Lipidic cubic phase crystallization of the adenosine A_2B receptor in complex with antagonists

Abstract

G protein-coupled receptors (GPCRs) are membrane-bound signaling proteins that are fundamental to human life. Through their signal transduction, they can affect virtually all aspects of human physiology and are consequently highly associated with pathophysiological processes. Therefore, they represent attractive drug targets for numerous diseases, yet the great potential of pharmacological intervention by targeting GPCRs is by far not fully exhausted. One interesting, emerging drug target is the G protein-coupled adenosine A_2B receptor (A_2BAR), a unique member of the GPCR subfamily of adenosine receptors (ARs). It is implicated in several diseases, such as inflammatory diseases, cancer and pain. Currently, the largest clinical potential for A_2BAR ligands is seen specifically for the application of its antagonists in the immunotherapy of cancer with numerous active clinical trials. <br /> In the beginning of this project, structural insights into the A_2BAR were non-existent. Since then, agonist-bound active state structures of the receptor have been elucidated by cryogenic electron microscopy, yet structural information on the clinically relevant antagonist-bound inactive state of the receptor has still been missing. To this day, inactive state structures of GPCRs are typically solved by X-ray crystallography, which is still a challenging endeavor for membrane proteins, due to their characteristically low stability in aqueous solutions, low expression and scarcity of hydrophilic surface available for forming crystal contacts. Therefore, extensive protein engineering is often necessary in order to obtain a receptor construct that is suitable for crystallization experiments. <br /> In the course of this study, the A_2BAR was successfully engineered for crystallization experiments through characterization of 174 different receptor constructs, which were modified through various point mutations, linker sequences, truncations and fusion proteins. Of those, 17 receptor constructs, in complex with several different A_2BAR antagonists, were utilized in 49 crystallization experiments using the lipidic ubic phase method. The crystallization experiments gave rise to numerous protein crystals which reached sizes of up to 50 μm and diffracted up to 3.5 Å. Eventually, a crystal structure of an antagonist-bound A_2BAR was solved at 4.0 Å resolution, which revealed insights into its inactive state, and illuminated a strategy for future structural biology projects on this AR subtype.