Structural and functional study of vesicular acetylcholine transporter (VAChT)

The need to study novel protein targets for insecticide research is paramount as insecticide-resistant pests are putting global food production at risk. Acetylcholine (ACh) is a major neurotransmitter in the insect’s central nervous system. Proteins in the ACh signalling pathway, such as the nicotinic acetylcholine receptor and acetylcholinesterase, have been heavily exploited as insecticide targets. The vesicular acetylcholine transporter (VAChT) is an integral membrane protein (IMP) belonging to the Major Facilitator Superfamily (MFS) and is an unexplored target in the ACh pathway. The aim of the project was to study VAChT structurally using X-ray crystallography, and to biochemically characterise the protein using ligand binding and transport assays. The production of VAChT in high enough quantities and purity for these studies was a significant challenge. Therefore, VAChT genes of various species have been expressed in yeast, bacterial and insect cell systems to increase the possibility of success. Yeast membrane containing VAChT was extracted from Saccharomyces cerevisiae and had the ability to bind a ligand, as confirmed by a radioligand binding assay. VAChT from Tribolium castaneum & Acyrthosiphon pisum displayed a high affinity towards the insecticidal compound – SYN01, with Kd values of 6 nM & 2.4 nM respectively. However, the purification of VAChT proved to be difficult and degradation was observed at various stages of the process. To overcome this issue, several methods to improve the stability of this membrane protein were investigated, including fusion partner proteins, transport deficient mutations and the use of high affinity ligands.Open Acces

Cryo-EM structure of the agonist-bound Hsp90-XAP2-AHR cytosolic complex

International audienceThe aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates a broad spectrum of (patho)physiological processes in response to numerous substances including pollutants, natural products and metabolites. However, the scarcity of structural data precludes understanding of how AHR is activated by such diverse compounds. Our 2.85 Å structure of the human indirubin-bound AHR complex with the chaperone Hsp90 and the co-chaperone XAP2, reported herein, reveals a closed conformation Hsp90 dimer with AHR threaded through its lumen and XAP2 serving as a brace. Importantly, we disclose the long-awaited structure of the AHR PAS-B domain revealing a unique organisation of the ligand-binding pocket and the structural determinants of ligand-binding specificity and promiscuity of the receptor. By providing structural details of the molecular initiating event leading to AHR activation, our study rationalises almost forty years of biochemical data and provides a framework for future mechanistic studies and structure-guided drug design

Experimental phasing opportunities for macromolecular crystallography at very long wavelengths

Despite recent advances in cryo-electron microscopy and artificial intelligence-based model predictions, a significant fraction of structure determinations by macromolecular crystallography still requires experimental phasing, usually by means of single-wavelength anomalous diffraction (SAD) techniques. Most synchrotron beamlines provide highly brilliant beams of X-rays of between 0.7 and 2 Å wavelength. Use of longer wavelengths to access the absorption edges of biologically important lighter atoms such as calcium, potassium, chlorine, sulfur and phosphorus for native-SAD phasing is attractive but technically highly challenging. The long-wavelength beamline I23 at Diamond Light Source overcomes these limitations and extends the accessible wavelength range to λ = 5.9 Å. Here we report 22 macromolecular structures solved in this extended wavelength range, using anomalous scattering from a range of elements which demonstrate the routine feasibility of lighter atom phasing. We suggest that, in light of its advantages, long-wavelength crystallography is a compelling option for experimental phasing