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Nucleotide exchange factors Fes1 and HspBP1 mimic substrate to release misfolded proteins from Hsp70

Nature Structural & Molecular Biology volume 25, pages 83–89 (2018)Cite this article

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Abstract

Protein quality control depends on the tight regulation of interactions between molecular chaperones and polypeptide substrates. Substrate release from the chaperone Hsp70 is triggered by nucleotide-exchange factors (NEFs) that control folding and degradation fates via poorly understood mechanisms. We found that the armadillo-type NEFs budding yeast Fes1 and its human homolog HspBP1 employ flexible N-terminal release domains (RDs) with substrate-mimicking properties to ensure the efficient release of persistent substrates from Hsp70. The RD contacts the substrate-binding domain of the chaperone, competes with peptide substrate for binding and is essential for proper function in yeast and mammalian cells. Thus, the armadillo domain engages Hsp70 to trigger nucleotide exchange, whereas the RD safeguards the release of substrates. Our findings provide fundamental mechanistic insight into the functional specialization of Hsp70 NEFs and have implications for the understanding of proteostasis-related disorders, including Marinesco–Sjögren syndrome.

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Fig. 1: The N terminus of Fes1 is highly flexible and reaches into the substrate-binding pocket of Hsp70 Ssa1.
Fig. 2: The RD is required for Fes1 function in vivo.
Fig. 3: The function of the RD is transferable to the structurally unrelated NEF BAG.
Fig. 4: The RD contacts SBDβ of Hsp70 and prevents substrate association.
Fig. 5: RD function is conserved in the human Fes1 ortholog HspBP1.
Fig. 6: Model for RD function of armadillo types NEFs.

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Acknowledgements

We thank S. Büttner, M. Ott and P.O. Ljungdahl (Stockholm University) for productive discussions and comments during the study. We also acknowledge support from the Imaging Facility at Stockholm University (IFSU). This work was supported by Swedish Research Council grants 2015-05094 (C.A.) and K2013-66X-20702-06-4 (M.Ö.), the Swedish Cancer Society grant CAN 2016/361 (C.A.), Carl Tryggers Stiftelse för Vetenskaplig Forskning (C.A.), and Deutsche Forschungsgemeinschaft grant MA1278/4-3 (M.P.M.).

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  1. Naveen K. C. Gowda and Jayasankar M. Kaimal contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden

    Naveen K. C. Gowda, Jayasankar M. Kaimal, Chammiran Daniel, Marie Öhman & Claes Andréasson

  2. Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Heidelberg, Germany

    Roman Kityk & Matthias P. Mayer

  3. Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden

    Jobst Liebau

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Contributions

N.K.C.G., J.M.K., R.K., C.D., M.Ö., M.P.M. and C.A. designed the experiments and conceptualized the data. N.K.C.G., J.M.K., R.K., C.D. and J.L. carried out experiments. C.A. wrote the manuscript together with N.K.C.G., J.M.K, M.P.M. and M.Ö. The work was supervised by C.A, M.P.M. and M.Ö.

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Correspondence to Claes Andréasson.

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Supplementary Figure 1 Removal of RD does not impact on structural stability of Fes1

a. CD measurements of purified Fes1 and ΔRD in phosphate buffer during heating of the samples from 20 °C to 80 °C followed by chilling to 20 °C. b. Data from A showing α-helical content before and after heating the proteins to 80°C. c. Melting temperature (Tm) of Fes1 and ΔRD determined from b at 222 nm. Data from three independent experiments are presented with their mean values and error bars representing SD.

Supplementary Figure 2 Fes1 interacts with SBDβ of Ssa1 via its RD domain

a. Coomassie stained gel from SDS-page analysis of 800 ng of the purified proteins GST, Fes1, ΔRD and RD-GST. A small amount of GST with somewhat slower gel migration was copurifed with GST and RD-GST (*). b. Representative Western blot of the experiment in Fig 4a. c. Experiment as in Fig 4a but side-by-side elution of parallel samples was performed with ATP and glutathione (GSH). Input and non-bound (NB) proteins are shown. A minor fraction of unprocessed 6×His-SUMO-Ssa1 was present from the purification of Ssa1 (*). After normalization to the amount of GST and RD-GST eluted with GSH, the relative Western blot signal from Ssa1 eluted with ATP or GSH from RD-GST was calculated from two independent experiments (below gel). d. Cell-free lysates (2 mg/ml) prepared from WT cells expressing Ssa1E423BPa-HA or carrying VC were supplemented with 1 mM ATP and 10 μM GST or GST-APPY. UV-A dependent crosslinking products were analyzed after 60 min irradiation (UV +) using α-HA and α-GST antibodies. A specific crosslink between Ssa1E423BPa-HA and GST-APPY is labeled with an arrow. e. Merged two-color exposures of the first four lanes from experiment in Fig. 4d. Ssa1E423BPa-HA was detected with α-HA antibodies (green signal) and Fes1 with α-Fes1 serum (white signal). Arrows indicate the position of migration of the crosslinked species detected exclusively by α-Fes1 serum (Fes1) and α-HA antibodies (Ssa1E423BPa-HA), respectively. f. Fes1 crosslinks specifically to Ssa1E423BPa-HA. Experiment as in Fig. 4d but the crosslinking was performed both in cells that carried the Ssa1E423BPa-HA expressing plasmid and vector control (VC). The uncropped blot and gel images are shown in Supplementary Data Set 1. In each case, representative data from three independent experiments are shown.

Supplementary Figure 3 The RD functions concomitant nucleotide exchange by the armadillo domain

a. Stimulation of nucleotide release by Fes1 and ∆RD. Preformed complexes of Ssa1 (0.5 µM) and MABA-ADP (0.5 µM) were rapidly mixed with 1 mM ATP in the absence or presence of Fes1 or ∆RD (2 µM). Exemplary traces of three independent experiments are shown. b and c. Dissociation rate constants of complexes of Ssa1 and a fluorescently labeled peptide in the presence of increasing concentrations of Fes1 in the absence of added nucleotides (b) and the presence of ATP (c). Ssa1 (1 µM) with bound ADP was pre-incubated for 30 min with the dansylated NRLLLTG peptide (1 µM) and then mixed with Fes1 at the indicated concentration (concentration after 1:1 mixing) in the absence of added nucleotide or in the presence of 1 mM ATP. Differences in (b) are not statistically significant (Sidak’s multiple comparison test). Data from three independent experiments are presented with their mean values. d. ATP binding to Ssa1 is accelerated by Fes1. Observed association rates of MABA-ATP to Ssa1 in the absence and presence of Fes1. MABA-ATP (4 and 8 µM; indicated are final concentration) was rapidly mixed 1:1 with nucleotide-free Ssa1 (1 µM) in the absence and presence of Fes1 (1 µM). ATP associates with Ssa1 with biexponential kinetics corresponding to the initial encounter complex (concentration dependent rate) and a subsequent conformational change (concentration independent) that leads to opening of the substrate binding domain and substrate release. Data from three independent experiments are presented with their mean values. ANOVA with Sidak's multiple comparisons test was performed; **** p<0.0001.

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Gowda, N.K.C., Kaimal, J.M., Kityk, R. et al. Nucleotide exchange factors Fes1 and HspBP1 mimic substrate to release misfolded proteins from Hsp70. Nat Struct Mol Biol 25, 83–89 (2018). https://doi.org/10.1038/s41594-017-0008-2

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