The highly conserved 90 kDa heat shock protein (Hsp90) chaperones use ATP to regulate the stability and activity of many signalling molecules like protein kinases and transcription factors. Studies using crystallography, electron microscopy and small-angle X-ray scattering yielded controversial results for the conformational states that these dimeric multidomain proteins assume while progressing through the ATPase cycle. To better understand the molecular mechanism of Hsp90 proteins, we studied the conformational dynamics of the Escherichia coli homologue HtpG in solution using amide hydrogen exchange mass spectrometry (HX-MS) and fluorescence spectroscopy. A conformation-sensitive fluorescent probe allowed to elucidate the ATPase cycle of HtpG. Continuous-labelling and pulse-labelling HX-MS experiments revealed major ATP-induced conformational changes throughout the protein that do not occur simultaneously, but progress surprisingly slow from the immediate nucleotide-binding site towards the N terminus and the middle domain. The conversion between the different conformational states is rate limiting for ATP hydrolysis, and the nucleotide-coordinating residue, Glu34, is important for the rate constant of conversion. Our findings, for the first time, allow to kinetically resolve changes in the conformational dynamics of individual structural elements of Hsp90.

The E3 ubiquitin ligase CHIP (C‐terminus of Hsc70‐interacting protein) is believed to be a central player in the cellular triage decision, as it links the molecular chaperones Hsp70/Hsc70 and Hsp90 to the ubiquitin proteasomal degradation pathway. To better understand the decision process, we determined the affinity of CHIP for Hsp70 and Hsp90 using isothermal titration calorimetry. We analyzed the influence of CHIP on the ATPase cycles of both chaperones in the presence of co‐chaperones and a substrate, and determined the ubiquitination efficacy of CHIP in the presence of the chaperones. We found that CHIP has a sixfold higher affinity for Hsp90 compared with Hsc70. CHIP had no influence on ADP dissociation or ATP association, but reduced the Hsp70 cochaperone Hdj1‐stimulated single‐turnover ATPase rates of Hsc70 and Hsp70. CHIP did not influence the ATPase cycle of Hsp90 in the absence of co‐chaperones or in the presence of the Hsp90 cochaperones Aha1 or p23. Polyubiquitination of heat‐denatured luciferase and the native substrate p53 was much more efficient in the presence of Hsc70 and Hdj1 than in the presence of Hsp90, indicating that CHIP preferentially ubiquitinates Hsp70‐bound substrates. Structured digital abstract  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7904367: CHIP (uniprotkb:http://www.uniprot.org/uniprot/Q9UNE7) and HSP 90‐beta (uniprotkb:http://www.uniprot.org/uniprot/P08238) physically interact (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915) by molecular sieving (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071)  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7904785: HSP 90‐beta (uniprotkb:http://www.uniprot.org/uniprot/P08238) and p23 (uniprotkb:http://www.uniprot.org/uniprot/Q15185) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by molecular sieving (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071)  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7904047: CHIP (uniprotkb:http://www.uniprot.org/uniprot/Q9UNE7), HSP 90‐beta (uniprotkb:http://www.uniprot.org/uniprot/P08238) and p23 (uniprotkb:http://www.uniprot.org/uniprot/Q15185) physically interact (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915) by molecular sieving (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071)  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7903424: Alpha‐lactalbumin (uniprotkb:http://www.uniprot.org/uniprot/P00711), HSP70 (uniprotkb:http://www.uniprot.org/uniprot/P08107) and CHIP (uniprotkb:http://www.uniprot.org/uniprot/Q9UNE7) physically interact (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915) by molecular sieving (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071)  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7903354: CHIP (uniprotkb:http://www.uniprot.org/uniprot/Q9UNE7) and HSC70 (uniprotkb:http://www.uniprot.org/uniprot/P11142) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by isothermal titration calorimetry (h...

The dimeric E3 ubiquitin ligase CHIP binds with its tetratricopeptide repeat (TPR) domain the C-terminus of molecular chaperones Hsp70 and Hsp90 and with its U-box region E2 ubiquitin-conjugating enzymes. By ubiquitinating chaperone-bound polypeptides, CHIP thus links the chaperone machinery to the proteasomal degradation pathway. The molecular mechanism of how CHIP discriminates between folding and destruction of chaperone substrates is not yet understood. Two recently published crystal structures of mouse and zebrafish CHIP truncation constructs differ substantially, showing either an asymmetric assembly or a symmetric assembly with a highly ordered middle domain. To characterize the conformational properties of the intact full-length protein in solution, we performed amide hydrogen exchange mass spectrometry (HX-MS) with human CHIP. In addition, we monitored conformational changes in CHIP upon binding of Hsp70, Hsp90, and their respective C-terminal EEVD peptides, and in complex with the different E2 ubiquitin-conjugating enzymes UbcH5a and Ubc13. Solution HX-MS data suggest a symmetric dimer assembly with highly flexible parts in the middle domain contrasting both the asymmetric and the symmetric crystal structure. CHIP exhibited an extraordinary flexibility with a largely unprotected N-terminal TPR domain. Formation of a complex with intact Hsp70 and Hsp90 or their respective C-terminal octapeptides induced folding of the TPR domain to a defined, highly stabilized structure with protected amide hydrogens. Interaction of CHIP with two different E2 ubiquitin-conjugating enzymes, UbcH5a and Ubc13, had distinct effects on the conformational dynamics of CHIP, suggesting different roles of the CHIP-E2 interaction in the ubiquitination of substrates and interaction with chaperones.

The expansion of weed species is a major problem in agriculture, especially when the number of herbicide‐resistant biotypes is rising continuously. The major ecological questions associated with the evolution of herbicide resistance involve an intricate understanding of the interplay between gene frequency, fitness, inheritance and gene flow. In this study, the RAPD (Random Amplified Polymorphic DNA) technique, which facilitates detection of variability at DNA level, was used to examine the spread of Solanum nigrum L. populations. Twenty‐five populations, from Poland, France and the UK, were analysed. Six populations from Poland and one from France showed target site‐based triazine resistance. The genetic relationship between individuals was studied using the RAPD technique. It was found that some resistant populations from the Gabin and Grojec areas show very high affinity levels compared with individuals from France. Three groups of populations in which resistance had developed independently were distinguished. The results of the present investigation suggest that migratory birds, such as Turdus pilaris L. and Sturnus vulgaris L., play an important in spreading S. nigrum seeds.

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Molecular chaperones are key components in the maintenance of cellular homeostasis and survival, not only during stress but also under optimal growth conditions. Among the ATP-dependent chaperones, heat shock proteins (Hsp90) proteins play a special role. While Hsp90s can interact with unfolded and misfolded proteins, their main (and in eukaryotic cells essential) function appears to involve interactions with a limited number of protein clients at late steps of maturation or in "alter-native" conformations for regulating their stability and activity. Because Hsp90 clients are hubs of diverse signaling networks and participate in nearly every cellular function, Hsp90s interconnect many regulatory circuits and link them to environmental impacts. The availability and activity of Hsp90 may thus influence complex physiological and pathophysiological processes, such as differentiation, development, aging, cancer, neurodegeneration, and infectious diseases. Furthermore, through homeostatic effects on differentiation and development, Hsp90s act as capacitors of phenotypic evolution. In this review, we discuss recent insights in the structure and chaperone cycle of Hsp90s, the mechanisms underlying Hsp90 binding to clients, and potential reasons why client proteins specifically require the assistance of Hsp90s. Moreover, the current views on Hsp90-cochaperone interactions and regulation of Hsp90 proteins via posttranslational modifications are summarized. The second half of this article is devoted to the role of Hsp90 proteins in health and disease, aging, and evolution.

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