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Classical hormone receptors reversibly and non-covalently bind active hormone molecules, which are generated by biosynthetic enzymes, to trigger signal transduction. The / hydrolase DWARF14 (D14), which hydrolyses the plant branching hormone strigolactone and interacts with the F-box protein D3/MAX2, is probably involved in strigolactone detection. However, the active form of strigolactone has yet to be identified and it is unclear which protein directly binds the active form of strigolactone, and in which manner, to act as the genuine strigolactone receptor. Here we report the crystal structure of the strigolactone-induced AtD14D3ASK1 complex, reveal that Arabidopsis thaliana (At)D14 undergoes an open-to-closed state transition to trigger strigolactone signalling, and demonstrate that strigolactone is hydrolysed into a covalently linked intermediate molecule (CLIM) to initiate a conformational change of AtD14 to facilitate interaction with D3. Notably, analyses of a highly branched Arabidopsis mutant d14-5 show that the AtD14(G158E) mutant maintains enzyme activity to hydrolyse strigolactone, but fails to efficiently interact with D3/MAX2 and loses the ability to act as a receptor that triggers strigolactone signalling in planta. These findings uncover a mechanism underlying the allosteric activation of AtD14 by strigolactone hydrolysis into CLIM, and define AtD14 as a non-canonical hormone receptor with dual functions to generate and sense the active form of strigolactone., Author(s): Ruifeng Yao [1]; Zhenhua Ming [2]; Liming Yan [2]; Suhua Li [1]; Fei Wang [1]; Sui Ma [1]; Caiting Yu [1]; Mai Yang [1]; Li Chen [1]; Linhai Chen [...]

DNA methylation is an important epigenetic modification (1-3). Ten-eleven translocation (TET) proteins are involved in DNA demethylation through iteratively oxidizing 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) (4-8). Here we show that human TET1 and TET2 are more active on 5mC-DNA than 5hmC/5fC-DNA substrates. We determine the crystal structures of TET2-5hmC-DNA and TET25fC-DNA complexes at 1.80 Å and 1.97 Å resolution, respectively. The cytosine portion of 5hmC/5fC is specifically recognized by TET2 in a manner similar to that of 5mC in the TET2-5mC-DNA structure (9), and the pyrimidine base of 5mC/5hmC/5fC adopts an almost identical conformation within the catalytic cavity. However, the hydroxyl group of 5hmC and carbonyl group of 5fC face towards the opposite direction because the hydroxymethyl group of 5hmC and formyl group of 5fC adopt restrained conformations through forming hydrogen bonds with the 1-carboxylate of NOG and N4 exocyclic nitrogen of cytosine, respectively. Biochemical analyses indicate that the substrate preference of TET2 results from the different efficiencies of hydrogen abstraction in TET2-mediated oxidation. The restrained conformation of 5hmC and 5fC within the catalytic cavity may prevent their abstractable hydrogen(s) adopting a favourable orientation for hydrogen abstraction and thus result in low catalytic efficiency. Our studies demonstrate that the substrate preference of TET2 results from the intrinsic value of its substrates at their 5mC derivative groups and suggest that 5hmC is relatively stable and less prone to further oxidation by TET proteins. Therefore, TET proteins are evolutionarily tuned to be less reactive towards 5hmC and facilitate the generation of 5hmC as a potentially stable mark for regulatory functions., Previous studies have shown that 5hmC is 10- to 100-fold more abundant than 5fC/5caC and that its level is relatively high in neurons, self-renewing and pluripotent stem cells, and greatly [...]

The heterotrimeric influenza virus polymerase, containing the PA, PB1 and PB2 proteins, catalyses viral RNA replication and transcription in the nucleus of infected cells. PBI holds the polymerase active site (1) and reportedly harbours endonuclease activity (2), whereas PB2 is responsible for cap binding (2-4). The PA amino terminus is understood to be the major functional part of the PA protein and has been implicated in several roles, including endonuclease (5) and protease activities (6) as well as viral RNA/complementary RNA promoter binding (7). Here we report the 2.2 angstrom (Å) crystal structure of the N-terminal 197 residues of PA, termed [PA.sub.N], from an avian influenza H5N1 virus. The [PA.sub.N] structure has an α/β architecture and reveals a bound magnesium ion coordinated by a motif similar to the (P)[DX.sub.N](D/E)XK motif characteristic of many endonucleases. Structural comparisons and mutagenesis analysis of the motif identified in PAN provide further evidence that [PA.sub.N] holds an endonuclease active site. Furthermore, functional analysis with in vivo ribonucleoprotein reconstitution and direct in vitro endonuclease assays strongly suggest that PAN holds the endonuclease active site and has critical roles in endonuclease activity of the influenza virus polymerase, rather than PB1. The high conservation of this endonuclease active site among influenza strains indicates that [PA.sub.N] is an important target for the design of new anti-influenza therapeutics., Highly pathogenic avian influenza A virus strains with H5N1 subtype are entrenched in poultry worldwide and pose a growing threat to human health. Of the 387 reported human cases of [...]

The recent emergence of highly pathogenic avian influenza A virus strains with subtype H5N1 pose a global threat to human health (1). Elucidation of the underlying mechanisms of viral replication [...]

The heterotrimeric influenza virus polymerase, containing the PA, PB1 and PB2 proteins, catalyses viral RNA replication and transcription in the nucleus of infected cells. PB1 holds the polymerase active site and reportedly harbours endonuclease activity, whereas PB2 is responsible for cap binding. The PA amino terminus is understood to be the major functional part of the PA protein and has been implicated in several roles, including endonuclease and protease activities as well as viral RNA/complementary RNA promoter binding. Here we report the 2.2 ångström (Å) crystal structure of the N-terminal 197 residues of PA, termed PAN, from an avian influenza H5N1 virus. The PAN structure has an α/β architecture and reveals a bound magnesium ion coordinated by a motif similar to the (P)DXN(D/E)XK motif characteristic of many endonucleases. Structural comparisons and mutagenesis analysis of the motif identified in PAN provide further evidence that PAN holds an endonuclease active site. Furthermore, functional analysis with in vivo ribonucleoprotein reconstitution and direct in vitro endonuclease assays strongly suggest that PAN holds the endonuclease active site and has critical roles in endonuclease activity of the influenza virus polymerase, rather than PB1. The high conservation of this endonuclease active site among influenza strains indicates that PAN is an important target for the design of new anti-influenza therapeutics. [ABSTRACT FROM AUTHOR]