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The β(1)-adrenergic receptor (β(1)AR) is a G-protein-coupled receptor whose inactive state structure was determined using a thermostabilized mutant (β(1)AR-M23). However, it was not thought to be in a fully inactivated state because there was no salt bridge between Arg139 and Glu285 linking the cytoplasmic ends of transmembrane helices 3 and 6 (the R(3.50) - D/E(6.30) "ionic lock"). Here we compare eight new structures of β(1)AR-M23, determined from crystallographically independent molecules in four different crystals with three different antagonists bound. These structures are all in the inactive R state and show clear electron density for cytoplasmic loop 3 linking transmembrane helices 5 and 6 that had not been seen previously. Despite significantly different crystal packing interactions, there are only two distinct conformations of the cytoplasmic end of helix 6, bent and straight. In the bent conformation, the Arg139-Glu285 salt bridge is present, as in the crystal structure of dark-state rhodopsin. The straight conformation, observed in previously solved structures of β-receptors, results in the ends of helices 3 and 6 being too far apart for the ionic lock to form. In the bent conformation, the R(3.50)-E(6.30) distance is significantly longer than in rhodopsin, suggesting that the interaction is also weaker, which could explain the high basal activity in β(1)AR compared to rhodopsin. Many mutations that increase the constitutive activity of G-protein-coupled receptors are found in the bent region at the cytoplasmic end of helix 6, supporting the idea that this region plays an important role in receptor activation.

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 A(2a) 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 degrees C and 9 degrees 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.

There are approximately 350 non-odorant G protein-coupled receptors (GPCRs) encoded by the human genome, many of which are predicted to be potential therapeutic targets, but there are only two structures available to represent the whole of the family. We hypothesized that improving the detergent stability of these receptors and simultaneously locking them into one preferred conformation will greatly improve the chances of crystallization. We developed a generic strategy for the isolation of detergent-solubilized thermostable mutants of a GPCR, the beta1-adrenergic receptor. The most stable mutant receptor, betaAR-m23, contained six point mutations that led to an apparent T(m) 21 degrees C higher than the native protein, and, in the presence of bound antagonist, betaAR-m23 was as stable as bovine rhodopsin. In addition, betaAR-m23 was significantly more stable in a wide range of detergents ideal for crystallization and was preferentially in an antagonist conformation in the absence of ligand.