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Acetylcholinesterase (AChE), a key enzyme in the central and peripheral nervous systems, is the principal target of organophosphorus nerve agents. Quaternary oximes can regenerate AChE activity by displacing the phosphyl group of the nerve agent from the active site, but they are poorly distributed in the central nervous system. A promising reactivator based on tetrahydroacridine linked to a nonquaternary oxime is also an undesired submicromolar reversible inhibitor of AChE. X-ray structures and molecular docking indicate that structural modification of the tetrahydroacridine might decrease inhibition without affecting reactivation. The chlorinated derivative was synthesized and, in line with the prediction, displayed a 10-fold decrease in inhibition but no significant decrease in reactivation efficiency. X-ray structures with the derivative rationalize this outcome. We thus show that rational design based on structural studies permits the refinement of new-generation pyridine aldoxime reactivators that may be more effective in the treatment of nerve agent intoxication.

Background:In animal cells, nine aminoacyl-tRNA synthetases form a supramolecular assembly. Results: The low resolution solution structure of this complex is determined by SAXS. Conclusion: This complex exhibits a non-compact, elongated, and multi-armed shape. Significance: This type of structural organization provides new insights to understand the structure-function relationship and spatiotemporal regulation of the activity of its multiple components.

Cytosolic and mitochondrial lysyl-tRNA synthetases (LysRS) are encoded by a single gene and can be distinguished only according to their very N-terminal sequences. It was believed that cytosolic LysRS is packaged into HIV-1 virions via its association with Gag. Using monospecific antibodies, it was later shown that only the mitochondrial LysRS is taken up in viral particles along with tRNA(3)(Lys), the primer for reverse transcription of the HIV-1 genome. In this work, we re-analyzed the interaction between LysRS and GagPol to determine whether the particular N-terminal sequence of mitochondrial LysRS triggers a specific recognition with GagPol, or if differential routing of the two LysRS species in vivo could explain specific and exclusive packaging of the mitochondrial species. Here, we show that LysRS associates with the Pol domain of GagPol. More specifically, the transframe (TF or p6) and integrase (IN) domain proteins of Pol interact with the catalytic domain of LysRS. A model of the assembly of the LysRS-tRNA(3)(Lys)-GagPol packaging complex is proposed, which is consistent with the release of its different components after maturation of GagPol in the virions. The cytoplasmic and mitochondrial LysRS species share an identical catalytic domain. Accordingly, we found that both enzymes have the intrinsic capacity to bind to GagPol in vitro. In addition, both enzymes interact with p38 in vitro, the scaffold protein of the cytoplasmic multi-aminoacyl-tRNA synthetase complex, even though only the cytoplasmic species of LysRS is a bona fide component of this complex. These results suggest that the different LysRS species are strictly targeted in vivo, and open new perspectives for the search of a new class of inhibitors of the HIV-1 development cycle that would block the packaging of tRNA(3)(Lys) into viral particles.

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Donepezil-based reactivators 1–3 show a better ability (8 fold higher) than pralidoxime to reactivate VX-hAChE, and oxime 2 is 5 to 11 fold more efficient than pralidoxime and HI-6 respectively to reactivate of VX-hBChE.

The cytoplasmic and mitochondrial species of human lysyl-tRNA synthetase are encoded by a single gene by means of alternative splicing of the KARS1 gene. The cytosolic enzyme possesses a eukaryote-specific N-terminal polypeptide extension that confers on the native enzyme potent tRNA binding properties required for the vectorial transfer of tRNA from the synthetase to elongation factor EF1A within the eukaryotic translation machinery. The mitochondrial enzyme matures from its precursor upon being targeted to that organelle. To understand how the cytosolic and mitochondrial enzymes are adapted to participate in two distinct translation machineries, of eukaryotic or bacterial origin, we characterized the mitochondrial LysRS species. Here we report that cleavage of the precursor of mitochondrial LysRS leads to a mature enzyme with reduced tRNA binding properties compared to those of the cytoplasmic counterpart. This adaptation mechanism may prevent inhibition of translation through sequestration of lysyl-tRNA on the synthetase in a compartment where the bacterial-like elongation factor EF-Tu could not assist in its dissociation from the synthetase. We also observed that the RxxxKRxxK tRNA-binding motif of mitochondrial LysRS is not functional in the precursor form of that enzyme and becomes operational after cleavage of the mitochondrial targeting sequence. The finding that maturation of the precursor is needed to reveal the potent tRNA binding properties of this enzyme has strong implications for the spatiotemporal regulation of its activities and is consistent with previous studies suggesting that the only LysRS species able to promote packaging of tRNA(Lys) into HIV-1 viral particles is the mature form of the mitochondrial enzyme.