The project will investigate the dynamics of enzyme action and inhibition, and address questions of structure and function, which go beyond the static picture provided by classical X-ray diffraction techniques. In particular, it will use time-resolved, and ultra-rapid freesing X-ray crystallography, to obtain structural information on transient enzyme-ligand complexes. The information gained will considerably advance our knowledge of, and ability to manipulate, protein ligand interactions. The enzyme chosen for these studies, Acetylcholinesterase (AChE, EC 18.104.22.168), is a highly efficient hydrolase whose biological role is the termination of impulse transmission at cholinergic synapses. Its high catalytic efficiency provides a challenge for new method development and improved data collection procedures, which should be widely applicable to the study of other enzymes.
The only crystal structure of AChE known to date is that of the Torpedo enzyme. This reveals many unexpected features; the active site is buried at the bottom of a deep and narrow gorge (which seems incompatible with the high catalytic efficiency); the enzyme has a very large dipole moment oriented along the gorge (which might attract cationic substrates into the active site); and binding to both charged and hydrophobic substrates and inhibitors is mediated largely by aromatic residues. These observations raise important questions about the AChE mechanism of action; the traffic of substrate and products through the active-site gorge and the modes of inhibitor binding.
An important aim of the project is to determine new X-ray structures of AChE enzymes from both vertebrate and insect sources. In combination with biochemical studies, which will be carried out on enzymes kinetically distinct from the Torpedo AChE, this will substantially improve understanding of structure-function relationships in this system.
The large quantities of pure enzyme required by the project will be produced using Baculovirus expression systems. This will include the expression of genes from insects with natural resistance to AChE inhíbitors. This gene pool will be used as a natural source of functionally altered AChE enzymes. In addition, site directed mutagenesis will be used to produce modified enzymes in a rational manner.
Molecular mechanic and molecular dynamic modelling techniques will be employed to analyse structural data on AChE and its complexes with inhibitors, to investigate the thermodynamic and dynamic principles involved, and the role of electrostatic interactions. Innovative use will be made of molecular dynamics techniques to improve the speed of exploring conformational space. In addition, molecular modelling will be used to build the 3D structures of related enzymes from other species, of enzymes with designed sequence modifications, and of complexes with various inhibitors. The knowledge acquired will be applied to design novel selective inhibitors. To support this work, crystal doping experiments will be carried out to obtain structural information on selected enzyme-inhibitor complexes. This combined approach may lead to the discovery of novel insecticides or pharmaceutical agents. The involvement of an industrial partner will optimise the chances of making exploitable discoveries. To achieve the set goals, the project brings together a unique group of laboratories, which are highly qualified in their disciplines and have a track record of interdisciplinary and international collaboration.
Funding SchemeCSC - Cost-sharing contracts
AL5 2JQ Harpenden
3584 CH Utrecht