RESUMEN
Folding of stringent clients requires transfer from Hsp70 to Hsp90. The co-chaperone Hop physically connects the chaperone machineries. Here, we define its role from the remodeling of Hsp70/40-client complexes to the mechanism of client transfer and the conformational switching from stalled to active client-processing states of Hsp90. We show that Hsp70 together with Hsp40 completely unfold a stringent client, the glucocorticoid receptor ligand-binding domain (GR-LBD) in large assemblies. Hop remodels these for efficient transfer onto Hsp90. As p23 enters, Hsp70 leaves the complex via switching between binding sites in Hop. Current concepts assume that to proceed to client folding, Hop dissociates and the co-chaperone p23 stabilizes the Hsp90 closed state. In contrast, we show that p23 functionally interacts with Hop, relieves the stalling Hsp90-Hop interaction, and closes Hsp90. This reaction allows folding of the client and is thus the key regulatory step for the progression of the chaperone cycle.
Asunto(s)
Pliegue de Proteína , Piridinolcarbamato , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Unión Proteica , Receptores de Glucocorticoides/metabolismoRESUMEN
p53, the guardian of the genome, requires chaperoning by Hsp70 and Hsp90. However, how the two chaperone machineries affect p53 conformation and regulate its function remains elusive. We found that Hsp70, together with Hsp40, unfolds p53 in an ATP-dependent reaction. This unfolded state of p53 is susceptible to aggregation after release induced by the nucleotide exchange factor Bag-1. However, when Hsp90 and the adaptor protein Hop are present, p53 is transferred from Hsp70 to Hsp90, allowing restoration of the native state upon ATP hydrolysis. Our results suggest that the p53 conformation is constantly remodeled by the two major chaperone machineries. This connects p53 activity to stress, and the levels of free molecular chaperones are important factors regulating p53 activity. Together, our findings reveal an intricate interplay and cooperation of Hsp70 and Hsp90 in regulating the conformation of a client.
Asunto(s)
Proteínas HSP70 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/química , Conformación Proteica , Proteína p53 Supresora de Tumor/química , Adenosina Trifosfato/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/genética , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Agregado de Proteínas/genética , Unión Proteica/genética , Pliegue de Proteína , Factores de Transcripción/química , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genéticaRESUMEN
Heat shock protein 90 (Hsp90) is a dimeric molecular chaperone that undergoes large conformational changes during its functional cycle. It has been established that conformational switch points exist in the N-terminal (Hsp90-N) and C-terminal (Hsp90-C) domains of Hsp90, however information for switch points in the large middle-domain (Hsp90-M) is scarce. Here we report on a tryptophan residue in Hsp90-M as a new type of switch point. Our study shows that this conserved tryptophan senses the interaction of Hsp90 with a stringent client protein and transfers this information via a cation-π interaction with a neighboring lysine. Mutations at this position hamper the communication between domains and the ability of a client protein to affect the Hsp90 cycle. The residue thus allows Hsp90 to transmit information on the binding of a client from Hsp90-M to Hsp90-N which is important for progression of the conformational cycle and the efficient processing of client proteins.
Asunto(s)
Adenosina Trifosfatasas/química , Proteínas HSP90 de Choque Térmico/química , Lisina/química , Mutación , Triptófano/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Caenorhabditis elegans/química , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Ligandos , Lisina/metabolismo , Ratones , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/química , Alineación de Secuencia , Homología Estructural de Proteína , Triptófano/metabolismo , Pez Cebra/metabolismoRESUMEN
Hsp90 is the most abundant molecular chaperone in the eukaryotic cell. One of the most stringent clients is the glucocorticoid receptor (GR), whose in vivo function strictly depends on the interaction with the Hsp90 machinery. However, the molecular mechanism of this interaction has been elusive. Here we have reconstituted the interaction of Hsp90 with hormone-bound GR using purified components. Our biochemical and structural analyses define the binding site for GR on Hsp90 and reveal that binding of GR modulates the conformational cycle of Hsp90. FRET experiments demonstrate that a partially closed form of the Hsp90 dimer is the preferred conformation for interaction. Consistent with this, the conformational cycle of Hsp90 is decelerated, and its ATPase activity decreases. Hsp90 cochaperones differentially affect formation of the Hsp90-GR complex, serving as control elements for cycle progression and revealing an intricate interplay of client and cochaperones as molecular modulators of the Hsp90 machine.
