Nucleoside binding

Abstract: INTRODUCTION Plants and animals respond to pathogen invasion through intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) that directly interact with pathogen proteins or indirectly detect pathogen-derived alterations in the host proteome. Upon recognition of pathogen invasion, NLRs trigger an immune response that resolves in a variety of ways depending on the type of NLR being activated. The overall architecture of NLRs is highly conserved, consisting of a C-terminal leucine-rich repeat (LRR) platform that determines substrate specificity and a central nucleotide-binding oligomerization domain. The N-terminal domain varies between NLRs and determines the mechanism used by the host to activate the immune response. Thus, NLRs in plants have been classified according to their N-terminal domain into Toll/interleukin 1 receptor (TIR) NLRs (TNLs), coiled-coil NLRs (CNLs), and RPW8-like coiled-coil NLRs (RNLs). Pathogen detection and oligomerization of the NLR activates these N-terminal domains by bringing them in close contact. In all three cases, association of the N-terminal domain leads to localized cell death and expression of disease resistance. The TIR domains of TNLs have been shown to have oligomerization-dependent NADase activity that is required for promoting cell death, but it is not understood how the interactions between TIR domains renders them catalytically active. RATIONALE The structure of the ROQ1 (recognition of XopQ 1)–XopQ (Xanthomonas outer protein Q) complex, an immune receptor bound to its pathogen substrate, was used as a model to study the mechanism of direct binding, oligomerization, and TIR domain activation of TNLs. ROQ1 has been shown to physically interact with the Xanthomonas effector XopQ, causing it to oligomerize and trigger a TIR-dependent hypersensitive cell death response. We coexpressed, extracted, and purified the assembled ROQ1-XopQ complex from ROQ1’s native host, Nicotiana benthamiana, and solved its structure by cryo–electron microscopy to 3.8-A resolution. The interactions described in our structure were further confirmed by in vivo mutational analysis. RESULTS Our structure reveals that ROQ1 forms a tetrameric resistosome upon recognizing XopQ. The LRR and a post-LRR domain named the C-terminal jelly-roll/Ig-like domain (C-JID), form a horseshoe-shaped scaffold that curls around the pathogen effector, thereby recognizing multiple regions of the substrate. Binding of the ROQ1 LRR to XopQ occurs through surface-exposed residues that make up the scaffold of the domain, as well as an elongated loop between two LRRs that forms a small amphipathic α-helix at the site of interaction. The mode of substrate recognition by the C-JID is reminiscent of that used by immunoglobulins to bind their antigen. Similar to the complementary-determining regions of antibodies, interconnecting loops emerging from the C-JID β-sandwich structure make substrate-specific contacts with XopQ. In particular, an extended loop of the C-JID dives into the active-site cleft of XopQ and interacts with conserved residues required for nucleoside binding, suggesting that ROQ1 not only recognizes its substrate but also inhibits its ligand-binding function. The nucleotide-binding domain (NBD), helical domain 1 (HD1) and the winged-helix domain (WHD), termed NB-ARC because of their presence in Apaf-1, R proteins, and CED-4 (ARC), are responsible for ROQ1 oligomerization in an ATP-bound state. Individual protomers intercalate in a similar fashion as found in other NLR structures, promoting association between the N-terminal TIR domains. The TIR domains bind to each other through two distinct interfaces (called AE and BE), causing them to form a dimer of dimers. BE-interface contacts cause a conformational rearrangement in a loop, called the BB-loop, at the periphery of the TIR domain active site that exposes the putative catalytic glutamate that is suggested to cleave NAD+. These results provide a rationale for the previously determined oligomerization dependence of TIR domain NADase activity. CONCLUSION We propose a step-by-step mechanism for ROQ1 immune signaling based on our structure of the activated complex and on previous biochemical studies. The LRR and C-JID of ROQ1 recognize the pathogen effector through direct contacts with its surface and active-site residues. Detection of the substrate releases autoinhibitory contacts between the NB-ARC domain and the LRR, allowing the NB-ARC domain to transition to an ATP-bound, oligomerization-prone state. Complex assembly brings the TIR domains in close contact, leading to opening of the NADase active site in an interface-dependent manner. Cleavage of NAD+ by the TIR domain results in the release of adenosine diphosphate ribose, a signaling molecule that triggers cytosolic Ca2+ influx, a widely used chemical cue in response to various biotic and abiotic stresses, leading to downstream activation of localized cell death and disease resistance.

Abstract: Deoxyribonucleoside kinases phosphorylate deoxyribonucleosides and activate a number of medically important nucleoside analogs. Here we report the structure of the Drosophila deoxyribonucleoside kinase with deoxycytidine bound at the nucleoside binding site and that of the human deoxyguanosine kinase with ATP at the nucleoside substrate binding site. Compared to the human kinase, the Drosophila kinase has a wider substrate cleft, which may be responsible for the broad substrate specificity of this enzyme. The human deoxyguanosine kinase is highly specific for purine substrates; this is apparently due to the presence of Arg 118, which provides favorable hydrogen bonding interactions with the substrate. The two new structures provide an explanation for the substrate specificity of cellular deoxyribonucleoside kinases.

Abstract: Dasabuvir (ABT-333) is a nonnucleoside inhibitor of the RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) NS5B gene. Dasabuvir inhibited recombinant NS5B polymerases derived from HCV genotype 1a and 1b clinical isolates, with 50% inhibitory concentration (IC 50 ) values between 2.2 and 10.7 nM, and was at least 7,000-fold selective for the inhibition of HCV genotype 1 polymerases over human/mammalian polymerases. In the HCV subgenomic replicon system, dasabuvir inhibited genotype 1a (strain H77) and 1b (strain Con1) replicons with 50% effective concentration (EC 50 ) values of 7.7 and 1.8 nM, respectively, with a 13-fold decrease in inhibitory activity in the presence of 40% human plasma. This level of activity was retained against a panel of chimeric subgenomic replicons that contained HCV NS5B genes from 22 genotype 1 clinical isolates from treatment-naive patients, with EC 50 s ranging between 0.15 and 8.57 nM. Maintenance of replicon-containing cells in medium containing dasabuvir at concentrations 10-fold or 100-fold greater than the EC 50 resulted in selection of resistant replicon clones. Sequencing of the NS5B coding regions from these clones revealed the presence of variants, including C316Y, M414T, Y448C, Y448H, and S556G, that are consistent with binding to the palm I site of HCV polymerase. Consequently, dasabuvir retained full activity against replicons known to confer resistance to other polymerase inhibitors, including the S282T variant in the nucleoside binding site and the M423T, P495A, P495S, and V499A single variants in the thumb domain. The use of dasabuvir in combination with inhibitors targeting HCV NS3/NS4A protease (ABT-450 with ritonavir) and NS5A (ombitasvir) is in development for the treatment of HCV genotype 1 infections.