Structural and functional studies of an insect membrane transporter

Background
The selection of target protein insensitive to insecticide by insect is reducing the efficiency of existing pest control toward food crops. Therefore, novel protein target for insecticide research is paramount for future food security (HEMINGWAY et al, 2002).
Acetylcholine (ACh) is synthesised in the nerve terminal cytoplasm by choline acetyltransferase (ChAT). It is then pumped into storage vesicles by vesicular acetylcholine transporter (VAChT) to be released into the synaptic clefts upon neuronal depolarisation (PRADO et al, 2002). The role of acetylcholine is restricted to the CNS in insect. VAChT inhibiting compounds containing the spiroindoline scaffold have shown to possess insecticidal activities in several insect species such as D. melanogaster (SLUDER et al, 2012).
VAChT is an antiporter, which utilises the proton gradient generated by ATPase, transporting one acetylcholine molecule for two proton (figure 1) (NGUYEN et al, 1998). Hydropathy studies predicts that VAChT have 12 transmembrane domains and a large intravesicular loop between domain I and II (ERICKSON et al, 1994). There is currently no crystal structural and functional assay available for VAChT.

Figure 1. Acetylcholine accumulation by VAChT
Aim
The aim of the project is to resolve the crystal structure of VAChT with known inhibitors and develop a functional assay. Identifying the amino acid residues important for substrate binding would be an impetus in driving the design of insecticidal compound for this protein.

Biochemical Characterisation
The expression and optimisation will be performed in S. cerevisiae initially. If no satisfactory yield of stable VAChT is obtained from yeast, the search will be extended to insect cell culture.
Once optimised expression condition have been established, VAChT will be characterised by binding assay with vesamicol (known inhibitor). A functional assay will be designed to further characterise VAChT activities by reconstituting the transporter and ATPase into a proteoliposome. Since the transport of ACh via VAChT involves no enzymatic reaction, an indirect detection method to detect the substrate will be required.

Biophysical Characterisation
The physical structure of VAChT will be resolved by X-ray crystallography with data collection conducted at the Diamond Light Source. Purified VAChT will be subjected to crystal trials to ascertain conditions suitable for protein crystallisation. MemGold sparse matrix screen will be the starting point of the investigation, in conjunction with detergents screening (i.e. DDM, LMNG etc.), crystallisation in lipid cubic phase (LCP) will also be considered. Crystallisation of the protein with and without inhibitor (vesamicol) will be attempted. Ideally the Apo and vesamicol bound structure can be resolved. Assay with novel inhibitors will also be performed in collaboration with Syngenta.

References

ERICKSON, J. D. et al. Functional identification of a vesicular acetylcholine transporter and its expression from a "cholinergic" gene locus. J Biol Chem, v. 269, n. 35, p. 21929-32, Sep 1994. ISSN 0021-9258.
HEMINGWAY, J.; FIELD, L.; VONTAS, J. An overview of insecticide resistance. Science, v. 298, n. 5591, p. 96-7, Oct 2002. ISSN 1095-9203.
NGUYEN, M. L.; COX, G. D.; PARSONS, S. M. Kinetic parameters for the vesicular acetylcholine transporter: two protons are exchanged for one acetylcholine. Biochemistry, v. 37, n. 38, p. 13400-10, Sep 1998. ISSN 0006-2960.
PRADO, M. A. et al. Regulation of acetylcholine synthesis and storage. Neurochem Int, v. 41, n. 5, p. 291-9, Nov 2002. ISSN 0197-0186.
SLUDER, A. et al. Spiroindolines identify the vesicular acetylcholine transporter as a novel target for insecticide action. PLoS One, v. 7, n. 5, p. e34712, 2012. ISSN 1932-6203.