RESUMEN
Other than more widely used methods, the use of styrene maleic acid allows the direct extraction of membrane proteins from the lipid bilayer into SMALPs keeping it in its native lipid surrounding. Here we present the combined use of SMALPs and LILBID-MS, allowing determination of oligomeric states of membrane proteins of different functionality directly from the native nanodiscs.
Asunto(s)
Lípidos/química , Maleatos/química , Proteínas de la Membrana/análisis , Estireno/química , Membrana Dobles de Lípidos/química , Espectrometría de Masas , Modelos Moleculares , Tamaño de la PartículaRESUMEN
Ion channel gating is essential for cellular homeostasis and is tightly controlled. In some eukaryotic and most bacterial ligand-gated K+ channels, RCK domains regulate ion fluxes. Until now, a single regulatory mechanism has been proposed for all RCK-regulated channels, involving signal transduction from the RCK domain to the gating area. Here, we present an inactive ADP-bound structure of KtrAB from Vibrio alginolyticus, determined by cryo-electron microscopy, which, combined with EPR spectroscopy and molecular dynamics simulations, uncovers a novel regulatory mechanism for ligand-induced action at a distance. Exchange of activating ATP to inactivating ADP triggers short helical segments in the K+-translocating KtrB dimer to organize into two long helices that penetrate deeply into the regulatory RCK domains, thus connecting nucleotide-binding sites and ion gates. As KtrAB and its homolog TrkAH have been implicated as bacterial pathogenicity factors, the discovery of this functionally relevant inactive conformation may advance structure-guided drug development.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Proteínas de Transporte de Catión/metabolismo , Proteínas de Transporte de Catión/ultraestructura , Vibrio alginolyticus/enzimología , Vibrio alginolyticus/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/química , Proteínas de Transporte de Catión/química , Microscopía por Crioelectrón , Espectroscopía de Resonancia por Spin del Electrón , Simulación de Dinámica MolecularRESUMEN
The superfamily of K+ transporters unites proteins from plants, fungi, bacteria, and archaea that translocate K+ and/or Na+ across membranes. These proteins are key components in osmotic regulation, pH homeostasis, and resistance to high salinity and dryness. The members of the superfamily are closely related to K+ channels such as KcsA but also show several striking differences that are attributed to their altered functions. This review highlights these functional differences, focusing on the bacterial superfamily members KtrB, TrkH, and KdpA. The functional variations within the family and comparison to MPM-type K+ channels are discussed in light of the recently solved structures of the Ktr and Trk systems.