title: Neurotoxic phospholipase A2 from rattlesnake as a new ligand and new regulator of prokaryotic receptor GLIC (proton-gated ion channel from G. violaceus).
abstract: Neurotoxic phospholipases A2 (sPLA2) from snake venoms interact with various protein targets with high specificity and potency. They regulate function of multiple receptors or channels essential to life processes including neuronal or neuromuscular chemoelectric signal transduction. These toxic sPLA2 exhibit high pharmacological potential and determination of PLA2-receptor binding sites represents challenging part in the receptor-channel biochemistry and pharmacology. To investigate the mechanism of interaction of neurotoxic PLA2 with its neuronal receptor at the molecular level, we used as a model crotoxin, a heterodimeric sPLA2 from rattlesnake venom and proton-gated ion channel GLIC, a bacterial homolog of pentameric ligand-gated ion channels. The three-dimensional structures of both partners, crotoxin and GLIC have been solved by X-ray crystallography and production of full-length pentameric GLIC (with ECD and TM domains) is well established. In the present study, for the first time, we demonstrated physical and functional interaction of full-length purified and solubilized GLIC with CB, (PLA2 subunit of crotoxin). We identified GLIC as a new protein target of CB and CB as a new ligand of GLIC, and showed that this non covalent interaction (PLA2-GLIC) involves the extracellular domain of GLIC. We also determined a novel function of CB as an inhibitor of proton-gated ion channel activity. In agreement with conformational changes observed upon formation of the complex, CB appears to be negative allosteric modulator (NAM) of GLIC. Finally, we proposed a possible stoichiometric model for CB - GLIC interaction based on analytical ultracentrifugation.
authors: Institut Pasteur, Unité Récepteurs-Canaux, CNRS-UMR 3571, 25, rue du Dr. Roux, F-75015 Paris, France; Department of Biochemistry, Nicolaus Copernicus University, Torun, Poland.
authors: Maciej
authors: M
authors: Ostrowski
authors: Institut Pasteur, Unité Récepteurs-Canaux, CNRS-UMR 3571, 25, rue du Dr. Roux, F-75015 Paris, France; Department of Biochemistry, Nicolaus Copernicus University, Torun, Poland.
authors: Dorota
authors: D
authors: Porowinska
authors: Institut Pasteur, Unité Récepteurs-Canaux, CNRS-UMR 3571, 25, rue du Dr. Roux, F-75015 Paris, France.
authors: Tomasz
authors: T
authors: Prochnicki
authors: Institut Pasteur, Unité Récepteurs-Canaux, CNRS-UMR 3571, 25, rue du Dr. Roux, F-75015 Paris, France.
authors: Marie
authors: M
authors: Prevost
authors: Institu Pasteur, Plate-Forme de Biophysique des Macromolecules et de leurs Interactions, 75015 Paris, France.
authors: Bertrand
authors: B
authors: Raynal
authors: Institu Pasteur, Plate-Forme de Biophysique des Macromolecules et de leurs Interactions, 75015 Paris, France.
authors: Bruno
authors: B
authors: Baron
authors: Institut Pasteur, Unité Récepteurs-Canaux, CNRS-UMR 3571, 25, rue du Dr. Roux, F-75015 Paris, France.
authors: Ludovic
authors: L
authors: Sauguet
authors: Institut Pasteur, Unité Récepteurs-Canaux, CNRS-UMR 3571, 25, rue du Dr. Roux, F-75015 Paris, France.
authors: Pierre-Jean
authors: PJ
authors: Corringer
authors: Institut Pasteur, Unité Récepteurs-Canaux, CNRS-UMR 3571, 25, rue du Dr. Roux, F-75015 Paris, France. Electronic address: grazyna.faure-kuzminska@pasteur.fr.
authors: Grazyna
authors: G
authors: Faure
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D003435:Crotalid Venoms / Q000737:chemistry*; D000458:Cyanobacteria / Q000378:metabolism; D004594:Electrophysiology; D058446:Ligand-Gated Ion Channels / Q000378:metabolism*; D008024:Ligands; D054467:Phospholipases A2 / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D001426:Bacterial Proteins; D003435:Crotalid Venoms; D058446:Ligand-Gated Ion Channels; D008024:Ligands; D011993:Recombinant Fusion Proteins; D054467:Phospholipases A2
keywords: Catalytic activity; Neurotoxic snake venom phospholipase A(2); Proton-gated ion channel GLIC; SPR-binding