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Studying Binding Pocket Dynamics in the Human A$_{2A}$ Receptor Using Synthetic Photoswitches and Serial Crystallography


Glover, Hannah. Studying Binding Pocket Dynamics in the Human A$_{2A}$ Receptor Using Synthetic Photoswitches and Serial Crystallography. 2024, University of Zurich, Faculty of Science.

Abstract

The human adenosine 2A receptor (A2AR) is a class A G protein coupled receptor responsible for reg- ulation of dopamine signalling in the basal ganglia. The A2AR has been extensively studied and structures of it have been determined in a number of ligand-bound states. However, for a deeper un- derstanding of receptor function, knowledge on protein dynamics is required. One method that has emerged to study these dynamics is time-resolved serial crystallography, where a series of snapshots along a reaction pathway can be collected, allowing motions in a protein to be followed. Time-resolved serial crystallography employs a pump-probe regime using a trigger system to initiate reactions syn- chronously, which for many experiments has been laser illumination of endogenous photoswitches. Synthetic photoswitches provide a way of expanding the utility of this technique to proteins without endogenous photoactivity. Here photoswitches targeting the A2AR have been selected and a pipeline for characterisation was established. Results from these experiments were used to justify the selection of two synthetic photoswitches to be studied with serial crystallography. Experiments were carried out at synchrotrons and X-Ray free electron lasers (XFELs) to deter- mine the response of the A2AR to photoswitch isomerisation. Analysis revealed key movements in the orthosteric binding pocket, such as opening of the canonical binding pocket gate formed by an ionic interaction between E169 and H264 [3]. Structural rearrangements were transmitted through the receptor, resulting in the dissociation of allosteric cholesterol molecules and rearrangement of the helix bundle. Comparison of the structural changes following isomerisation of two different pho- toswitches revealed the influence small chemical modifications can have on ligand behaviour in the binding pocket. Helping to explain differing results observed during biophysical characterisations of the ligands.

Abstract

The human adenosine 2A receptor (A2AR) is a class A G protein coupled receptor responsible for reg- ulation of dopamine signalling in the basal ganglia. The A2AR has been extensively studied and structures of it have been determined in a number of ligand-bound states. However, for a deeper un- derstanding of receptor function, knowledge on protein dynamics is required. One method that has emerged to study these dynamics is time-resolved serial crystallography, where a series of snapshots along a reaction pathway can be collected, allowing motions in a protein to be followed. Time-resolved serial crystallography employs a pump-probe regime using a trigger system to initiate reactions syn- chronously, which for many experiments has been laser illumination of endogenous photoswitches. Synthetic photoswitches provide a way of expanding the utility of this technique to proteins without endogenous photoactivity. Here photoswitches targeting the A2AR have been selected and a pipeline for characterisation was established. Results from these experiments were used to justify the selection of two synthetic photoswitches to be studied with serial crystallography. Experiments were carried out at synchrotrons and X-Ray free electron lasers (XFELs) to deter- mine the response of the A2AR to photoswitch isomerisation. Analysis revealed key movements in the orthosteric binding pocket, such as opening of the canonical binding pocket gate formed by an ionic interaction between E169 and H264 [3]. Structural rearrangements were transmitted through the receptor, resulting in the dissociation of allosteric cholesterol molecules and rearrangement of the helix bundle. Comparison of the structural changes following isomerisation of two different pho- toswitches revealed the influence small chemical modifications can have on ligand behaviour in the binding pocket. Helping to explain differing results observed during biophysical characterisations of the ligands.

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Additional indexing

Item Type:Dissertation (monographical)
Referees:Hennig Michael, Dutzler Raimund, Wachtveitl Josef, Standfuss Jörg
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry

UZH Dissertations
Dewey Decimal Classification:610 Medicine & health
570 Life sciences; biology
Language:English
Place of Publication:Zürich
Date:20 March 2024
Deposited On:20 Mar 2024 14:00
Last Modified:04 Apr 2024 07:04
Number of Pages:100
OA Status:Green
  • Content: Published Version
  • Language: English