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The human oxidative phosphorylation (OxPhos) system consists of approximately 90 proteins encoded by nuclear and mitochondrial genomes and serves as the primary cellular pathway for ATP biosynthesis. While the core protein machinery for OxPhos is well characterized, many of its assembly, maturation, and regulatory factors remain unknown. We exploited the tight transcriptional control of the genes encoding the core OxPhos machinery to identify novel regulators. We developed a computational procedure, which we call expression screening, which integrates information from thousands of microarray data sets in a principled manner to identify genes that are consistently co-expressed with a target pathway across biological contexts. We applied expression screening to predict dozens of novel regulators of OxPhos. For two candidate genes, CHCHD2 and SLIRP, we show that silencing with RNAi results in destabilization of OxPhos complexes and a marked loss of OxPhos enzymatic activity. Moreover, we show that SLIRP plays an essential role in maintaining mitochondrial-localized mRNA transcripts that encode OxPhos protein subunits. Our findings provide a catalogue of potential novel OxPhos regulators that advance our understanding of the coordination between nuclear and mitochondrial genomes for the regulation of cellular energy metabolism.

How drugs bind to their receptors--from initial association, through drug entry into the binding pocket, to adoption of the final bound conformation, or \"pose\"--has remained unknown, even for G-protein-coupled receptor modulators, which constitute one-third of all marketed drugs. We captured this pharmaceutically critical process in atomic detail using the first unbiased molecular dynamics simulations in which drug molecules spontaneously associate with G-protein-coupled receptors to achieve final poses matching those determined crystallographically. We found that several beta blockers and a beta agonist all traverse the same well-defined, dominant pathway as they bind to the β₁- and β₂-adrenergic receptors, initially making contact with a vestibule on each receptor's extracellular surface. Surprisingly, association with this vestibule, at a distance of 15 Å from the binding pocket, often presents the largest energetic barrier to binding, despite the fact that subsequent entry into the binding pocket requires the receptor to deform and the drug to squeeze through a narrow passage. The early barrier appears to reflect the substantial dehydration that takes place as the drug associates with the vestibule. Our atomic-level description of the binding process suggests opportunities for allosteric modulation and provides a structural foundation for future optimization of drug-receptor binding and unbinding rates.

The X-ray crystal structure of the M3 muscarinic acetylcholine receptor bound to the bronchodilator drug tiotropium is reported; comparison of this structure with that of the M2 muscarinic acetylcholine receptor reveals key differences that could potentially be exploited to develop subtype-selective drugs. Muscrarinic receptor structures compared The muscarinic acetylcholine receptors (mAChRs) constitute a family of G-protein-coupled receptors. These membrane proteins are targets for treatment of a broad range of conditions, including Alzheimer's disease, schizophrenia and chronic obstructive pulmonary disease. The five mAChR subtypes (M1–M5) share a high degree of sequence homology, but show marked differences in G-protein-coupling preference and physiological function. This pair of papers from Brian Kobilka's group presents the structures of two of the five subtypes. Haga et al . report the X-ray crystal structure of the M2 receptor, which is essential for the physiological control of cardiovascular function; Kruse et al . determine the structure of the M3 receptor, active in the bronchial airways and elsewhere. Comparison of the two structures reveals key differences that could potentially be exploited to develop subtype-selective drugs. Acetylcholine, the first neurotransmitter to be identified 1 , exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1–M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate 2 , 3 , 4 . Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences 5 , 6 . We describe here the structure of the G q/11 -coupled M3 mAChR (‘M3 receptor’, from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the G i/o -coupled M2 receptor 7 , offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.

G protein-coupled receptors (GPCRs) relay diverse extracellular signals into cells by catalyzing nucleotide release from heterotrimeric G proteins, but the mechanism underlying this quintessential molecular signaling event has remained unclear. Here we use atomic-level simulations to elucidate the nucleotide-release mechanism. We find that the G protein a subunit Ras and helical domains—previously observed to separate widely upon receptor binding to expose the nucleotide-binding site—separate spontaneously and frequently even in the absence of a receptor. Domain separation is necessary but not sufficient for rapid nucleotide release. Rather, receptors catalyze nucleotide release by favoring an internal structural rearrangement of the Ras domain that weakens its nucleotide affinity. We use double electron-electron resonance spectroscopy and protein engineering to confirm predictions of our computationally determined mechanism.

Protease-activated receptor 1 (PAR1) is the prototypical member of a family of G-protein-coupled receptors that mediate cellular responses to thrombin and related proteases. Thrombin irreversibly activates PAR1 by cleaving the amino-terminal exodomain of the receptor, which exposes a tethered peptide ligand that binds the heptahelical bundle of the receptor to affect G-protein activation. Here we report the 2.2-Å-resolution crystal structure of human PAR1 bound to vorapaxar, a PAR1 antagonist. The structure reveals an unusual mode of drug binding that explains how a small molecule binds virtually irreversibly to inhibit receptor activation by the tethered ligand of PAR1. In contrast to deep, solvent-exposed binding pockets observed in other peptide-activated G-protein-coupled receptors, the vorapaxar-binding pocket is superficial but has little surface exposed to the aqueous solvent. Protease-activated receptors are important targets for drug development. The structure reported here will aid the development of improved PAR1 antagonists and the discovery of antagonists to other members of this receptor family. The X-ray crystal structure of the human G-protein-coupled receptor protease-activated receptor 1 (PAR1) bound to the antagonist vorapaxar is solved, revealing an unusual method of drug binding that should facilitate the development of improved PAR1-selective antagonists. Thrombin receptor structure The X-ray crystal structure of the human protease-activated receptor 1 (PAR1) bound to vorapaxar, a PAR1 antagonist, has been determined at 2.2 Å resolution. PAR1, also known as the thrombin receptor, is a G protein-coupled receptor that mediates cellular responses to the coagulation protease thrombin and related proteases. Vorapaxar was recently shown to prevent myocardial infarction in at-risk patients, and knowledge of the PAR structure will be relevant to the design of PAR1 antagonists with better drug properties.

Binding modes and molecular mechanisms of several allosteric modulators of a prototypical G-protein-coupled receptor are revealed using atomic-level simulations and validated by the rational design of a modulator with substantially altered effects. Allosteric mechanism of GPCR modulation A third of clinically used drugs elicit their biological effects via a G-protein-coupled receptor (GPCR), usually by binding at the orthosteric (primary ligand-binding) site, in competition with the ligands that naturally regulate receptor signalling. The design of small molecules able to selectively modulate a GPCR by binding to an allosteric site is a desirable goal, but difficult to achieve because neither the binding modes nor the molecular mechanisms of such molecules are known. In this manuscript, the authors used molecular dynamics simulations, with experimental validation, to determine where and how structurally diverse allosteric modulators bind to the M2 muscarinic acetylcholine receptor, which is essential for the physiological control of cardiovascular function. Despite substantial structural diversity of the small molecule ligands, the molecules all formed cation-π interactions with clusters of aromatic residues in the receptor's extracellular vestibule, about 15 Å from the ligand-binding pocket. These findings may facilitate the rational design of allosteric modulators targeting muscarinic and related GPCRs. The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known 1 , 2 . Here we determine binding sites, bound conformations and specific drug–receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation–π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15 Å from the classical, ‘orthosteric’ ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator’s allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

β-adrenergic receptor structures Two papers by Brian Kobilka and colleagues describe the X-ray crystal structure of the human β 2 adrenergic receptor (β 2 AR) bound to various agonists. β 2 AR is a member of the G protein coupled receptor (GPCR) family of membrane-spanning receptors that sense molecules outside the cell and activate internal signalling pathways. With a ubiquitous role in human physiology, GPCRs are prime targets for drug discovery. A third paper by Christopher Tate and his team describes crystal structures of a similar GPCR, the turkey β 1 -adrenergic receptor (β 1 AR), bound to full and partial agonists. Together, these new structures reveal the subtle structural changes that accompany agonist binding, showing how binding events inside and outside the cell membrane stabilize the receptor's active state. Agonist binding to β 1 AR is shown to induce a contraction of the catecholamine-binding pocket relative to the antagonist-bound receptor, and molecular-dynamics simulations of the β 2 AR agonist complex suggest that the agonist-bound active state spontaneously relaxes to an inactive-like state in the absence of a G protein. The X-ray crystal structure of the human β 2 adrenergic receptor, a G-protein-coupled receptor (GPCR), covalently bound to a small-molecule agonist is solved. Comparison of this structure with structures of this GPCR in an inactive state and in an antibody-stabilized active state reveals how binding events at both the extracellular and intracellular surfaces stabilize the active conformation of the receptor. Molecular dynamics simulations suggest that the agonist-bound active state spontaneously relaxes to an inactive-like state in the absence of a G protein. G-protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that modulate biological function by initiating cellular signalling in response to chemically diverse agonists. Despite recent progress in the structural biology of GPCRs 1 , the molecular basis for agonist binding and allosteric modulation of these proteins is poorly understood. Structural knowledge of agonist-bound states is essential for deciphering the mechanism of receptor activation, and for structure-guided design and optimization of ligands. However, the crystallization of agonist-bound GPCRs has been hampered by modest affinities and rapid off-rates of available agonists. Using the inactive structure of the human β 2 adrenergic receptor (β 2 AR) as a guide, we designed a β 2 AR agonist that can be covalently tethered to a specific site on the receptor through a disulphide bond. The covalent β 2 AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystallized a covalent agonist-bound β 2 AR–T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method 2 , and determined its structure at 3.5 Å resolution. A comparison to the inactive structure and an antibody-stabilized active structure (companion paper 3 ) shows how binding events at both the extracellular and intracellular surfaces are required to stabilize an active conformation of the receptor. The structures are in agreement with long-timescale (up to 30 μs) molecular dynamics simulations showing that an agonist-bound active conformation spontaneously relaxes to an inactive-like conformation in the absence of a G protein or stabilizing antibody.

An enzymatic approach enables synthesis of a defined DNA sequence using TdT with reversibly tethered dNTPs. Oligonucleotides are almost exclusively synthesized using the nucleoside phosphoramidite method, even though it is limited to the direct synthesis of ∼200 mers and produces hazardous waste. Here, we describe an oligonucleotide synthesis strategy that uses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Each TdT molecule is conjugated to a single deoxyribonucleoside triphosphate (dNTP) molecule that it can incorporate into a primer. After incorporation of the tethered dNTP, the 3′ end of the primer remains covalently bound to TdT and is inaccessible to other TdT–dNTP molecules. Cleaving the linkage between TdT and the incorporated nucleotide releases the primer and allows subsequent extension. We demonstrate that TdT–dNTP conjugates can quantitatively extend a primer by a single nucleotide in 10–20 s, and that the scheme can be iterated to write a defined sequence. This approach may form the basis of an enzymatic oligonucleotide synthesizer.

Mitochondrial sensor The nutrient sensor molecule mTOR (mammalian target of rapamycin) is a kinase involved in the regulation of cell growth and proliferation. Its close links to the cell's energetics suggest that it might interact with the mitochondria, and a computational genomics study now confirms that it does. mTOR balances energy metabolism via transcriptional control of mitochondrial gene expression and oxidative function, with the transcriptional regulators PGC-1a and YY1 as mediators. This pathway opens new possibilities for therapeutic interventions in metabolic diseases in which mitochondrial activity is compromised. The nutrient sensor mTOR controls mitochondrial gene expression and oxidative function via an interaction between the transcriptional regulators PGC-1α and YY1. Transcriptional complexes that contain peroxisome-proliferator-activated receptor coactivator (PGC)-1α control mitochondrial oxidative function to maintain energy homeostasis in response to nutrient and hormonal signals 1 , 2 . An important component in the energy and nutrient pathways is mammalian target of rapamycin (mTOR), a kinase that regulates cell growth, size and survival 3 , 4 , 5 . However, it is unknown whether and how mTOR controls mitochondrial oxidative activities. Here we show that mTOR is necessary for the maintenance of mitochondrial oxidative function. In skeletal muscle tissues and cells, the mTOR inhibitor rapamycin decreased the gene expression of the mitochondrial transcriptional regulators PGC-1α, oestrogen-related receptor α and nuclear respiratory factors, resulting in a decrease in mitochondrial gene expression and oxygen consumption. Using computational genomics, we identified the transcription factor yin-yang 1 (YY1) as a common target of mTOR and PGC-1α. Knockdown of YY1 caused a significant decrease in mitochondrial gene expression and in respiration, and YY1 was required for rapamycin-dependent repression of those genes. Moreover, mTOR and raptor interacted with YY1, and inhibition of mTOR resulted in a failure of YY1 to interact with and be coactivated by PGC-1α. We have therefore identified a mechanism by which a nutrient sensor (mTOR) balances energy metabolism by means of the transcriptional control of mitochondrial oxidative function. These results have important implications for our understanding of how these pathways might be altered in metabolic diseases and cancer.

Enzymatic oligonucleotide synthesis methods based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) promise to enable the de novo synthesis of long oligonucleotides under mild, aqueous conditions. Intermediates with a 3′ terminal structure (hairpins) will inevitably arise during synthesis, but TdT has poor activity on these structured substrates, limiting its usefulness for oligonucleotide synthesis. Here, we described two parallel efforts to improve the activity of TdT on hairpins: (1) optimization of the concentrations of the divalent cation cofactors and (2) engineering TdT for enhanced thermostability, enabling reactions at elevated temperatures. By combining both of these improvements, we obtained a ~10-fold increase in the elongation rate of a guanine-cytosine hairpin.