The vesicular acetylcholine transporter (VAChT) contains six conserved sequence motifs that are rich in proline and glycine. Because these residues can have special roles in the conformation of polypeptide backbone, the motifs might have special roles in conformational changes during transport. Using published bioinformatics insights, the amino acid sequences of the 12 putative, helical, transmembrane segments of wild-type and mutant VAChTs were analyzed for propensity to form non-a-helical conformations and molecular notches. Many instances were found. In particular, high propensity for kinks and notches are robustly predicted for motifs D2, C and C¢. Mutations in these motifs either increase or decrease V max for transport, but they rarely affect the equilibrium dissociation constants for ACh and the allosteric inhibitor, vesamicol. The near absence of equilibrium effects implies that the mutations do not alter the backbone conformation. In contrast, the V max effects demonstrate that the mutations alter the difficulty of a major conformational change in transport. Interestingly, mutation of an alanine to a glycine residue in motif C significantly increases the rates for reorientation across the membrane. These latter rates are deduced from the kinetics model of the transport cycle. This mutation is also predicted to produce a more flexible kink and tighter tandem notches than are present in wild-type. For the full set of mutations, faster reorientation rates correlate with greater predicted propensity for kinks and notches. The results of the study argue that conserved motifs mediate conformational changes in the VAChT backbone during transport.

To confirm that the cytochrome bc 1 complex exists as a dimer with intertwining Rieske iron-sulfur proteins in solution, four Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc 1 complexes containing two pairs of cysteine substitutions, one in the interface between the head domain of iron-sulfur protein (ISP) and cytochrome b and the other between the tail domain of ISP and cytochrome b, were generated and characterized. The cytochrome bc 1 complex (also known as ubiquinol-cytochrome c reductase or Complex III) is an essential segment of the electron transfer chains of mitochondria and many respiratory and photosynthetic bacteria (1). This complex catalyzes electron transfer from ubiquinol to cytochrome c and concomitantly translocates protons across the membrane to generate a membrane potential and pH gradient for ATP synthesis. The polypeptide composition of cytochrome bc 1 complexes from different sources varies from three to eleven subunits. The redox subunits: cytochrome b, cytochrome c 1 , and the Rieske ironsulfur protein (ISP 1 ), are conserved in all cytochrome bc 1 complexes.Recently, mitochondrial cytochrome bc 1 complexes from beef (2, 3), chicken (4), and yeast (5) were crystallized and their three-dimensional structure determined. These three-dimensional structures not only established the location of the redox centers, transmembrane helices, and inhibitor binding sites, but also showed an unexpected dimeric structural arrangement of this complex in the crystal (2-5). The two ISPs from the two monomers are intertwined; the head domain of ISP in one monomer is close to the cytochrome b and cytochrome c 1 in the other monomer. The molecule of ISP can be divided into three domains: head, tail, and neck, with the 2Fe-2S cluster located at the tip of the head (6, 7). In tetragonal I4 1 22 crystals of native oxidized bovine cytochrome bc 1 complex, the 2Fe-2S cluster in one monomer is 27 Å from heme b L of the other monomer and 40 Å from the heme b L of the same monomer (2). This structural arrangement suggests that mitochondrial bc 1 complex functions as a dimer, because the distance between the 2Fe-2S of ISP of one monomer and the low potential cytochrome b (b L ) of the other monomer is less than that between these groups in the same monomer. The shorter distance accommodates fast electron transfer from QH 2 to ISP and b L . However, a complex that exists as a dimer in the crystal might exist and function as a monomer in solution. Evidence for the cytochrome bc 1 complex functioning as a dimer or a monomer has been reported (8 -11). Therefore, it is important to establish whether or not the structure of the dimeric cytochrome bc 1 complex observed in the crystal also exists in solution.One way to address this question is to generate a mutant bc 1 complex that forms two intersubunit disulfide bonds, one at the interface between the head domain of ISP and cytochrome b and the other at the proximity of the tail domain of ISP and cytochrome b. If the complex exists as a dimer with intertw...

Invariant E309 is in contact with critical and invariant D398 in a three-dimensional homology model of vesicular acetylcholine transporter (VAChT, TC# 2.A.1.2.13). In the work reported here, E309 and D398 in human VAChT were mutated singly and together to test their functions, assign pK values to them, and determine whether the residues are close to each other in three-dimensional space. Mutants were stably expressed in the PC12 A123.7 cell line, and transport and binding properties were characterized at different pH values using radiolabeled ligands and filtration assays. Contrary to a prior conclusion, the results demonstrate that most D398 mutants do not bind the allosteric inhibitor vesamicol even weakly. Earlier work showed that most D398 mutants do not transport ACh. D398 therefore probably is the residue that must deprotonate with pK = 6.5 for binding of vesamicol and with pK ~5.9 for transport of ACh. Because E309Q has no effect on VAChT functions at physiological pH, E309 has no apparent critical role. However, radical mutations in E309 cause decreases in ACh and vesamicol affinities and total loss of ACh transport. Unlike wild-type VAChT, which exhibits a peak of [ 3 H]vesamicol binding centered at pH 7.4, the mutants E309Q, E309D, E309A, and E309K all exhibit peaks of binding centered at pH ≥9. The combination of high pH and mutated E309 apparently produces a relaxed (in contrast to tense) conformation of VAChT that binds vesamicol exceptionally tightly. No compensatory interactions between E309 and D398 in double mutants were discovered. Proof of a close spatial relationship between E309 and D398 was not found. Nevertheless, the data are more consistent with the homology model than an alternative hydropathy model of VAChT that likely locates E309 far away in three-dimensional space from D398 and the ACh binding site. Also, a probable network of interactions involving E309 and an unknown residue having pK = 10 has been revealed. Vesicular acetylcholine transporter (VAChT, TC# 2.A.1.2.13) 1 is found in the membranes of synaptic vesicles inside of nerve terminals that release acetylcholine (ACh). It transports ACh from cytoplasm to the inside of the vesicles, thereby preparing ACh for exocytotic release. Protons translocate from inside the vesicle through VAChT to cytoplasm and drive the transport cycle (1,2). Residues that could mediate translocation of protons thus are of special interest in the ACh transport mechanism. A man-made compound called vesamicol binds with high affinity to a saturable allosteric site and thereby inhibits ACh binding and transport. It has been very useful in elucidating VAChT properties (3).Based on hydropathy analysis of the amino acid sequence, twelve transmembrane helices (TMs) are predicted for VAChT. A number of the TMs contain invariant ionic or potentially ionic residues (4). VAChT is closely related to vesicular monoamine transporters (VMATs) 1 † This research was supported by Grant NS15047 from the National Institute of Neurological Disorders and Stroke. * T...

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