Increased stability of the TM helix oligomer abrogates the apoptotic activity of the human Fas receptor.

Human death receptors control apoptotic events during cell differentiation, cell homeostasis and the elimination of damaged or infected cells. Receptor activation involves ligand-induced structural reorganizations of preformed receptor trimers. Here we show that the death receptor transmembrane domains only have a weak intrinsic tendency to homo-oligomerize within a membrane, and thus these domains potentially do not significantly contribute to receptor trimerization. However, mutation of Pro183 in the human CD95/Fas receptor transmembrane helix results in a dramatically increased interaction propensity, as shown by genetic assays. The increased interaction of the transmembrane domain is coupled with a decreased ligand-sensitivity of cells expressing the Fas receptor, and thus in a decreased number of apoptotic events. Mutation of Pro183 likely results in a substantial rearrangement of the self-associated Fas receptor transmembrane trimer, which likely abolishes further signaling of the apoptotic signal but may activate other signaling pathways. Our study shows that formation of a stable Fas receptor transmembrane helix oligomer does not per se result in receptor activation.

In vivo selection of heterotypically interacting transmembrane helices: Complementary helix surfaces, rather than conserved interaction motifs, drive formation of transmembrane hetero-dimers.

Single pass transmembrane proteins make up almost half of the whole transmembrane proteome. Contacts between such bitopic transmembrane proteins are common, and oligomerization of their single transmembrane helix is involved in triggering and regulation of signal transduction across cell membranes. In several recent analyses the distribution of amino acids at helix-helix contact sides has been analyzed, and e.g. a preference of amino acids with small side chains has been identified. Here we select amino acids, amino acid pairings and amino acid motifs, which mediate strong interactions of single-span transmembrane α-helices. Our analysis illustrates an architecture of TM helix dimers that is much more complex and diverse as might be expected from previous screens selecting homo-dimerizing TM helices. However, our findings are in excellent agreement with several previous computational analyses of existing transmembrane proteins and thus indicate that our screen nicely resembled the forces having guided evolution of transmembrane bundle structures. Furthermore, the results of this study indicate that helices do not per se have a strong propensity to interact via identical or highly similar helix surfaces, rather the geometries of two interacting helix surfaces "just" have to match to tightly pack and thereby form a stable transmembrane helix dimer. Finally, while evolution of transmembrane helix-helix interactions most likely was a compromise between formation of thermodynamically stable contact surfaces and protein function, our results suggest that "stability" was a major driving force during the evolution of α-helical transmembrane proteins.

Genetic systems for monitoring interactions of transmembrane domains in bacterial membranes.

In recent years several systems have been developed to study interactions of TM domains within the inner membrane of the Gram-negative bacterium Escherichia coli. Mostly, a transmembrane domain of interest is fused to a soluble DNA-binding domain, which dimerizes in E. coli cytoplasm after interactions of the transmembrane domains. The dimeric DNA-binding domain subsequently binds to a promoter/operator region and thereby activates or represses a reporter gene. In 1996 the first bacterial system has been introduced to measure interactions of TM helices within a bacterial membrane, which is based on fusion of a transmembrane helix of interest to the DNA-binding domain of the Vibrio cholerae ToxR protein. Interaction of a transmembrane helix of interest within the membrane environment results in dimerization of the DNA-binding domain in the bacterial cytoplasm, and the dimeric DNA-binding domain then binds to the DNA and activates a reporter gene. Subsequently, systems with improved features, such as the TOXCAT- or POSSYCCAT system, which allow screening of TM domain libraries, or the GALLEX system, which allows measuring heterotypic interactions of TM helices, have been developed and successfully applied. Here we briefly introduce the currently most applied systems and discuss their advantages together with their limitations.

Isolation of the silicatein-α interactor silintaphin-2 by a novel solid-phase pull-down assay.

The skeleton of siliceous sponges consists of amorphous biogenous silica (biosilica). Biosilica formation is driven enzymatically by means of silicatein(s). During this unique process of enzymatic polycondensation, skeletal elements (spicules) that enfold a central proteinaceous structure (axial filament), mainly comprising silicatein, are formed. However, only the concerted action of silicatein and other proteins can explain the genetically controlled diversity of spicular morphotypes, from simple rods with pointed ends to intricate structures with up to six rays. With the scaffold protein silintaphin-1, a first silicatein interactor that facilitates the formation of the axial filament and, consequently, of the growing spicule was discovered. In this study, a new interactor has been identified by both a conventional yeast two-hybrid library screening and a newly established pull-down assay. For the latter approach, silicatein-α has been bioengineered to carry a Glu tag, which confers binding affinity to hydroxyapatite. After immobilization on a solid-phase matrix (hydroxyapatite), the Glu-tagged silicatein was used as bait for the identification of interactors. Both approaches revealed a 15 kDa polypeptide, and its identity was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Colocalization of silintaphin-2 and silicatein-α within the axial filament and on the spicule surface was shown by immunohistological analyses. Subsequent autoradiography demonstrated the Ca(2+) binding affinity of this silicatein interactor. These findings indicate that both proteins operate in concert during spiculogenesis. Besides binding of calcium, silintaphin-2 shares several structural features with certain acidic, secreted extracellular matrix proteins that facilitate tissue mineralization in Metazoa. Hence, silintaphin-2 might mediate signal transduction during spiculogenesis or may play a more direct role during biosilica formation, in concert with silicatein.