RESUMEN
The 43 kDa subunit of the chloroplast signal recognition particle, cpSRP43, is an ATP-independent chaperone essential for the biogenesis of the light harvesting chlorophyll-binding proteins (LHCP), the most abundant membrane protein family on earth. cpSRP43 is activated by a stromal factor, cpSRP54, to more effectively capture and solubilize LHCPs. The molecular mechanism underlying this chaperone activation is unclear. Here, a combination of hydrogen-deuterium exchange, electron paramagnetic resonance, and NMR spectroscopy experiments reveal that a disorder-to-order transition of the ankyrin repeat motifs in the substrate binding domain of cpSRP43 drives its activation. An analogous coil-to-helix transition in the bridging helix, which connects the ankyrin repeat motifs to the cpSRP54 binding site in the second chromodomain, mediates long-range allosteric communication of cpSRP43 with its activating binding partner. Our results provide a molecular model to explain how the conformational dynamics of cpSRP43 enables regulation of its chaperone activity and suggest a general mechanism by which ATP-independent chaperones with cooperatively folding domains can be regulated.
Asunto(s)
Adenosina Trifosfato/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de la Membrana/genética , Partícula de Reconocimiento de Señal/genética , Secuencia de Aminoácidos/genética , Proteínas de Arabidopsis/ultraestructura , Sitios de Unión , Cloroplastos/genética , Complejos de Proteína Captadores de Luz/genética , Modelos Moleculares , Chaperonas Moleculares/genética , Unión Proteica/genética , Conformación Proteica , Pliegue de Proteína , Partícula de Reconocimiento de Señal/ultraestructuraRESUMEN
Integral membrane proteins are prone to aggregation and misfolding in aqueous environments and therefore require binding by molecular chaperones during their biogenesis. Chloroplast signal recognition particle 43 (cpSRP43) is an ATP-independent chaperone required for the biogenesis of the most abundant class of membrane proteins, the light-harvesting chlorophyll a/b-binding proteins (LHCPs). Previous work has shown that cpSRP43 specifically recognizes an L18 loop sequence conserved among LHCP paralogs. However, how cpSRP43 protects the transmembrane domains (TMDs) of LHCP from aggregation was unclear. In this work, alkylation-protection and site-specific cross-linking experiments found that cpSRP43 makes extensive contacts with all the TMDs in LHCP. Site-directed mutagenesis identified a class of cpSRP43 mutants that bind tightly to the L18 sequence but are defective in chaperoning full-length LHCP. These mutations mapped to hydrophobic surfaces on or near the bridging helix and the ß-hairpins lining the ankyrin repeat motifs of cpSRP43, suggesting that these regions are potential sites for interaction with the client TMDs. Our results suggest a working model for client protein interactions in this membrane protein chaperone.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Unión a Clorofila/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Mapas de Interacción de Proteínas , Partícula de Reconocimiento de Señal/metabolismo , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Unión a Clorofila/química , Modelos Moleculares , Complejo de Proteína del Fotosistema II/química , Mutación Puntual , Agregado de Proteínas , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Partícula de Reconocimiento de Señal/química , Partícula de Reconocimiento de Señal/genéticaRESUMEN
Membrane protein biogenesis poses enormous challenges to cellular protein homeostasis and requires effective molecular chaperones. Compared with chaperones that promote soluble protein folding, membrane protein chaperones require tight spatiotemporal coordination of their substrate binding and release cycles. Here we define the chaperone cycle for cpSRP43, which protects the largest family of membrane proteins, the light harvesting chlorophyll a/b-binding proteins (LHCPs), during their delivery. Biochemical and NMR analyses demonstrate that cpSRP43 samples three distinct conformations. The stromal factor cpSRP54 drives cpSRP43 to the active state, allowing it to tightly bind substrate in the aqueous compartment. Bidentate interactions with the Alb3 translocase drive cpSRP43 to a partially inactive state, triggering selective release of LHCP's transmembrane domains in a productive unloading complex at the membrane. Our work demonstrates how the intrinsic conformational dynamics of a chaperone enables spatially coordinated substrate capture and release, which may be general to other ATP-independent chaperone systems.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Cloroplastos/química , Complejos de Proteína Captadores de Luz/metabolismo , Chaperonas Moleculares/metabolismo , Partícula de Reconocimiento de Señal/química , Secuencia de Aminoácidos , Proteínas de Arabidopsis/metabolismo , Sitios de Unión , Proteínas de Cloroplastos/metabolismo , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Partícula de Reconocimiento de Señal/metabolismo , Solubilidad , Relación Estructura-Actividad , Proteínas de las Membranas de los Tilacoides/metabolismo , Tilacoides/metabolismoRESUMEN
A method for the Pd-catalyzed N-arylation of both aryl and alkyl amidines with a wide range of aryl bromides, chlorides, and triflates is described. The reactions proceed in short reaction times and with excellent selectivity for monoarylation. A one-pot synthesis of quinazoline derivatives, via addition of an aldehyde to the crude reaction mixture following Pd-catalyzed N-arylation, is also demonstrated.