alpha]IIb cytoplasmic domain is largely disordered, but it interacts with and influences the conformation of the [beta]3 cytoplasmic domain. The [beta]3 cytoplasmic domain consists of a stable proximal helix contiguous with the TM helix and two distal amphiphilic helices. To confirm the NMR structure in a membrane-like environment, we studied the [beta]3 cytoplasmic domain tethered to phospholipid bilayers. Hydrogen-deuterium exchange mass spectrometry, as well as circular dichroism spectroscopy, demonstrated that the [beta]3 cytoplasmic domain becomes more ordered and helical under these conditions, consistent with our NMR results. Further, these experiments suggest that the two distal helices associate with lipid bilayers but undergo fluctuations that would allow rapid binding of cytoplasmic proteins regulating integrin activation, such as talin and kindlin-3. Thus, these results provide a framework for understanding the kinetics and thermodynamics of protein interactions involving integrin cytoplasmic domains and suggest that such interactions act in a concerted fashion to influence integrin stalk separation and exposure of extracellular ligand binding sites. platelets | fibrinogen receptors | receptor regulation doi/ 10.1073/pnas.1015545107

Federal Circuit opinions have long espoused the maxim that patents can claim genera of antibodies, only to invalidate two such patents in Chiron Corp. v. Genentech, Inc. and Centocor Ortho Biotech, Inc. v. Abbott Laboratories, disputes over rights to multibillion-dollar pharmaceuticals. This Update resolves the intra-circuit split by characterizing an implicit distinction in Federal Circuit precedent: method-of-use patents may claim genera of antibodies while composition patents enjoy less freedom to make broad claims. This distinction fostered two new patenting strategies. A conservative approach claims specific antibodies rather than genera and threatens enforcement pursuant to the doctrine of equivalents, which cedes the patent's fate to the jury. An aggressive approach claims genera of antibodies in method-of-use patents, but risks patent invalidation should the Federal Circuit extend Chiron and Centocor to method-of-use patents. Both strategies, however, remain untested in the courts. [ABSTRACT FROM AUTHOR]

Homomeric and heteromeric interactions between the αllb and β3 transmembrane domains are involved in the regUlation of integrin αIIbβ3 function. These domains appear to interact in the inactivated state but separate upon integrin activation. Moreover, homomeric interactions may increase the level of αllβ3 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 αllbβ3 transmembrane domain. One mutation that enhanced the dimerization of the αllbβ transmembrane domain indeed induced constitutive αllbβ3 activation. However, a series of mutations that disrupted homodimerization also led to αllbβ3 activation. These results suggest that the homo- and heterodimerization motifs overlap in the αllbβ3 transmembrane domain, and that mutations that disrupt the αllbβ3 transmembrane domain heterodimer are sufficient to activate the integrin. The data also imply a mechanism for αllbβ3 regulation in which the integrin can be shifted from its inactive to its active state by destabilizing an αllbβ3 transmembrane domain heterodimer and by stabilizing the resulting αllb and β3 transmembrane domain homodimers. [ABSTRACT FROM AUTHOR]

The integrin αIIbβ3 is a transmembrane (TM) heterodimeric adhesion receptor that exists in equilibrium between resting and active ligand binding conformations. In resting αIIbβ3, the TM and cytoplasmic domains of αIIb and β3 form a heterodimer that constrains αIIbβ3 in its resting conformation. To study the structure and dynamics of the cytoplasmic domain heterodimer, we prepared a disulfide-stabilized complex consisting of portions of the TM domains and the full cytoplasmic domains. NMR and hydrogen-deuterium exchange of this complex in micelles showed that the αIIb cytoplasmic domain is largely disordered, but it interacts with and influences the conformation of the β3 cytoplasmic domain. The β3 cytoplasmic domain consists of a stable proximal helix contiguous with the TM helix and two distal amphiphilic helices. To confirm the NMR structure in a membrane-like environment, we studied the β3 cytoplasmic domain tethered to phospholipid bilayers. Hydrogen-deuterium exchange mass spectrometry, as well as circular dichroism spectroscopy, demonstrated that the β3 cytoplasmic domain becomes more ordered and helical under these conditions, consistent with our NMR results. Further, these experiments suggest that the two distal helices associate with lipid bilayers but undergo fluctuations that would allow rapid binding of cytoplasmic proteins regulating integrin activation, such as talin and kindlin-3. Thus, these results provide a framework for understanding the kinetics and thermodynamics of protein interactions involving integrin cytoplasmic domains and suggest that such interactions act in a concerted fashion to influence integrin stalk separation and exposure of extracellular ligand binding sites.