RESUMEN
Type III protein secretion is widespread in Gram-negative pathogens. It comprises the injectisome with a surface-exposed needle and an inner membrane translocase. The translocase contains the SctRSTU export channel enveloped by the export gate subunit SctV that binds chaperone/exported clients and forms a putative ante-chamber. We probed the assembly, function, structure and dynamics of SctV from enteropathogenic E. coli (EPEC). In both EPEC and E. coli lab strains, SctV forms peripheral oligomeric clusters that are detergent-extracted as homo-nonamers. Membrane-embedded SctV9 is necessary and sufficient to act as a receptor for different chaperone/exported protein pairs with distinct C-domain binding sites that are essential for secretion. Negative staining electron microscopy revealed that peptidisc-reconstituted His-SctV9 forms a tripartite particle of â¼22 nm with a N-terminal domain connected by a short linker to a C-domain ring structure with a â¼5 nm-wide inner opening. The isolated C-domain ring was resolved with cryo-EM at 3.1 Å and structurally compared to other SctV homologues. Its four sub-domains undergo a three-stage "pinching" motion. Hydrogen-deuterium exchange mass spectrometry revealed this to involve dynamic and rigid hinges and a hyper-flexible sub-domain that flips out of the ring periphery and binds chaperones on and between adjacent protomers. These motions are coincident with local conformational changes at the pore surface and ring entry mouth that may also be modulated by the ATPase inner stalk. We propose that the intrinsic dynamics of the SctV protomer are modulated by chaperones and the ATPase and could affect allosterically the other subunits of the nonameric ring during secretion.
Asunto(s)
Adenosina Trifosfatasas/química , Escherichia coli Enteropatógena/ultraestructura , Proteínas de Escherichia coli/química , Flagelos/ultraestructura , Canales de Translocación SEC/química , Sistemas de Secreción Tipo III/ultraestructura , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Regulación Alostérica , Sitios de Unión , Clonación Molecular , Microscopía por Crioelectrón , Medición de Intercambio de Deuterio , Escherichia coli Enteropatógena/genética , Escherichia coli Enteropatógena/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Flagelos/genética , Flagelos/metabolismo , Expresión Génica , Regulación Bacteriana de la Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Cinética , Espectrometría de Masas , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canales de Translocación SEC/genética , Canales de Translocación SEC/metabolismo , Especificidad por Sustrato , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismoRESUMEN
Previously we introduced peptidiscs as an alternative to detergents to stabilize membrane proteins in solution (Carlson et al., 2018). Here, we present 'on-gradient' reconstitution, a new gentle approach for the reconstitution of labile membrane-protein complexes, and used it to reconstitute Rhodobacter sphaeroides reaction center complexes, demonstrating that peptidiscs can adapt to transmembrane domains of very different sizes and shapes. Using the conventional 'on-bead' approach, we reconstituted Escherichia coli proteins MsbA and MscS and find that peptidiscs stabilize them in their native conformation and allow for high-resolution structure determination by cryo-electron microscopy. The structures reveal that peptidisc peptides can arrange around transmembrane proteins differently, thus revealing the structural basis for why peptidiscs can stabilize such a large variety of membrane proteins. Together, our results establish the gentle and easy-to-use peptidiscs as a potentially universal alternative to detergents as a means to stabilize membrane proteins in solution for structural and functional studies.
Asunto(s)
Proteínas Bacterianas/química , Membrana Celular/química , Proteínas de la Membrana/química , Nanoestructuras , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Microscopía por Crioelectrón , Regulación Bacteriana de la Expresión Génica , Procesamiento de Imagen Asistido por Computador , Proteínas de la Membrana/clasificación , Conformación Proteica , Rhodobacter sphaeroides/metabolismoRESUMEN
Membrane proteins are difficult to work with due to their insolubility in aqueous solution and quite often their poor stability in detergent micelles. Here, we present the peptidisc for their facile capture into water-soluble particles. Unlike the nanodisc, which requires scaffold proteins of different lengths and precise amounts of matching lipids, reconstitution of detergent solubilized proteins in peptidisc only requires a short amphipathic bi-helical peptide (NSPr) and no extra lipids. Multiple copies of the peptide wrap around to shield the membrane-exposed part of the target protein. We demonstrate the effectiveness of this 'one size fits all' method using five different membrane protein assemblies (MalFGK2, FhuA, SecYEG, OmpF, BRC) during 'on-column', 'in-gel', and 'on-bead' reconstitution embedded within the membrane protein purification protocol. The peptidisc method is rapid and cost-effective, and it may emerge as a universal tool for high-throughput stabilization of membrane proteins to advance modern biological studies.
