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1.
Nat Commun ; 15(1): 4751, 2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38834573

RESUMO

Intracellular potassium (K+) homeostasis is fundamental to cell viability. In addition to channels, K+ levels are maintained by various ion transporters. One major family is the proton-driven K+ efflux transporters, which in gram-negative bacteria is important for detoxification and in plants is critical for efficient photosynthesis and growth. Despite their importance, the structure and molecular basis for K+-selectivity is poorly understood. Here, we report ~3.1 Å resolution cryo-EM structures of the Escherichia coli glutathione (GSH)-gated K+ efflux transporter KefC in complex with AMP, AMP/GSH and an ion-binding variant. KefC forms a homodimer similar to the inward-facing conformation of Na+/H+ antiporter NapA. By structural assignment of a coordinated K+ ion, MD simulations, and SSM-based electrophysiology, we demonstrate how ion-binding in KefC is adapted for binding a dehydrated K+ ion. KefC harbors C-terminal regulator of K+ conductance (RCK) domains, as present in some bacterial K+-ion channels. The domain-swapped helices in the RCK domains bind AMP and GSH and they inhibit transport by directly interacting with the ion-transporter module. Taken together, we propose that KefC is activated by detachment of the RCK domains and that ion selectivity exploits the biophysical properties likewise adapted by K+-ion-channels.


Assuntos
Microscopia Crioeletrônica , Proteínas de Escherichia coli , Escherichia coli , Potássio , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Glutationa/metabolismo , Simulação de Dinâmica Molecular , Potássio/metabolismo , Antiportadores de Potássio-Hidrogênio/metabolismo , Antiportadores de Potássio-Hidrogênio/química , Antiportadores de Potássio-Hidrogênio/genética , Domínios Proteicos
2.
Int J Biol Macromol ; 112: 638-647, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29412176

RESUMO

Trehalase catalyzes hydrolysis of trehalose and plays a crucial role in insect metabolism. In the present study, phylogenetic analysis and multiple sequence alignment suggested that H. armigera trehalase-1 (HaTre-1) is closely related to other soluble trehalases with conserved signature features and functional sites. We have expressed and purified recombinant HaTre-1 having Vmax ~0.16mM/min and KM ~1.34mM. Inhibition kinetics and Microscale thermophoresis illustrated competitive inhibition of HaTre-1 by Validamycin A having Ki ~3nM and KD ~542nM, respectively. Docking studies of HaTre-1 with Validamycin A indicated that it binds at the active site with multiple hydrogen bonds. Ingestion of Validamycin A resulted in impediment of H. armigera growth and developmental defects. Treated larvae showed concentration dependent decrease in fecundity. It also led to total inhibition of ex-vivo trehalase activity and down-regulation of gene expression of HaTre-1. Relatively high insect mortality was observed on tomato plants sprayed with combination of Validamycin A with Azadirachta indica (neem) and Pongamia pinnata (karanj) oil as compared to the individual treatments. This report has re-emphasized trehalase inhibition as a potential insecticidal strategy and also recommends Validamycin A as a prospective value-added ingredient to commercial bio-pesticide formulations.


Assuntos
Inositol/análogos & derivados , Lepidópteros/enzimologia , Lepidópteros/fisiologia , Trealase/antagonistas & inibidores , Trealase/metabolismo , Sequência de Aminoácidos , Animais , Bioensaio , Composição de Medicamentos , Sinergismo Farmacológico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Comportamento Alimentar , Concentração de Íons de Hidrogênio , Inositol/farmacologia , Cinética , Lepidópteros/efeitos dos fármacos , Lepidópteros/crescimento & desenvolvimento , Filogenia , Óleos de Plantas/química , Pongamia/química , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Temperatura , Trealase/química
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