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Abstract:The glycophorin A transmembrane segment homodimerizes to a right-handed pair of a-helices. Here, we identified the amino acid motif mediating this interaction within a natural membrane environment. Critical residues were grafted onto two different hydrophobic host sequences in a stepwise manner and self-assembly of the hybrid sequences was determined with the ToxR transcription activator system. Our results show that the motif LIxxGxxxGxxxT elicits a level of self-association equivalent to that of the original glycophorin A transmembrane segment. This motif is very similar to the one previously established in detergent solution. Interestingly, the central GxxxG motif by itself already induced strong self-assembly of host sequences and the three-residue spacing between both glycines proved to be optimal for the interaction. The GxxxG element thus appears to be the most crucial part of the interaction motif.

Specific interactions between ␣-helical transmembrane segments are important for folding and/or oligomerization of membrane proteins. Previously, we have shown that most transmembrane helix-helix interfaces of a set of crystallized membrane proteins are structurally equivalent to soluble leucine zipper interaction domains. To establish a simplified model of these membrane-spanning leucine zippers, we studied the homophilic interactions of artificial transmembrane segments using different experimental approaches. Importantly, an oligoleucine, but not an oligoalanine, sequence efficiently self-assembled in membranes as well as in detergent solution. Self-assembly was maintained when a leucine zipper type of heptad motif consisting of leucine residues was grafted onto an alanine host sequence. Analysis of point mutants or of a random sequence confirmed that the heptad motif of leucines mediates self-recognition of our artificial transmembrane segments. Further, a data base search identified degenerate versions of this leucine motif within transmembrane segments of a variety of functionally different proteins. For several of these natural transmembrane segments, self-interaction was experimentally verified. These results support various lines of previously reported evidence where these transmembrane segments were implicated in the oligomeric assembly of the corresponding proteins.In any type of cell, a multitude of integral membrane proteins is simultaneously synthesized and integrated into various membranes followed by association to homo-or heterooligomeric complexes. To ensure specific assembly, their subunits must present complementary recognition domains to each other. These domains may be located on the ectodomains and/or the transmembrane segments (TMSs).1 Interactions between TMSs are currently intensely studied, since they usually form autonomous ␣-helices and have been found to direct subunit assembly or support correct folding of many membrane proteins (1, 2). Biochemical and functional analyses, molecular modeling, and structural studies indicated that the self-assembly of transmembrane helices is driven by a close packing of their characteristically shaped surfaces. These packing interactions may result in pairs of ␣-helices with a right-handed twist as exemplified by glycophorin A (3, 4) and probably by synaptobrevin II (5). Other TMS interactions involve a leucine zipper type of side-chain packing as known from certain soluble proteins. Within soluble leucine zippers, the interacting residues form repeated heptad (abcdefg) motifs. Residues at a-and d-positions constitute the hydrophobic core of the interfaces; side-chains at the e-and g-positions are frequently charged, form salt bridges to each other, and make hydrophobic contacts to the core (6). Heptad motifs were also suggested to form the TMS interfaces of phospholamban (7, 8) and the M2 proton channel (9). Based on a quantitative evaluation of high resolution structures, we recently confirmed previous observations (10, 11) in demonstrating that ...

Assembly of the SNARE proteins synaptobrevin/ VAMP, syntaxin, and SNAP-25 to binary and ternary complexes is important for docking and/or fusion of presynaptic vesicles to the neuronal plasma membrane prior to regulated neurotransmitter release. Despite the well characterized structure of their cytoplasmic assembly domains, little is known about the role of the transmembrane segments in SNARE protein assembly and function. Here, we identified conserved amino acid motifs within the transmembrane segments that are required for homodimerization of synaptobrevin II and syntaxin 1A. Minimal motifs of 6 -8 residues grafted onto an otherwise monomeric oligoalanine host sequence were sufficient for self-interaction of both transmembrane segments in detergent solution or membranes. These motifs constitute contiguous areas of interfacial residues assuming ␣-helical secondary structures. Since the motifs are conserved, they also contributed to heterodimerization of synaptobrevin II and syntaxin 1A and therefore appear to constitute interaction domains independent of the cytoplasmic coiled coil regions. Interactions between the transmembrane segments may stabilize the SNARE complex, cause its multimerization to previously observed multimeric superstructures, and/or be required for the fusogenic activity of SNARE proteins.Intracellular membrane fusion events, e.g. constitutive organelle traffic or Ca 2ϩ -regulated neurotransmitter release, require conserved sets of membrane proteins, designated SNAREs.1 The best characterized SNAREs are those mediating exocytosis of synaptic vesicles in neurons (reviewed in Refs. 1-4). In detergent extracts from presynaptic nerve terminals, the single-span integral membrane SNAREs synaptobrevin (also referred to as VAMP) and syntaxin together with the peripheral membrane SNARE protein SNAP-25 form a stable ternary complex that is disassembled in vitro after binding of soluble ␣-SNAP by the ATPase NSF (5, 6). According to the original SNARE hypothesis (7), interaction between these SNARE partners bridges opposing vesicular and plasma membranes. Therefore, assembly and disassembly of ternary SNARE complexes would proceed in a trans-configuration that is regarded essential for vesicle docking, priming and/or fusion (8 -12). On the other hand, SNARE complexes are also found on the vesicular (13, 14) as well as the plasma (15) membrane in a cis-configuration, i.e. side by side. Protein domains involved in the binary and ternary interactions leading to SNARE complex formation have been originally identified by in vitro binding studies using recombinant soluble fragments of synaptobrevin II, syntaxin 1A, and SNAP-25 as follows: (i) the cytoplasmic domain of synaptobrevin II; (ii) a carboxyl-terminal, membrane-proximal region of syntaxin 1A; and (iii) carboxyl-terminal plus amino-terminal regions of SNAP-25 (16 -21). More recently, structural studies confirmed that previously predicted cytoplasmic coiled coil domains of synaptobrevin II (H1, H2) and syntaxin 1A (H3) form a tightly packed parallel fo...

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