Methylxanthines, including caffeine and theophylline, are among the most widely consumed stimulant drugs in the world. These effects are mediated primarily via blockade of adenosine receptors. Xanthine analogs with improved properties have been developed as potential treatments for diseases such as Parkinson's disease. Here we report the structures of a thermostabilized adenosine A(2A) receptor in complex with the xanthines xanthine amine congener and caffeine, as well as the A(2A) selective inverse agonist ZM241385. The receptor is crystallized in the inactive state conformation as defined by the presence of a salt bridge known as the ionic lock. The complete third intracellular loop, responsible for G protein coupling, is visible consisting of extended helices 5 and 6. The structures provide new insight into the features that define the ligand binding pocket of the adenosine receptor for ligands of diverse chemotypes as well as the cytoplasmic regions that interact with signal transduction proteins.

There are Ϸ350 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 ␤1-adrenergic receptor. The most stable mutant receptor, ␤AR-m23, contained six point mutations that led to an apparent Tm 21°C higher than the native protein, and, in the presence of bound antagonist, ␤AR-m23 was as stable as bovine rhodopsin. In addition, ␤AR-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.G protein-coupled receptor ͉ membrane protein ͉ stabilization O ver the past 20 years the rate of determination of membrane protein structures has gradually increased, but most success has been in crystallizing membrane proteins from bacteria rather than from eukaryotes (1). Bacterial membrane proteins have been easier to overexpress using standard techniques in Escherichia coli than eukaryotic membrane proteins (2, 3), and the bacterial proteins are often far more stable in detergent, detergent stability being an essential prerequisite to purification and crystallization. However, of the 125 unique membrane protein structures that have been solved to date, there are only eight structures of mammalian integral membrane proteins; five of these membrane proteins were purified from natural sources and are stable in detergent solutions. Apart from the difficulties in overexpressing eukaryotic membrane proteins, they often have poor stability in detergent solutions, which severely restricts the range of crystallization conditions that can be explored without their immediate denaturation or precipitation. Ideally, membrane proteins should be stable for many days in any given detergent solution, but the detergents that are best suited to growing diffraction-quality crystals tend to be the most destabilizing detergents, i.e., those with short aliphatic chains and small or charged head groups. It is also the structures of human membrane proteins that we would like to solve, because these are required to help the development of therapeutic agents by the pharmaceutical industry; often there are substantial differences in the pharmacology of receptors, channels, and transporters from different mammals, whereas yeast and bacterial genomes may not include any homologous genes. There is thus an overwhelming need to develop a generic strategy that will allow the production of detergent-stable eukaryotic integral membrane proteins for crystallization and structure determination.Membrane proteins have evolved to be sufficiently stable in the membrane to ensure cell viability, b...

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...

NADPH oxidases (NOXs) are the only enzymes exclusively dedicated to reactive oxygen species (ROS) generation. Dysregulation of these polytopic membrane proteins impacts the redox signaling cascades that control cell proliferation and death. We describe the atomic crystal structures of the catalytic flavin adenine dinucleotide (FAD)- and heme-binding domains of NOX5. The two domains form the core subunit that is common to all seven members of the NOX family. The domain structures were then docked in silico to provide a generic model for the NOX family. A linear arrangement of cofactors (NADPH, FAD, and two membrane-embedded heme moieties) injects electrons from the intracellular side across the membrane to a specific oxygen-binding cavity on the extracytoplasmic side. The overall spatial organization of critical interactions is revealed between the intracellular loops on the transmembrane domain and the NADPH-oxidizing dehydrogenase domain. In particular, the C terminus functions as a toggle switch, which affects access of the NADPH substrate to the enzyme. The essence of this mechanistic model is that the regulatory cues conformationally gate NADPH-binding, implicitly providing a handle for activating/deactivating the very first step in the redox chain. Such insight provides a framework to the discovery of much needed drugs that selectively target the distinct members of the NOX family and interfere with ROS signaling.

Structural studies on G protein-coupled receptors (GPCRs) have been hampered for many years by their instability in detergent solution and by the number of potential conformations that receptors can adopt. Recently, the structures of the β1 and β2 adrenergic receptors and the adenosine A2a receptor were determined with antagonist bound, a receptor conformation that is thought to be more stable than the agonist-bound state. In contrast to these receptors, the neurotensin receptor NTS1 is much less stable in detergent solution. We have therefore used a systematic mutational approach coupled to activity assays to identify receptor mutants suitable for crystallisation, both alone and in complex with the peptide agonist, neurotensin. The best receptor mutant, NTS1-7m, contained 4 point mutations. It showed increased stability compared to the wild type receptor, in the absence of ligand, after solubilisation with a variety of detergents. In addition, NTS1-7m bound to neurotensin was more stable than unliganded NTS1-7m. Of the four thermostabilising mutations, only one residue (A86L) is predicted to be in the lipid environment. In contrast, I260A appears to be buried within the transmembrane helix bundle, F342A may form a distant part of the putative ligand binding site, whereas F358A is likely to be in a region important for receptor activation. NTS1-7m binds neurotensin with a similar affinity to the wild-type receptor. However, agonist dissociation was slower, and NTS1-7m activated G proteins poorly. The affinity of NTS1-7m for the antagonist SR48692 was also lower than that of the wild-type receptor. Thus we have successfully stabilised NTS1 in an agonist-binding conformation that does not efficiently couple to G proteins.

The serotonin transporter (SERT) is an integral membrane protein responsible for the clearance of serotonin from the synaptic cleft following the release of the neurotransmitter. SERT plays a prominent role in the regulation of serotoninergic neurotransmission and is a molecular target for multiple antidepressants as well as substances of abuse. Here we show that SERT associates with lipid rafts in both heterologous expression systems and rat brain and that the inclusion of the transporter into lipid microdomains is critical for serotonin uptake activity. SERT is present in a subpopulation of lipid rafts, which is soluble in Triton X-100 but insoluble in other non-ionic detergents such as Brij 58. Disaggregation of lipid rafts upon depletion of cellular cholesterol results in a decrease of serotonin transport capacity (V max ), due to the reduction of turnover number of serotonin transport. Our data suggest that the association of SERT with lipid rafts may represent a mechanism for regulating the transporter activity and, consequently, serotoninergic signaling in the central nervous system, through the modulation of the cholesterol content in the cell membrane. Furthermore, SERT-containing rafts are detected in both intracellular and cell surface fractions, suggesting that raft association may be important for trafficking and targeting of SERT. The serotonin transporter (SERT)1 is a member of a Na ϩ / Cl Ϫ -dependent family of integral membrane proteins that includes carriers for neurotransmitters, osmolytes, and nutrients (1-3). SERT, the norepinephrine transporter, and the dopamine transporter are most closely related, defining a subgroup characterized by a high degree of similarity both in sequence and pharmacological properties (4). SERT is responsible for the clearance of serotonin (5-hydroxytryptamine, 5HT) from the synaptic cleft following the release of the neurotransmitter (2).5HT is believed to accumulate inside the cell through cotransport with Na ϩ and Cl Ϫ and countertransport with K ϩ (4). SERT is an important pharmacological target for substances of abuse, such as cocaine, 3,4-methylenedioxymethamphetamine (Ecstasy), and p-chloramphetamine, and a variety of therapeutic antidepressants (1, 5, 6).Acute changes in SERT endogenous activity are likely to originate from local variations in 5HT concentration, cell surface distribution of SERT, interaction with regulatory proteins, or reversible post-translational modifications. To date SERT expression on the plasma membrane has been shown to be down-regulated by protein kinase C activation, following elevation of intracellular levels of Ca 2ϩ (7,8), or by phorbol 12-myristate 13-acetate treatment (9 -12). The SNARE protein syntaxin 1A (Syn 1A) is one of the few proteins known to modulate SERT function via protein-protein interaction by regulating the number of SERT molecules on the plasma membrane (13,14).To carry out clearance of extracellular 5HT, SERT must be inserted into the plasma membrane in an active form. This may require targeting the transport...

The rat serotonin transporter (rSERT) is an N-glycosylated integral membrane protein with 12 transmembrane regions; the N-glycans improve the ability of the SERT polypeptide chain to fold into a functional transporter, but they are not required for the transmembrane transport of serotonin per se. In order to define the best system for the expression, purification and structural analysis of serotonin transporter (SERT), we expressed SERT in Escherichia coli, Pichia pastoris, the baculovirus expression system and in four different stable mammalian cell lines. Two stable cell lines that constitutively expressed SERT (Imi270 and Coca270) were constructed using episomal plasmids in HEK293 cells expressing the EBNA-1 antigen. SERT expression in the three different inducible stable mammalian cell lines was induced either by a decrease in temperature (cell line pCytTS-SERT), the addition of tetracycline to the growth medium (cell line T-REx-SERT) or by adding DMSO which caused the cells to differentiate (cell line MEL-SERT). All the mammalian cell lines expressed functional SERT, but SERT expressed in E. coli or P. pastoris was nonfunctional as assessed by 5-hydroxytryptamine uptake and inhibitor binding assays. Expression of functional SERT in the mammalian cell lines was assessed by an inhibitor binding assay; the cell lines pCytTS-SERT, Imi270 and Coca270 contained levels of functional SERT similar to that of the standard baculovirus expression system (250,000 copies per cell). The expression of SERT in induced T-REx-SERT cells was 400,000 copies per cell, but in MEL-SERT it was only 80,000 copies per cell. All the mammalian stable cell lines expressed SERT at the plasma membrane as assessed by [3H]-5-hydroxytryptamine uptake into whole cells, but the V(max) for the T-Rex-SERT cell line was 10-fold higher than any of the other cell lines. It was noticeable that the cell lines that constitutively expressed SERT grew extremely poorly, compared to the inducible cell lines whose growth rates were similar to the parental cell lines when not induced. In addition, the cell lines MEL-SERT, Imi270 and T-REx-SERT all expressed fully N-glycosylated SERT and no unglycosylated inactive protein, in contrast to the baculovirus expression system where the vast majority of expressed SERT was unglycosylated and nonfunctional.

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