Co-evolving stability and conformational homogeneity of the human adenosine A _2a receptor

Abstract

Structural studies on mammalian integral membrane proteins have long been hampered by their instability in detergent. This is particularly true for the agonist conformation of G protein-coupled receptors (GPCRs), where it is thought that the movement of helices that occurs upon agonist binding results in a looser and less stable packing in the protein. Here, we show that mutagenesis coupled to a specific selection strategy can be used to stabilize the agonist and antagonist conformations of the adenosine A2a receptor. Of the 27 mutations identified that improve the thermostability of the agonist conformation, only three are also present in the 17 mutations identified that improve the thermostability of the antagonist conformation, suggesting that the selection strategies used were specific for each conformation. Combination of the stabilizing mutations for the antagonist-or agonist-binding conformations resulted in mutants that are more stable at higher temperatures than the wild-type receptor by 17°C and 9°C, respectively. The mutant receptors both showed markedly improved stability in short-chain alkyl-glucoside detergents compared with the wild-type receptor, which will facilitate their structural analysis.conformational thermostabilization ͉ G protein-coupled receptor ͉ membrane protein G protein-coupled receptors (GPCRs) represent one of the largest single families of integral membrane proteins in the human genome and bind multifarious ligands that mediate many physiological processes, which explains why GPCRs represent a major proportion of drug targets (1). The binding of an extracellular ligand to a GPCR promotes coupling of the receptor to trimeric G proteins, situated on the intracellular side of the plasma membrane, triggering a signaling cascade. Despite sharing common features such as seven transmembrane helices and conserved signatures in their amino acid sequence (2), the sequence homology between different GPCRs is rather low. Detailed structure determination of GPCRs is therefore required to elucidate the mechanism of receptor activation to improve the design of both agonist and antagonist ligands of medical relevance.For many years, crystallographic studies of GPCRs were limited to rhodopsin because of its abundance in native sources (retina) and its intrinsic stability in the dark state (3, 4). However, even rhodopsin has been shown to be structurally more dynamic in detergent solution than in lipid bilayers (5), and such flexibility is more pronounced for GPCRs that bind to diffusible ligands. Another obstacle to structural analysis, and especially to the formation of well ordered crystals, arises from the conformational heterogeneity of these receptors (6, 7). The active agonist-bound state of GPCRs is normally found to be intrinsically less stable than the inactive antagonist-bound state, probably reflecting the receptor requirement for higher flexibility in its active state (8). Agonist binding to the receptor triggers the recruitment of G ␣ protein binding to the intracellular side of the...

Fluorine NMR‐Based Screening on Cell Membrane Extracts

“…Often, modification of membrane enzymes with fusion partners, such as maltose binding protein (MBP), or point mutations are required to enhance the level of expression of these proteins, their solubility and stability in solution. [13][14][15][16][17][18] Here, we show that n-FABS method can be extended to cell extracts, therefore using the enzyme of interest in more physiological conditions. As a model protein, we used fatty acid amide hydrolase (FAAH), a membrane-bound serine hydrolase responsible for the catabolism of a class of endogenous bioactive lipids called fatty acid ethanolamides (FAEs).…”

International Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature and Classification of Adenosine Receptors—An Update

“…Magnani et al (2008) elegantly addressed this issue by mutating the adenosine A 2A receptor sequentially to improve its thermostability. They managed to create relatively stable receptor variants with up to five amino acid substitutions, albeit with different patterns for either agonist or antagonist binding.…”

Naturally evolved G protein-coupled receptors adopt metastable conformations

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