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Class C G protein-coupled receptors encompass a range of promising therapeutic targets for a variety of diseases, yet to date only two members of this sub-family of GPCRs have been drugged. Recent advances in structural biology have revealed the X-ray crystallographic structures of allosteric ligands bound to two Class C metabotropic glutamate (mGlu) receptors, mGlu1 and mGlu5. Herein, we review how this information can be leveraged to help understand some of the historical challenges of mGlu receptor allosteric modulator drug discovery, and discuss how the structural enablement can be prospectively used for structurebased drug discovery approaches across Class C GPCR targets in general.

Neurokinins (or tachykinins) are peptides that modulate a wide variety of human physiology through the neurokinin G protein-coupled receptor family, implicated in a diverse array of pathological processes. Here we report high-resolution crystal structures of the human NK1 receptor (NK1R) bound to two small-molecule antagonist therapeutics – aprepitant and netupitant and the progenitor antagonist CP-99,994. The structures reveal the detailed interactions between clinically approved antagonists and NK1R, which induce a distinct receptor conformation resulting in an interhelical hydrogen-bond network that cross-links the extracellular ends of helices V and VI. Furthermore, the high-resolution details of NK1R bound to netupitant establish a structural rationale for the lack of basal activity in NK1R. Taken together, these co-structures provide a comprehensive structural basis of NK1R antagonism and will facilitate the design of new therapeutics targeting the neurokinin receptor family., Neurokinin receptors are G protein-coupled receptors. Here the authors present three crystal structures of the neurokinin 1 receptor (NK1R) in complex with small-molecule antagonists including aprepitant and netupitant and observe that these clinically approved compounds induce a conformational change in the receptor.

The metabotropic glutamate receptor family includes many potential therapeutic targets for a wide range of neurological disorders however to date no approved drugs have progressed to market. For some receptor subtypes it has been difficult to separate therapeutic benefit from undesirable side effects. For others finding suitable drug like molecules has been challenging. Chemotypes identified from screening have been limited and difficult to optimise away from undesirable groups. Frequently within related series, compounds have switched from agonist to antagonists. Recently the structures of the transmembrane domain of mGlu1 and mGlu5 have been solved revealing the binding site of allosteric modulators which provides an understanding of the difficulties to date and an opportunity for future structure based approaches to drug design.

Structural analysis of class B G-protein-coupled receptors (GPCRs), cell-surface proteins that respond to peptide hormones, has been restricted to the amino-terminal extracellular domain, thus providing little understanding of the membrane-spanning signal transduction domain. The corticotropin-releasing factor receptor type 1 is a class B receptor which mediates the response to stress and has been considered a drug target for depression and anxiety. Here we report the crystal structure of the transmembrane domain of the human corticotropin-releasing factor receptor type 1 in complex with the small-molecule antagonist CP-376395. The structure provides detailed insight into the architecture of class B receptors. Atomic details of the interactions of the receptor with the non-peptide ligand that binds deep within the receptor are described. This structure provides a model for all class B GPCRs and may aid in the design of new small-molecule drugs for diseases of brain and metabolism.

10 páginas, 5 figuras -- PAGS nros. 1380-1389, The DNA ligase IV–Xrcc4 complex is responsible for the ligation of broken DNA ends in the non-homologous end-joining (NHEJ) pathway of DNA double strand break repair in mammals. Mutations in DNA ligase IV (Lig4) lead to immunodeficiency and radiosensitivity in humans. Only partial structural information for Lig4 and Xrcc4 is available, while the structure of the full-length proteins and their arrangement within the Lig4–Xrcc4 complex is unknown. The C-terminal domain of Xrcc4, whose structure has not been solved, contains phosphorylation sites for DNA-PKcs and is phylogenetically conserved, indicative of a regulatory role in NHEJ. Here, we have purified full length Xrcc4 and the Lig4–Xrcc4 complex, and analysed their structure by single-particle electron microscopy. The three-dimensional structure of Xrcc4 at a resolution of ∼37 Å reveals that the C-terminus of Xrcc4 forms a dimeric globular domain connected to the N-terminus by a coiled-coil. The N- and C-terminal domains of Xrcc4 locate at opposite ends of an elongated molecule. The electron microscopy images of the Lig4–Xrcc4 complex were examined by two-dimensional image processing and a double-labelling strategy, identifying the site of the C-terminus of Xrcc4 and the catalytic core of Lig4 within the complex. The catalytic domains of Lig4 were found to be in the vicinity of the N-terminus of Xrcc4. We provide a first sight of the structural organization of the Lig4–Xrcc4 complex, which suggests that the BRCT domains could provide the link of the ligase to Xrcc4 while permitting some movements of the catalytic domains of Lig4. This arrangement may facilitate the ligation of diverse configurations of damaged DNA, This work has been supported by project SAF2008-00451 from the Spanish Ministry of Science and Innovation (OL), project RD06/0020/1001 of the “Red Temática de Investigación Cooperativa en Cáncer (RTICC)” financed by the “Instituto de Salud Carlos III” (OL) and a Programme Grant from Cancer Research UK (LHP). AR-C was supported by an I3P fellowship from CSIC and the European Social Fund. MAR-C is supported by the Spanish Ministry of Science by a FPI grant. EA-P is supported by the Autonomous Region of Madrid. Llorca's group is additionally supported by the Human Frontiers Science Program Organization (RGP39/2008)

Rad9, Rad1, and Hus1 form a heterotrimeric complex (9-1-1) that is loaded onto DNA at sites of DNA damage. DNA-loaded 9-1-1 activates signaling through the Chk1 arm of the DNA damage checkpoint response via recruitment and stimulation of ATR. Additionally, 9-1-1 may play a direct role in facilitating DNA damage repair via interaction with a number of DNA repair enzymes. We have now determined the crystal structure of the human 9-1-1 complex, revealing a toroidal structure with a similar architecture to the homotrimeric PCNA DNA-binding clamp. The structure explains the formation of a unique heterotrimeric arrangement and reveals significant differences among the three subunits in the sites implicated in binding to the clamp loader and to ligand proteins. Biochemical analysis reveals a single repair enzyme-binding site on 9-1-1 that can be blocked competitively by the PCNA-binding cell-cycle regulator p21(cip1/waf1).

DNA ligase IV catalyses the final ligation step in the non-homologous end-joining (NHEJ) DNA repair pathway and requires interaction of the ligase with the Xrcc4 'genome-guardian', an essential NHEJ factor. Here we report the 3.9 A crystal structure of the Saccharomyces cerevisiae Xrcc4 ortholog ligase interacting factor 1 (Lif1p) complexed with the C-terminal BRCT domains of DNA ligase IV (Lig4p). The structure reveals a novel mode of protein recognition by a tandem BRCT repeat, and in addition provides a molecular basis for a human LIG4 syndrome clinical condition.

Cellular life depends upon the preservation and transmission of genetic material. Double stranded DNA breaks (DSBs) cause catastrophic gene loss in cell division and must be promptly and accurately repaired. In eukaryotes DSBs may be repaired by either non-homologous end-joining (NHEJ), single strand annealing or homologous recombination (HR). Vertebrate NHEJ has been shown to depend upon the DNA-dependent protein kinase (DNA-PK) consisting of the phosphatidylinositol 3 (PI 3)-kinase like (PIKK) catalytic sub-unit (DNA-PKcs) and the DNA targeting factor Ku. Our analysis of recently completed genomes found several novel PIKKs in Anopheles gambiae and Drosophila melanogaster including a novel mosquito DNA-PKcs orthologue, the first non-vertebrate DNA-PKcs described to date. We also detected a DNA-PKcs fragment in the high quality EST set of Apis mellifera ligustica (honey bee) suggesting that DNA-PK is a far older and more important eukaryotic complex than previously thought.

G protein-coupled receptors (GPCRs) play a crucial role in many diseases and are the site of action of 25–30 % of current drugs (Overington et al., Nat Rev Drug Discov 5(12):993–996, 2006). As such GPCRs represent a major area of interest for the pharmaceutical industry. Despite the rich history of this target class there remain many opportunities for clinical intervention and there is a scarcity of high quality drug-like molecules for many receptors. High-throughput screening has often failed to unlock the potential of members of this superfamily and new, complementary approaches to GPCR drug discovery are required. However, the instability of GPCRs when removed from the cell membrane has severely limited the application of the techniques of structure-based and fragment-based drug discovery. The Heptares approach is successfully overcoming this fundamental challenge and facilitates both biophysical and biochemical fragment screening and also the generation of structural information. Heptares uses its StaR® technology to thermostabilise GPCRs using mutations in precisely defined biologically-relevant conformations (Robertson et al., Neuropharmacology 60(1):36–44, 2011). StaR proteins are amenable to techniques that cannot be readily used with wild-type GPCRs, including fragment screening, biophysical kinetic profiling and X-ray crystallography. Crystal structures of multiple GPCRs have been solved using this approach in the last 5 years (Dore et al., Structure 19(9):1283–1293, 2011; Dore et al., Nature 511:557–562, 2014; Hollenstein et al., Nature 499(7459):438–443, 2013).

Schizosaccharomyces pombe Crb2 is a checkpoint mediator required for the cellular response to DNA damage. Like human 53BP1 and Saccharomyces cerevisiae Rad9 it contains Tudor2 and BRCT2 domains. Crb2-Tudor2 domain interacts with methylated H4K20 and is required for recruitment to DNA dsDNA breaks. The BRCT2 domain is required for dimerization, but its precise role in DNA damage repair and checkpoint signaling is unclear. The crystal structure of the Crb2–BRCT2 domain, alone and in complex with a phosphorylated H2A.1 peptide, reveals the structural basis for dimerization and direct interaction with γ-H2A.1 in ionizing radiation-induced foci (IRIF). Mutational analysis in vitro confirms the functional role of key residues and allows the generation of mutants in which dimerization and phosphopeptide binding are separately disrupted. Phenotypic analysis of these in vivo reveals distinct roles in the DNA damage response. Dimerization mutants are genotoxin sensitive and defective in checkpoint signaling, Chk1 phosphorylation, and Crb2 IRIF formation, while phosphopeptide-binding mutants are only slightly sensitive to IR, have extended checkpoint delays, phosphorylate Chk1, and form Crb2 IRIF. However, disrupting phosphopeptide binding slows formation of ssDNA-binding protein (Rpa1/Rad11) foci and reduces levels of Rad22(Rad52) recombination foci, indicating a DNA repair defect.

The archaeal/eukaryotic proliferating cell nuclear antigen (PCNA) toroidal clamp interacts with a host of DNA modifying enzymes, providing a stable anchorage and enhancing their respective processivities. Given the broad range of enzymes with which PCNA has been shown to interact, relatively little is known about the mode of assembly of functionally meaningful combinations of enzymes on the PCNA clamp. We have determined the X-ray crystal structure of the Sulfolobus solfataricus PCNA1–PCNA2 heterodimer, bound to a single copy of the flap endonuclease FEN1 at 2.9 Å resolution. We demonstrate the specificity of interaction of the PCNA subunits to form the PCNA1–PCNA2–PCNA3 heterotrimer, as well as providing a rationale for the specific interaction of the C-terminal PIP-box motif of FEN1 for the PCNA1 subunit. The structure explains the specificity of the individual archaeal PCNA subunits for selected repair enzyme ‘clients’, and provides insights into the co-ordinated assembly of sequential enzymatic steps in PCNA-scaffolded DNA repair cascades.

DNA-dependent protein kinase (DNA-PK) is part of the eukaryotic DNA double strand break repair pathway and as such is crucial for maintenance of genomic stability, as well as for V(D)J (variable-diversity-joining) recombination. The catalytic subunit of DNA-PK (DNA-PKcs) belongs to the phosphatidylinositol-3 (PI-3) kinase-like kinase (PIKK) superfamily and is comprised of approximately 4100 amino acids. We have used a novel repeat detection method to analyse this enormous protein and have identified two different types of helical repeat motifs in the N-terminal region of the sequence, as well as other previously unreported features in this repeat region. A comparison with the ATMs, ATRs, and TORs show that the features identified are likely to be conserved throughout the PIKK superfamily. Homology modelling of parts of the DNA-PKcs sequence has been undertaken and we have been able to fit the models to previously obtained electron microscopy data. This work provides an insight into the overall architecture of the DNA-PKcs protein and identifies regions of interest for further experimental studies.