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Murine norovirus (MNoV) is closely related to human norovirus (HNoV), an infectious agent responsible for acute gastroenteritis worldwide. Here we report the X-ray crystal structure of the dimeric MNoV VP1 protruding (P) domain in complex with its cellular receptor CD300lf. CD300lf binds the P domain with a 2:2 stoichiometry, engaging a cleft between the AB and DE loops of the P2 subdomain at a site that overlaps the epitopes of neutralizing antibodies. We also identify that bile acids are cofactors enhancing MNoV cell-binding and infectivity. Structures of CD300lf–P domain in complex with glycochenodeoxycholic acid (GCDCA) and lithocholic acid (LCA) reveal two bile acid binding sites at the P domain dimer interface distant from receptor binding sites. The structural determinants for receptor and bile acid binding are supported by numerous biophysical assays utilizing interface residue mutations. We find that the monomeric affinity of CD300lf for the P domain is low and is divalent cation dependent. We have also determined the crystal structure of CD300lf in complex with phosphocholine, revealing that MNoV engages its receptor in a manner mimicking host ligands including similar metal coordination. Docking of the cocomplex structures onto a cryo-EM–derived model of MNoV suggests that each virion can make multiple CD300lf engagements, and thus, infection may be driven by the avidity of cell surface clustered CD300lf. These studies identify multiple potential modulators of norovirus infection that may act to regulate the interaction between the viral capsid P domain and its cognate cellular receptor.

Poly(ADP-ribose)ylation (PARylation) by PAR polymerase 1 (PARP1) and PARylation removal by poly(ADP-ribose) glycohydrolase (PARG) critically regulate DNA damage responses; yet, conflicting reports obscure PARG biology and its impact on cancer cell resistance to PARP1 inhibitors. Here, we found that PARG expression is upregulated in many cancers. We employed chemical library screening to identify and optimize methylxanthine derivatives as selective bioavailable PARG inhibitors. Multiple crystal structures reveal how substituent positions on the methylxanthine core dictate binding modes and inducible-complementarity with a PARG-specific tyrosine clasp and arginine switch, supporting inhibitor specificity and a competitive inhibition mechanism. Cell-based assays show selective PARG inhibition and PARP1 hyperPARylation. Moreover, our PARG inhibitor sensitizes cells to radiation-induced DNA damage, suppresses replication fork progression and impedes cancer cell survival. In PARP inhibitor-resistant A172 glioblastoma cells, our PARG inhibitor shows comparable killing to Nedaplatin, providing further proof-of-concept that selectively inhibiting PARG can impair cancer cell survival. PARG catalyzes the removal of poly(ADP-ribose) (PAR) from target proteins and executes critical functions in the DNA damage response. Here the authors provide structural and biological insight with small molecule PARG inhibitors and show that PARG inhibition sensitizes cells to ionizing radiation and kills cancer cells through replication fork defects.

Poly ADP-ribosylation regulates cellular processes such as genomic stability maintenance, transcription and cell death. The structure of a mammalian poly(ADP-ribose) glycohydrolase gives insight into the enzyme's endoglycosidase activity and provides a basis for the development of therapeutic inhibitors. Reversible post-translational modification by poly(ADP-ribose) (PAR) regulates chromatin structure, DNA repair and cell fate in response to genotoxic stress. PAR glycohydrolase (PARG) removes PAR chains from poly ADP-ribosylated proteins to restore protein function and release oligo(ADP-ribose) chains to signal damage. Here we report crystal structures of mammalian PARG and its complex with a substrate mimic that reveal an open substrate-binding site and a unique 'tyrosine clasp' enabling endoglycosidic cleavage of branched PAR chains.

PROSTAGLANDINSand glucocorticoids are potent mediators of inflammation. Non-steroidal anti-inflammatory drugs (NSAIDs) exert their effects by inhibition of prostaglandin production. The pharmacological target of NSAIDs is cyclooxygenase (COX, also known as PGH synthase), which catalyses the first committed step in arachidonic-acid metabolism 1,2 . Two isoforms of the membrane protein COX are known 3 : COX-1, which is constitu-tively expressed in most tissues, is responsible for the physiological production of prostaglandins 4 ; and COX-2, which is induced by cytokines, mitogens and endotoxins in inflammatory cells 5 , is responsible for the elevated production of prostaglandins during inflammation 6,7 . The structure of ovine COX-1 complexed with several NSAIDs has been determined 8–10 . Here we report the structures of unliganded murine COX-2 and complexes with flurbiprofen, indomethacin and SC-558, a selective COX-2 inhibitor, determined at 3.0 to 2.5 Åresolution. These structures explain the structural basis for the selective inhibition of COX-2, and demonstrate some of the conformational changes associated with time-dependent inhibition.

Cyclooxygenases are bifunctional enzymes that catalyse the first committed step in the synthesis of prostaglandins, thromboxanes and other eicosanoids 1 , 2 , 3 . The two known cyclooxygenases isoforms share a high degree of amino-acid sequence similarity 1 , 2 , 3 , 4 , structural topology 5 , 6 , 7 and an identical catalytic mechanism 1 , 2 , 3 . Cyclooxygenase enzymes catalyse two sequential reactions in spatially distinct, but mechanistically coupled active sites 8 , 9 , 10 , 11 . The initial cyclooxygenase reaction converts arachidonic acid (which is achiral) to prostaglandin G 2 (which has five chiral centres). The subsequent peroxidase reaction reduces prostaglandin G 2 to prostaglandin H 2 . Here we report the co-crystal structures of murine apo-cyclooxygenase-2 in complex with arachidonic acid and prostaglandin. These structures suggest the molecular basis for the stereospecificity of prostaglandin G 2 synthesis.

HERPESVIRUSES encode a serine protease 1,2 that specifically cleaves assembly protein 3 . This protease is critical for replication 4 , and represents a new target for antiviral drug design 5 . Here we report the three-dimensional structure of the protease from human cytomegalovirus (hCMV) at 2.27 Å resolution. The structure reveals a unique fold and new catalytic strategy for cleavage. The monomer fold of the enzyme, a seven-stranded β-barrel encircled by a chain of helices that form the carboxy terminus of the molecule, is unrelated to those observed in classic serine proteases such as chymotrypsin and subtilisin. The serine nucleo-phile at position 132 is activated by two juxtaposed histidine residues at positions 63 and 157. Dimerization, which seems to be necessary for activity 6,7 , is observed in the crystals. Correlations of the structure with the sequences of herpesvirus proteases 1,5,8 suggest that dimerization may confer specificity and recognition in substrate binding.

5-enol-Pyruvylshikimate -3-phosphate synthase (EPSP synthase; phosphoenolpyruvate:3-phosphoshikimate 1-carboxyvinyltransferase, EC 2.5.1.19) is an enzyme on the pathway toward the synthesis of aromatic amino acids in plants, fungi, and bacteria and is the target of the broad-spectrum herbicide glyphosate. The three-dimensional structure of the enzyme from Escherichia coli has been determined by crystallographic techniques. The polypeptide backbone chain was traced by examination of an electron density map calculated at 3-A resolution. The two-domain structure has a distinctive fold and appears to be formed by 6-fold replication of a protein folding unit comprising two parallel helices and a four-stranded sheet. Each domain is formed from three of these units, which are related by an approximate threefold symmetry axis; in each domain three of the helices are completely buried by a surface formed from the three beta-sheets and solvent-accessible faces of the other three helices. The domains are related by an approximate dyad, but in the present crystals the molecule does not display pseudo-symmetry related to the symmetry of point group 32 because its approximate threefold axes are almost normal. A possible relation between the three-dimensional structure of the protein and the linear sequence of its gene will be described. The topological threefold symmetry and orientation of each of the two observed globular domains may direct the binding of substrates and inhibitors by a helix macrodipole effect and implies that the active site is located near the interdomain crossover segments. The structure also suggests a rationale for the glyphosate tolerance conferred by sequence alterations

Herpesviruses encode a serine protease that specifically cleaves assembly protein. This protease is critical for replication, and represents a new target for antiviral drug design. Here we report the three-dimensional structure of the protease from human cytomegalovirus (hCMV) at 2.27 angstroms resolution. The structure reveals a unique fold and new catalytic strategy for cleavage. The monomer fold of the enzyme, a seven-stranded beta-barrel encircled by a chain of helices that form the carboxy terminus of the molecule, is unrelated to those observed in classic serine proteases such as chymotrypsin and subtilisin. The serine nucleophile at position 132 is activated by two juxtaposed histidine residues at positions 63 and 157. Dimerization, which seems to be necessary for activity, is observed in the crystals. Correlations of the structure with the sequences of herpesvirus proteases suggest that dimerization may confer specificity and recognition in substrate binding.