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Structural insights into a unique Hsp70-Hsp40 interaction in the eukaryotic ribosome-associated complex

Nature Structural & Molecular Biology volume 24, pages 144–151 (2017)Cite this article

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Abstract

Cotranslational chaperones assist de novo folding of nascent polypeptides, prevent them from aggregating and modulate translation. The ribosome-associated complex (RAC) is unique in that the Hsp40 protein Zuo1 and the atypical Hsp70 chaperone Ssz1 form a stable heterodimer, which acts as a cochaperone for the Hsp70 chaperone Ssb. Here we present the structure of the Chaetomium thermophilum RAC core comprising Ssz1 and the Zuo1 N terminus. We show how the conserved allostery of Hsp70 proteins is abolished and this Hsp70–Hsp40 pair is molded into a functional unit. Zuo1 stabilizes Ssz1 in trans through interactions that in canonical Hsp70s occur in cis. Ssz1 is catalytically inert and cannot adopt the closed conformation, but the substrate binding domain β is completed by Zuo1. Our study offers insights into the coupling of a special Hsp70–Hsp40 pair, which evolved to link protein folding and translation.

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Figure 1: Characterization of the Ssz1-Zuo1 complex.
Figure 2: Structure of the Ssz1 linker region.
Figure 3: Zuo1N completes Ssz1 SBDβ.
Figure 4: RAC binding at the ribosomal tunnel exit.

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Acknowledgements

We thank J. Kopp, C. Siegmann and G. Müller from the BZH-Cluster of Excellence:CellNetworks crystallization platform for protein crystallization, and acknowledge access to the beamlines at the European Synchrotron Radiation Facility (ESRF) in Grenoble and the support of the beamline scientists. We acknowledge A. Hendricks for expert technical assistance and K. Wild for discussions. We would like to thank E. Hurt, E. Thomson and M. Thoms (Heidelberg University Biochemistry Center) for supplying the C. thermophilum cDNA and S. cerevisiae strains, and G. Stier (Heidelberg University Biochemistry Center) for the modified pET24d and pET16b expression vectors and the SUMO protease. We thank R. Kityk and M. Mayer (Heidelberg University Molecular Biology Center) for providing purified DnaK, for advice for fluorescence anisotropy experiments and for discussions. This work was supported by the Deutsche Forschungs-gemeinschaft (DFG) (through FOR967, GRK1188 and the Leibniz programme) and by HBIGS fellowships (to G.V.G. and F.A.W.). I.S. is an investigator of the Cluster of Excellence:CellNetworks.

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  1. Andrea Gumiero

    Present address: Present address: Istituto Poligrafico e Zecca dello Stato S.p.A., Roma, Italy.,

  2. Felix Alexander Weyer and Andrea Gumiero: These authors contributed equally to this work.

Authors and Affiliations

  1. Heidelberg University Biochemistry Center (BZH), Heidelberg, Germany

    Felix Alexander Weyer, Andrea Gumiero, Genís Valentín Gesé, Karine Lapouge & Irmgard Sinning

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Contributions

F.A.W., A.G., G.V.G., K.L. and I.S. designed the experiments, analyzed the data and wrote the manuscript. F.A.W., A.G., G.V.G. and K.L. performed experiments. All authors read and commented on the manuscript.

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Correspondence to Irmgard Sinning.

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Integrated supplementary information

Supplementary Figure 1 Pulldown analysis of Ssz1 with Zuo1 or Zuo1N and purification of the Ssz1–Zuo1N complex for crystallization.

(a) MBP-pulldown analysis of Ssz1 with MBP-Zuo1 or MBP-Zuo1N and of the coexpressed Ssz1-MBP-Zuo1N complex. (b) Uncropped SDS-PAGE of Fig. 1c showing the SEC fractions of the Ssz1-Zuo1N complex.

Supplementary Figure 2 Packing of the two molecules in the asymmetric unit.

Within the two molecules (Ssz1: gray and purple, Zuo1N: orange; cartoon representation) in the asymmetric unit, several regions are disordered. The disordered regions are depicted as dotted lines and the residues numbered. The N-, C-terminus and domains are indicated. ATP is represented as sticks and Mg2+ is shown as a pink sphere.

Supplementary Figure 3 Electron density map of typical regions in the Ssz1–Zuo1N complex and B factor plot for the Ssz1–Zuo1N structure.

(a) Stereoview of the Zuo1N EPVG motif (orange, yellow sticks). The 2Fo-Fc electron density map is contoured at 1 σ. (b) Stereoview of the nucleotide bound to Ssz1. ATP (sticks), Mg2+ (green sphere) and water molecules (red spheres). The 2Fo-Fc electron density map is contoured at 2 σ. (c) Ribbon representation of the Ssz1-Zuo1N complex with Ssz1 (left) and Zuo1N (right) colored according to B-factors, scaled from 50 (blue) to 250 (red) Å2. ATP is represented as sticks. NBD and SBDβ are indicated.

Supplementary Figure 4 Surface analyses of Hsp70 proteins (with bound ATP).

Comparison of electrostatic surface potentials of (a) Ssz1, (b) Ssb1 (Gumiero, A. et al. Interaction of the cotranslational Hsp70 Ssb with ribosomal proteins and rRNA depends on its lid domain. Nat. Commun. 7,13563 (2016)) and (c) DnaK (Kityk, R., Kopp, J., Sinning, I. & Mayer, M.P. Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones. Mol. Cell 48, 863-74 (2012)). The structures have been superimposed on the NBD and are shown side by side. The positively charged patch observed in Ssz1 is absent in canonical Hsp70s. (d) Close-up view of the positively charged patch in Ssz1. Surface charge spans from -4 kT/e (deep red) to +4kT/e (deep blue). (e) and (f) Conservation surface mapping of the Ssz1 patch shown in (d) as reported by ConSurf (Landau, M. et al. ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures. Nucleic Acids Res. 33, W299-302 (2005)), from variable (cyan) to conserved (deep red). Figures were produced using PDB2PQR (Dolinsky, T.J. et al. PDB2PQR: expanding and upgrading automated preparation of biomolecular structures for molecular simulations. Nucleic Acids Res. 35, W522-5 (2007); Dolinsky, T.J., Nielsen, J.E., McCammon, J.A. & Baker, N.A. PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations. Nucleic Acids Res. 32, W665-7 (2004)) and APBS (Baker, N.A., Sept, D., Joseph, S., Holst, M.J. & McCammon, J.A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci. U S A 98, 10037-41 (2001)) in PyMOL (Schrödinger, L.L.C. The PyMOL Molecular Graphics System, Version 1.8. (2015)).

Supplementary Figure 5 Comparison of Ssz1–Zuo1N and DnaK to show the different domain arrangement.

Ribbon representation of (a) the Ssz1 inter-domain linker (dark blue), Ssz1 SBDb (pale cyan), Zuo1N (orange), and (b)DnaK SBDβ (green, 4B9Q (Kityk, R., Kopp, J., Sinning, I. & Mayer, M.P. Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones. Mol. Cell 48, 863-74 (2012)). Both structures have been superimposed on the NBD. NBD and SBDβ are indicated and the NBDs are shown in surface representation (gray). SBDb binds to different lobes of the NBD.

Supplementary Figure 6 Superposition of the SBDs of Ssz1 (with bound Zuo1N) and DnaK (ADP, substrate).

Ssz1-Zuo1N and DnaK are shown in ribbon representation, with Zuo1N in orange and Ssz1 SBDβ in pale cyan, and DnaK in green (bound-substrate is in yellow; 1DKY (Zhu, X. et al. Structural analysis of substrate binding by the molecular chaperone DnaK. Science 272, 1606-14 (1996)). The Ssz1 specific insertion in β4–β5 would interfere with the lid closure observed in canonical Hsp70s.

Supplementary Figure 7 Comparison of the substrate binding domains from Ssz1, DnaK (closed and open) and Ssb.

(a) Schematic representation of the twisted β-sandwich, (b) ribbon representation of the structures of Ssz1 SBDβ–Zuo1N (pale cyan, orange), DnaK-ADP SBDβ (Zhu, X. et al. Structural analysis of substrate binding by the molecular chaperone DnaK. Science 272, 1606-14 (1996); green, sand) with bound substrate (yellow), DnaK-ATP SBDβ (Kityk, R., Kopp, J., Sinning, I. & Mayer, M.P. Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones. Mol. Cell 48, 863-74 (2012); green, sand), and Ssb1-ATP SBDβ (Gumiero, A. et al. Interaction of the cotranslational Hsp70 Ssb with ribosomal proteins and rRNA depends on its lid domain. Nat. Commun. 7, 13563 (2016); dark red). (c) Corresponding residues in the putative substrate-binding pocket of Ssz1 SBDβ and the SBDs of DnaK and Ssb1 are shown. The β-strands are labeled, the residue are shown as sticks.

Supplementary Figure 8 Binding assays of RAC to known Hsp70 substrates.

Fluorescence anisotropy binding assay of (a) EcDnaK, Ssz1 / Zuo1N or RAC with the NR peptide (NRLLLTG) (Schlecht, R., Erbse, A.H., Bukau, B. & Mayer, M.P. Mechanics of Hsp70 chaperones enables differential interaction with client proteins. Nat. Struct. Mol. Biol. 18, 345-51 (2011); Gragerov, A. & Gottesman, M.E. Different peptide binding specificities of hsp70 family members. J Mol Biol 241, 133-5 (1994)) and (b) CtSsb, Ssz1 / Zuo1N or RAC with the CoxIV peptide (Pfund, C. et al. The molecular chaperone Ssb from Saccharomyces cerevisiae is a component of the ribosome-nascent chain complex. EMBO J. 17, 3981-3989 (1998)). (c) Binding constants (KD) values for peptide substrates binding with EcDnaK or CtSsb. Each value is the average of three technical replicates ± standard deviation.

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Weyer, F., Gumiero, A., Gesé, G. et al. Structural insights into a unique Hsp70-Hsp40 interaction in the eukaryotic ribosome-associated complex. Nat Struct Mol Biol 24, 144–151 (2017). https://doi.org/10.1038/nsmb.3349

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