scholarly journals The second metal-binding site of 70 kDa heat-shock protein is essential for ADP binding, ATP hydrolysis and ATP synthesis

2004 ◽  
Vol 378 (3) ◽  
pp. 793-799 ◽  
Author(s):  
Xueji WU ◽  
Mihiro YANO ◽  
Hiroyo WASHIDA ◽  
Hiroshi KIDO
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Binding Site ◽  
Metal Binding ◽  
Atp Hydrolysis ◽  
Atp Synthesis ◽  
Site Directed Mutagenesis ◽  
Atp Binding ◽  
Binding Motif ◽  
Metal Binding Site

The chaperone activity of Hsp70 (70 kDa heat-shock protein) in protein folding and its conformational switch, including oligomeric and monomeric interconversion, are regulated by the hydrolysis of ATP and the ATP–ADP exchange cycle. The crystal structure of human ATPase domain shows two metal-binding sites, the first for ATP binding and a second, in close proximity to the first, whose function remains unknown [Sriram, Osipiuk, Freeman, Morimoto and Joachimiak (1997) Structure 5, 403–414]. In this study, we have characterized the second metal-binding motif by site-directed mutagenesis and the kinetics of ATP and ADP binding, and found that the second metal-binding site, comprising a loop co-ordinated by His-227, Glu-231 and Asp-232, participates both in ATP hydrolysis and ATP-synthetic activities, in co-operation with the first metal-binding site. The first metal-binding site, a catalytic centre, is essential for ATP binding and the second site for ADP binding in the reactions of ATP hydrolysis and ATP synthesis.

scholarly journals ClpE from Lactococcus lactis Promotes Repression of CtsR-Dependent Gene Expression

2003 ◽  
Vol 185 (17) ◽  
pp. 5117-5124 ◽  
Author(s):  
Pekka Varmanen ◽  
Finn K. Vogensen ◽  
Karin Hammer ◽  
Airi Palva ◽  
Hanne Ingmer
Keyword(s):  
Gene Expression ◽  
High Temperature ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Lactococcus Lactis ◽  
Blot Analysis ◽  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Adaptation Period ◽  
Bacterial Cells

ABSTRACT The heat shock response in bacterial cells is characterized by rapid induction of heat shock protein expression, followed by an adaptation period during which heat shock protein synthesis decreases to a new steady-state level. In this study we found that after a shift to a high temperature the Clp ATPase (ClpE) in Lactococcus lactis is required for such a decrease in expression of a gene negatively regulated by the heat shock regulator (CtsR). Northern blot analysis showed that while a shift to a high temperature in wild-type cells resulted in a temporal increase followed by a decrease in expression of clpP encoding the proteolytic component of the Clp protease complex, this decrease was delayed in the absence of ClpE. Site-directed mutagenesis of the zinc-binding motif conserved in ClpE ATPases interfered with the ability to repress CtsR-dependent expression. Quantification of ClpE by Western blot analysis revealed that at a high temperature ClpE is subjected to ClpP-dependent processing and that disruption of the zinc finger domain renders ClpE more susceptible. Interestingly, this domain resembles the N-terminal region of McsA, which was recently reported to interact with the CtsR homologue in Bacillus subtilis. Thus, our data point to a regulatory role of ClpE in turning off clpP gene expression following temporal heat shock induction, and we propose that this effect is mediated through CtsR.

scholarly journals Mutational analysis of the two ATP-binding sites in ClpB, a heat shock protein with protein-activated ATPase activity in Escherichia coli

1998 ◽  
Vol 333 (3) ◽  
pp. 671-676 ◽  
Author(s):  
Keun Il KIM ◽  
Kee Min WOO ◽  
Ihn Sik SEONG ◽  
Zee-Won LEE ◽  
Sung Hee BAEK ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Atpase Activity ◽  
Binding Sites ◽  
Mutational Analysis ◽  
Site Directed Mutagenesis ◽  
Atp Binding ◽  
Saturation Curve ◽  
Mutant Proteins

The 93 kDa ClpB (ClpB93) is a heat shock protein and has a protein-activated ATPase activity. To define the role of the two ATP-binding sites in ClpB93, site-directed mutagenesis was performed to replace Lys212 or Lys611 with Thr or Glu. All of the mutant proteins hydrolysed ATP at a higher rate than that seen with ClpB93 at ATP concentrations up to 2 mM. However, ClpB93 carrying mutations in both of the ATP-binding sites could not cleave ATP. Thus any of the two ATP-binding sites seems to be capable of supporting the ATPase activity of ClpB93. The ATPase activities of both ClpB93/K212T and ClpB93/K212E were gradually decreased when ATP concentrations were increased above 2 mM, unlike those of ClpB93, ClpB93/K611T and ClpB93/K611E, which showed a typical saturation curve. Furthermore ADP inhibited ATP hydrolysis by ClpB93/K212T and ClpB93/K212E more effectively than that by the latter proteins, suggesting that the mutations in the first ATP-binding site result in an increase in the affinity of ADP for the second site in ClpB93. In addition, all of the purified ClpB93 and its mutant forms behaved as an oligomer of 400–450 kDa on a Sephacryl S-300 gel-filtration column, whether or not ATP was present. Thus the binding of ATP to either of the two sites seems not to be essential for oligomerization of ClpB93. Although a low-copy plasmid carrying clpB93 could rescue the sensitivity of a clpB-null mutant cell at 52 °C, none of the plasmids carrying the mutations in the ATP-binding sites could. Furthermore, incubation at 52 °C resulted in a gradual loss of the ATPase activity of ClpB93 carrying the mutations in either of the two ATP-binding sites, but not of the parental ClpB93, indicating that the mutant proteins have a greater tendency to denature at this temperature than the parental ClpB93. These results suggest that both of the ATP-binding sites in ClpB have an important role in maintaining the thermotolerance of the protein and hence in the survival of Escherichia coli at high temperatures.

scholarly journals Spotlight on the microbes that produce heat shock protein 90-targeting antibiotics

Open Biology ◽  
2012 ◽  
Vol 2 (12) ◽  
pp. 120138 ◽  
Author(s):  
Peter W. Piper ◽  
Stefan H. Millson
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Binding Site ◽  
Cancer Progression ◽  
Heat Shock Protein 90 ◽  
Hsp90 Inhibitor ◽  
Cancer Drug ◽  
Atp Binding ◽  
Hsp90 Inhibition ◽  
Atp Binding Site

Heat shock protein 90 (Hsp90) is a promising cancer drug target as a molecular chaperone critical for stabilization and activation of several of the oncoproteins that drive cancer progression. Its actions depend upon its essential ATPase, an activity fortuitously inhibited with a very high degree of selectivity by natural antibiotics: notably the actinomycete-derived benzoquinone ansamycins (e.g. geldanamycin) and certain fungal-derived resorcyclic acid lactones (e.g. radicicol). The molecular interactions made by these antibiotics when bound within the ADP/ATP-binding site of Hsp90 have served as templates for the development of several synthetic Hsp90 inhibitor drugs. Much attention now focuses on the clinical trials of these drugs. However, because microbes have evolved antibiotics to target Hsp90, it is probable that they often exploit Hsp90 inhibition when interacting with each other and with plants. Fungi known to produce Hsp90 inhibitors include mycoparasitic, as well as plant-pathogenic, endophytic and mycorrhizal species. The Hsp90 chaperone may, therefore, be a prominent target in establishing a number of mycoparasitic (interfungal), fungal pathogen–plant and symbiotic fungus–plant relationships. Furthermore the Hsp90 family proteins of the microbes that produce Hsp90 inhibitor antibiotics are able to reveal how drug resistance can arise by amino acid changes in the highly conserved ADP/ATP-binding site of Hsp90.

scholarly journals Mebendazole’s Conformational Space and its Predicted Binding to Human Heat-Shock Protein 90

2021 ◽  
Author(s):  
Walter Fiedler ◽  
Fabian Freisleben ◽  
Jasmin Wellbrock ◽  
Karl Kirschner
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Binding Site ◽  
Leukemia Cell ◽  
Heat Shock Protein 90 ◽  
Md Simulations ◽  
Conformational Space ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Experimental Crystal

Recent experimental evidence suggest that mebendazole, a popular antiparasitic drug, binds to heat shock protein 90 (Hsp90) and inhibit acute myeloid leukemia cell growth. In this study we use quantum mechanics (QM), molecular similarity and molecular dynamics (MD) calculations to predict possible binding poses of mebendazole to the adenosine triphosphate (ATP) binding site of Hsp90. Extensive conformational searches and minimization of the five tautomers of mebendazole using MP2/aug-cc-pVTZ theory level resulting in 152 minima being identified. Mebendazole-Hsp90 complex models were created using the QM optimized conformations and protein coordinates obtained from experimental crystal structures that were chosen through similarity calculations. Nine different poses were identified from a total of 600 ns of explicit solvent, all-atom MD simulations using two different force fields. All simulations support the hypothesis that mebendazole is able to bind to the ATP binding site of Hsp90.

Unraveling the Substrate−Metal Binding Site of Ferrochelatase:  An X-ray Absorption Spectroscopic Study†

Biochemistry ◽  
2002 ◽  
Vol 41 (15) ◽  
pp. 4809-4818 ◽  
Author(s):  
Gloria C. Ferreira ◽  
Ricardo Franco ◽  
Arianna Mangravita ◽  
Graham N. George
Keyword(s):  
Spectroscopic Study ◽  
Binding Site ◽  
Metal Binding ◽  
Metal Binding Site ◽  
X Ray ◽  
Substrate Metal ◽  
X Ray Absorption
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