RESUMO
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.
Assuntos
Dobramento de Proteína , Piridinolcarbamato , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Ligação Proteica , Receptores de Glucocorticoides/metabolismoRESUMO
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.
Assuntos
Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP90/química , Conformação Proteica , Proteína Supressora de Tumor p53/química , Trifosfato de Adenosina/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP90/genética , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Agregados Proteicos/genética , Ligação Proteica/genética , Dobramento de Proteína , Fatores de Transcrição/química , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genéticaRESUMO
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.
Assuntos
Adenosina Trifosfatases/química , Regulação Fúngica da Expressão Gênica , Proteínas de Choque Térmico HSP90/química , Modelos Moleculares , Receptores de Glucocorticoides/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Secundária de Proteína , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de SinaisRESUMO
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.