Diamond’s eBIC provides novel insights into Parkinson’s disease

Artistic montage showing the atomic model of TH in the centre surrounded by dopamine molecules, which stabilise the N-terminal α-helices (highlighted in dark blue) in the four active centres of TH (only three of which are visible), blocking the active sites. The moving α-helix (marked by the orange arrow) illustrates how the four α-helices “float” outside the active centre in the absence of dopamine.

TH plays a pivotal role in the conversion of amino acid tyrosine into dopamine. It is known that dopamine interaction with TH inhibits the activity of TH, whilst phosphorylation at specific site reactivates the enzyme. However, understanding precisely how this regulatory mechanism functions at the molecular level requires a deeper understanding of the structure of full-length TH. This is key to developing therapies to target malfunctioning TH in Parkinsonisms. The structure of full-length TH had been elusive until now, remaining an analytical challenge.

Professor José Valpuesta explains:

Cryo-electron microscopy (cryo-EM) enables researchers to study proteins at atomic or near-atomic spatial resolutions, whilst also keeping the sample in a frozen-hydrated state. This minimises any chemical or structural changes to the sample which may occur during preparation. The latest advancements in cryo-EM are enabling researchers to significantly improve data quality.

Professor Valpuesta explains:

In this work, the team utilised the 300 kV Titan Krios cryo-electron microscope at Diamond’s eBIC facility to analyse the structure of recombinant human TH. The central structure of TH was shown to be formed from four sub-units (tetramer) with regulatory and catalytic domains (distinct functional regions of the enzyme structure) separated by a distance of 1.5 nanometres. This distance is approximately 200,000 times smaller than the diameter of a grain of salt!

The team also investigated differences in the structure of TH in the presence and absence of dopamine, noting some interesting changes at the enzyme’s active site. When in the presence of dopamine, an alpha helix protein structure, detached from the main TH complex in the absence of dopamine, was shown to penetrate the active site of TH. This enabled both visualisation of the alpha helix using cryo-EM and strong dopamine binding at the active site.

More information on the interactions between TH and dopamine were revealed using molecular dynamics simulations. The team showed that when dopamine is chemically bound to TH by the alpha helix, the function of the enzyme is inhibited. When TH is phosphorylated at a specific location, this releases the alpha helix which was blocking the active site, thereby reactivating the enzyme. These two processes are instrumental in regulating the function of TH in the brain.

This study showcases how Diamond is facilitating access and support for researchers from all over the world to make use of cutting-edge cryo-EM facilities.

Professor Valpuesta says:

By revealing the full-length structure of TH and the pivotal role played by the alpha helix in regulating TH activity, this work constitutes a leap forward in understanding the structure-based mechanism underpinning some Parkinsonisms. A better structural understanding may also support the development of novel stabilising/chaperoning therapies to target malfunctioning TH, very much needed by patients who are unresponsive to conventional medications for Parkinson’s.

To find out more about eBIC or discuss potential applications, please contact Principal Electron Microscopist Daniel Clare: daniel.clare@diamond.ac.uk