RESUMEN
Hsp90 couples ATP hydrolysis to large conformational changes essential for activation of client proteins. The structural transitions involve dimerization of the N-terminal domains and formation of 'closed states' involving the N-terminal and middle domains. Here, we used Hsp90 mutants that modulate ATPase activity and biological function as probes to address the importance of conformational cycling for Hsp90 activity. We found no correlation between the speed of ATP turnover and the in vivo activity of Hsp90: some mutants with almost normal ATPase activity were lethal, and some mutants with lower or undetectable ATPase activity were viable. Our analysis showed that it is crucial for Hsp90 to attain and spend time in certain conformational states: a certain dwell time in open states is required for optimal processing of client proteins, whereas a prolonged population of closed states has negative effects. Thus, the timing of conformational transitions is crucial for Hsp90 function and not cycle speed.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/genética , Modelos Moleculares , Mutación Puntual , Conformación Proteica , Dominios Proteicos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone responsible for the activation, maturation, and trafficking of several hundred client proteins in the cell. It is well known that (but not understood how) residues far away from Hsp90's nucleotide binding pocket can regulate its ATPase activity, a phenomenon called allosteric regulation. Here, the computational design of allosteric mutations was combined with in vitro and in vivo experiments to unravel nucleotide-responsive hot spots in the regulation of Hsp90. With this approach, we identified both activating and inhibiting regulation points and show that changes in those amino acids affect the conformational dynamics and ATPase activity of Hsp90 in vitro. Our observations that activating mutations loosen and inhibiting mutations rigidify the protein explain for the first time how Hsp90 changes in response to allosteric mutations. Additionally, mutations of these allosteric regulation points can be controlled by the interplay with Hsp90 co-chaperones, thus providing cells with an efficient mechanism of modifying Hsp90's intrinsic properties via different layers of regulation. Altogether, our results show that a framework for transmitting conformational information exists in the Hsp90 structure.
Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Saccharomyces cerevisiae/enzimología , Regulación Alostérica , Análisis Mutacional de ADN , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
The molecular chaperone Hsp90 undergoes an ATP-driven cycle of conformational changes in which large structural rearrangements precede ATP hydrolysis. Well-established small-molecule inhibitors of Hsp90 compete with ATP-binding. We wondered whether compounds exist that can accelerate the conformational cycle. In a FRET-based screen reporting on conformational rearrangements in Hsp90 we identified compounds. We elucidated their mode of action and showed that they can overcome the intrinsic inhibition in Hsp90 which prevents these rearrangements. The mode of action is similar to that of the co-chaperone Aha1 which accelerates the Hsp90 ATPase. However, while the two identified compounds influence conformational changes, they target different aspects of the structural transitions. Also, the binding site determined by NMR spectroscopy is distinct. This study demonstrates that small molecules are capable of triggering specific rate-limiting transitions in Hsp90 by mechanisms similar to those in protein cofactors.
Asunto(s)
Proteínas HSP90 de Choque Térmico/química , Transferencia Resonante de Energía de Fluorescencia , Resonancia Magnética Nuclear Biomolecular , Conformación ProteicaRESUMEN
Heat shock protein 90 (Hsp90) is an essential molecular chaperone in eukaryotes that facilitates the conformational maturation and function of a diverse protein clientele, including aberrant and/or over-expressed proteins that are involved in cancer growth and survival. A role for Hsp90 in supporting the protein homeostasis of cancer cells has buoyed interest in the utility of Hsp90 inhibitors as anti-cancer drugs. Despite the fact that all clinically evaluated Hsp90 inhibitors target an identical nucleotide-binding pocket in the N domain of the chaperone, the precise determinants that affect drug binding in the cellular environment remain unclear, and it is possible that chemically distinct inhibitors may not share similar binding preferences. Here we demonstrate that two chemically unrelated Hsp90 inhibitors, the benzoquinone ansamycin geldanamycin and the purine analog PU-H71, select for overlapping but not identical subpopulations of total cellular Hsp90, even though both inhibitors bind to an amino terminal nucleotide pocket and prevent N domain dimerization. Our data also suggest that PU-H71 is able to access a broader range of N domain undimerized Hsp90 conformations than is geldanamycin and is less affected by Hsp90 phosphorylation, consistent with its broader and more potent anti-tumor activity. A more complete understanding of the impact of the cellular milieu on small molecule inhibitor binding to Hsp90 should facilitate their more effective use in the clinic.
Asunto(s)
Benzodioxoles/metabolismo , Benzodioxoles/farmacología , Benzoquinonas/metabolismo , Benzoquinonas/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/metabolismo , Lactamas Macrocíclicas/metabolismo , Lactamas Macrocíclicas/farmacología , Procesamiento Proteico-Postraduccional , Purinas/metabolismo , Purinas/farmacología , Benzodioxoles/química , Benzoquinonas/química , Sitios de Unión , Línea Celular Tumoral , Células HEK293 , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/genética , Humanos , Lactamas Macrocíclicas/química , Fosforilación , Unión Proteica , Conformación Proteica , Purinas/química , Transfección , Células Tumorales CultivadasRESUMEN
The ATPase-driven dimeric molecular Hsp90 (heat shock protein 90) and its cofactor Cdc37 (cell division cycle 37 protein) are crucial to prevent the cellular depletion of many protein kinases. In complex with Hsp90, Cdc37 is thought to bind an important lid structure in the ATPase domain of Hsp90 and inhibit ATP turnover by Hsp90. As different interaction modes have been reported, we were interested in the interaction mechanism of Hsp90 and Cdc37. We find that Cdc37 can bind to one subunit of the Hsp90 dimer. The inhibition of the ATPase activity is caused by a reduction in the closing rate of Hsp90 without obviously bridging the two subunits or affecting nucleotide accessibility to the binding site. Although human Cdc37 binds to the N-terminal domain of Hsp90, nematodal Cdc37 preferentially interacts with the middle domain of CeHsp90 and hHsp90, exposing two Cdc37 interaction sites. A previously unreported site in CeCdc37 is utilized for the middle domain interaction. Dephosphorylation of CeCdc37 by the Hsp90-associated phosphatase PPH-5, a step required during the kinase activation process, proceeds normally, even if only the new interaction site is used. This shows that the second interaction site is also functionally relevant and highlights that Cdc37, similar to the Hsp90 cofactors Sti1 and Aha1, may utilize two different attachment sites to restrict the conformational freedom and the ATP turnover of Hsp90.
Asunto(s)
Adenosina Trifosfatasas/química , Proteínas de Ciclo Celular/química , Chaperoninas/química , Proteínas HSP90 de Choque Térmico/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Sitios de Unión , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/genética , Chaperoninas/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiologíaRESUMEN
In the N2 domain of the gene-3-protein of phage fd, two consecutive beta-strands are connected by a mobile loop of seven residues (157-163). The stability of this loop is low, and the Asp160-Pro161 bond at its tip shows conformational heterogeneity with 90% being in the cis and 10% in the trans form. The refolding kinetics of N2 are complex because the molecules with cis or trans isomers at Pro161 both fold to native-like conformations, albeit with different rates. We employed consensus design to shorten the seven-residue irregular loop around Pro161 to a four-residue type I' turn without a proline. This increased the conformational stability of N2 by almost 10 kJ mol(-1) and abolished the complexity of the folding kinetics. Turn sequences obtained from in vitro selections for increased stability strongly resembled those derived from the consensus design. Two other type I' turns of N2 could also be stabilized by consensus design. For all three turns, the gain in stability originates from an increase in the rate of refolding. The turns form native-like structures early during refolding and thus stabilize the folding transition state. The crystal structure of the variant with all three stabilized turns confirms that the 157-163 loop was in fact shortened to a type I' turn and that the other turns maintained their type I' conformation after sequence optimization.