The core aim of this project is the development of a comprehensive model of the transport cycle of the human dopamine transporter (DAT) and of the bacterial small amino acid transporter LeuT. While LeuT has been crystallised in three conformations, no crystal or cyro-EM structures of the human DAT is available. The availability of structures of the closely related human serotonin transporter and the drosophila DAT allow for building reliable models.
First, DAT models will be evaluated using structure quality assessment methods. These will then be tested against modern force fields using extensive molecular dynamics (MD) simulations, as is was shown that simulation are able to detect also small structural deficiencies. Simulations will be carried out in close collaboration with the Projects of NeuroTrans, which are experimentally investigating on the same transporter. Access to HPC clusters allows for extensive use of advanced simulation techniques. DAT and LeuT will be studied in parallel, because sharing the transport mechanics. Comparison allows for separating transporter specific properties from general valid principles. The main objective of Project 9 are:
- to study the conformational changes of the transport cycle. Advanced simulation methods including Metadynamics, Accelerated Weight Histogram and the Potential of Mean Force calculations will be used to study transporter dynamics.
- to identify energy barriers, which separate states along the path of substrate transport.
- to characterize ligand binding in DAT, which in case of substrate (dopamine) leads to translocation, in presence of inhibitors (cocaine) leads to an arrest, while releasers (amphetamines) invert the mode of action and lead to substrate efflux.
- to elucidate the effect of disease causing mutation on DAT structure, dynamics and function, studied in silico through µs long unbiased simulations. Theoretical studies will be carried out in close collaboration with the Projects experimentally studying the same mutations.