scholarly journals Dynamic Aha1 Co-Chaperone Binding to Human Hsp90

2019 ◽  
Author(s):  
Javier Oroz ◽  
Laura J. Blair ◽  
Markus Zweckstetter
Keyword(s):  
Nmr Spectroscopy ◽  
Atpase Activity ◽  
Binding Site ◽  
Solution Structure ◽  
Structural Changes ◽  
Nucleotide Binding ◽  
Nucleotide Binding Site ◽  
Binding Mechanism ◽  
Chaperone Binding ◽  
Multiple Conformations

AbstractHsp90 is an essential chaperone that requires large allosteric changes to determine its ATPase activity and client binding. Because of the inherent low ATPase activity of human Hsp90, the co-chaperone Aha1, which is the only known ATPase stimulator in eukaryotes, is important for regulation of Hsp90’s allosteric timing. Little is known, however, about the structure of the Hsp90/Aha1 full-length complex. Here, we characterize the solution structure of unmodified human Hsp90 in complex with Aha1 using NMR spectroscopy. We show that the 214 kDa complex adopts multiple conformations in the absence of nucleotide. Interaction with Aha1 induces structural changes near the nucleotide-binding site in Hsp90’s N-terminal domain, providing a basis for its ATPase-enhancing activity. Moreover, the E67K mutation in Aha1 strongly diminishes the interaction, supporting a two-step binding mechanism. Our data reveal important aspects of this pivotal chaperone/co-chaperone interaction and emphasize the relevance of characterizing dynamic chaperone structures in solution.

scholarly journals Effects of Mg2+, anions and cations on the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum

1987 ◽  
Vol 245 (3) ◽  
pp. 723-730 ◽  
Author(s):  
H I Stefanova ◽  
R M Napier ◽  
J M East ◽  
A G Lee
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Kinetic Model ◽  
Atpase Activity ◽  
Binding Site ◽  
Conformational Transition ◽  
Nucleotide Binding ◽  
Nucleotide Binding Site ◽  
Phosphorylated Form ◽  
Low Concentrations ◽  
Anions And Cations

In a previous paper [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227] we presented a kinetic model for the activity of the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum. Here we extend the model to account for the effects on ATPase activity of Mg2+, cations and anions. We find that Mg2+ concentrations in the millimolar range inhibit ATPase activity, which we attribute to competition between Mg2+ and MgATP for binding to the nucleotide-binding site on the E1 and E2 conformations of the ATPase and on the phosphorylated forms of the ATPase. Competition is also suggested between Mg2+ and MgADP for binding to the phosphorylated form of the ATPase. ATPase activity is increased by low concentrations of K+, Na+ and NH4+, but inhibited by higher concentrations. It is proposed that these effects follow from an increase in the rate of dephosphorylation but a decrease in the rate of the conformational transition E1′PCa2-E2′PCa2 with increasing cation concentration. Li+ and choline+ decrease ATPase activity. Anions also decrease ATPase activity, the effects of I- and SCN- being more marked than that of Cl-. These effects are attributed to binding at the nucleotide-binding site, with a decrease in binding affinity and an increase in ‘off’ rate constant for the nucleotide.

scholarly journals Stimulation of the Ca2+-ATPase of sarcoplasmic reticulum by disulfiram

1996 ◽  
Vol 320 (1) ◽  
pp. 101-105 ◽  
Author(s):  
Anthony P STARLING ◽  
J. Malcolm EAST ◽  
Anthony G LEE
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Binding Site ◽  
Equilibrium Constant ◽  
Nucleotide Binding ◽  
Nucleotide Binding Site ◽  
Single Site ◽  
Stimulation Of

Disulfiram [bis(diethylthiocarbamoyl)disulphide] has been found to stimulate reversibly the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum. At pH 7.2, 2.1 mM ATP and 25 °C, ATPase activity was found to double on addition of 120 µM disulfiram. Stimulation fitted to binding of disulfiram at a single site with a Kd of 61 µM. Disulfiram had no effect on the Ca2+ affinity of the ATPase or on the rate of phosphorylation of the ATPase by ATP, but increased the rate of dissociation of Ca2+ from the phosphorylated ATPase (the transport step) and increased the rate of dephosphorylation of the phosphorylated ATPase. It also decreased the level of phosphorylation of the ATPase by Pi, consistent with a 7.5-fold decrease in the equilibrium constant of the phosphorylated to non-phosphorylated forms (E2PMg/E2PiMg) at 80 µM disulfiram. Disulfiram had no significant effect on the concentration of ATP resulting in stimulation of ATPase activity, suggesting that it does not bind to the empty nucleotide-binding site on the phosphorylated ATPase. Studies of the effects of mixtures of disulfiram and jasmone (another molecule that stimulates the ATPase) suggest that they bind to separate sites on the ATPase.

Characterization of Nucleotide Binding ­Site-Encoding Genes in Sweetpotato, Ipomoea batatas(L.) Lam., and Their Response to Biotic and Abiotic Stresses

2021 ◽  
pp. 1-15
Author(s):  
Zengzhi Si ◽  
Yake Qiao ◽  
Kai Zhang ◽  
Zhixin Ji ◽  
Jinling Han
Keyword(s):  
Binding Site ◽  
Abiotic Stresses ◽  
Ipomoea Batatas ◽  
Expression Profiles ◽  
Nucleotide Binding ◽  
Regulatory Elements ◽  
Nucleotide Binding Site ◽  
Biotic And Abiotic Stresses ◽  
Encoding Genes

Sweetpotato, <i>Ipomoea batatas</i> (L.) Lam., is an important and widely grown crop, yet its production is affected severely by biotic and abiotic stresses. The nucleotide binding site (NBS)-encoding genes have been shown to improve stress tolerance in several plant species. However, the characterization of NBS-encoding genes in sweetpotato is not well-documented to date. In this study, a comprehensive analysis of NBS-encoding genes has been conducted on this species by using bioinformatics and molecular biology methods. A total of 315 NBS-encoding genes were identified, and 260 of them contained all essential conserved domains while 55 genes were truncated. Based on domain architectures, the 260 NBS-encoding genes were grouped into 6 distinct categories. Phylogenetic analysis grouped these genes into 3 classes: TIR, CC (I), and CC (II). Chromosome location analysis revealed that the distribution of NBS-encoding genes in chromosomes was uneven, with a number ranging from 1 to 34. Multiple stress-related regulatory elements were detected in the promoters, and the NBS-encoding genes’ expression profiles under biotic and abiotic stresses were obtained. According to the bioinformatics analysis, 9 genes were selected for RT-qPCR analysis. The results revealed that <i>IbNBS75</i>, <i>IbNBS219</i>, and <i>IbNBS256</i> respond to stem nematode infection; <i>Ib­NBS240</i>, <i>IbNBS90</i>, and <i>IbNBS80</i> respond to cold stress, while <i>IbNBS208</i>, <i>IbNBS71</i>, and <i>IbNBS159</i> respond to 30% PEG treatment. We hope these results will provide new insights into the evolution of NBS-encoding genes in the sweetpotato genome and contribute to the molecular breeding of sweetpotato in the future.

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