Conformational Propensities of Peptides Mimicking Transmembrane Helix 5 and Motif C in Wild-type and Mutant Vesicular Acetylcholine Transporters.

The work reported here investigates an important transmembrane helix (TM) and motif in the vesicular acetylcholine transporter (VAChT), which is a member of the major facilitator superfamily (MFS) (1). VAChT takes up acetylcholine (ACh) synthesized in the cytoplasm of nerve terminals and stores it for evoked secretion (2). Most members of the MFS, including VAChT, contain 12 TMs that are predominantly α-helical. There is no crystal structure of VAChT. However, nearly atomic resolution structures have been obtained for three bacterial members of the MFS, namely, lactose permease (LacY; PDB id 1PV6; 3.5 A resolution) (3), glycerol-3-phosphate transporter (GlpT; PDB id 1PW4; 3.3 A resolution) (4), and the bacterial multidrug resistance transporter EmrD (PDB id 2GFP; 3.5 A resolution) (5). Despite low sequence identity for these proteins, the three-dimensional structures of the polypeptide backbones are highly similar. This observation led to the hypothesis that all members of the MFS containing 12 TMs share the same fold (6−12). Accordingly, a homology model of VAChT has been proposed based on the GlpT structure (7). In the model, TMs 1−6 cluster together, as do TMs 7−12 (Figure ​(Figure1).1). Because the template is distantly related to VAChT, the model is approximate. A transport channel for the substrate lies between the two clusters approximately perpendicular to the plane of the membrane. The binding site for ACh lies somewhere in the channel, and it is hypothesized to access each side of the membrane in alternating fashion when the two clusters of TMs rock back and forth against each other. This type of conformational change has been termed the “rocker-switch”. Figure 1 VAChT homology model. The left frame is a side view from inside the membrane, and the right frame is a view from inside the synaptic vesicle looking through the bottom of the transport channel toward cytoplasm. The first cluster of six TMs is cyan, and ... The MFS includes a family of bacterial multidrug resistance transporters containing six conserved sequence “motifs” 7−13 residues long (13−16). All of the motifs are present in VAChT (17). Four of them occur in TMs, and three of those are rich in proline and glycine residues. Proline and glycine promote formation of “defects,” such as hinges that mediate kinking and wobbling, and formation of short segments of 310- and π-helix in otherwise α-helical TMs (18,19). The defects often mediate special functions (20−24). Motif C in TM 5 is especially interesting, because its consensus sequence is gxxxGPxxGGxl (capital ≥70% conservation, lower case ≥40% conservation, and x any residue). The consensus sequence for motif C in VAChT is G224SLVAPPFGGIL235 (boldface = invariant), where the numbering applies to human VAChT (hVAChT). This very highly conserved sequence contains invariant A228, which usually is G in the bacterial multidrug resistance transporter family. In a previous study from this laboratory, A228 in motif C of VAChT was mutated to glycine. The change leads to >3-fold increase in the turnover rate for ACh transport (17). Equilibrium affinities for binding of ACh and an allosteric inhibitor called vesamicol are not affected, indicating that the resting conformation of VAChT is not affected by the mutation. The results suggest that the mutation lowers the energy barrier for the rocker-switch conformational change. Notably, motif C is located at an interface with the second bundle of TMs, and it probably participates in rocker-switch reorientation of the ACh binding site (Figure ​(Figure11). Molecular dynamics (MD) simulation of intact VAChT would be of interest to assess the conformational properties of TM 5 and motif C in situ, but as noted above, only an approximate homology model of VAChT is available. MD simulation of an approximate model would be of limited utility. However, analysis of a peptide having the TM 5 and motif C sequence should be useful. MD simulation of polyalanine mimicking a TM systematically and sparsely substituted with proline and glycine residues predicts that the flexibility of motif C is increased by the A228G mutation (17,25) Statistical correlations of sequence pattern descriptors with atomic-resolution structures of integral membrane proteins also do (26). In the study reported here, MD techniques were used to characterize the intrinsic conformational propensities for peptides corresponding to TM 5 and motif C in wild-type and mutant hVAChTs.