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Multidomain structure and correlated dynamics determined by self-consistent FRET networks

Nature Methods volume 14, pages 174–180 (2017)Cite this article

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Abstract

We present an approach that enables us to simultaneously access structure and dynamics of a multidomain protein in solution. Dynamic domain arrangements are experimentally determined by combining self-consistent networks of distance distributions with known domain structures. Local structural dynamics are correlated with the global arrangements by analyzing networks of time-resolved single-molecule fluorescence parameters. The strength of this hybrid approach is shown by an application to the flexible multidomain protein Hsp90. The average solution structure of Hsp90's closed state resembles the known X-ray crystal structure with Angstrom precision. The open state is represented by an ensemble of conformations with interdomain fluctuations of up to 25 Å. The data reveal a state-specific suppression of the submillisecond fluctuations by dynamic protein–protein interaction. Finally, the method enables localization and functional characterization of dynamic elements and domain interfaces.

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Figure 1: Global domain arrangement of Hsp90's open state.
Figure 2: Analyzing networks of distances and fluorescence parameters.
Figure 3: Network of time-correlated distance distributions.
Figure 4: Time-correlated structural ensembles and dynamics of Hsp90.
Figure 5: Structural insights from unrestrained MD simulations.

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Acknowledgements

We thank J. Michaelis, C. Seidel, S. Schmid, M. Götz, M. Jahn, J. Buchner, S. Andrade and O. Einsle for helpful discussion. We thank J. Michaelis and C. Seidel for software support. We thank D. Elnatan, D. Agard and M. Götz for help with Δ131Δ expression and purification. This work is funded by the German Research Foundation SFB863 to T.H. (A4) and M.Z. (A10) and the European Research Council through grant agreement 681891 (T.H.).

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Authors and Affiliations

  1. Institute of Physical Chemistry, University of Freiburg, Freiburg, Germany

    Björn Hellenkamp, Philipp Wortmann & Thorsten Hugel

  2. Physics Department, Technische Universität München, Munich, Germany

    Florian Kandzia & Martin Zacharias

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  1. Björn Hellenkamp
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Contributions

B.H., P.W. and T.H. designed the research; B.H. and P.W. performed the research; B.H. developed analytic tools and the software; F.K. and M.Z. performed MD simulations; B.H. analyzed the data in consultation with all authors; B.H. and T.H. wrote the manuscript in consultation with all authors.

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Correspondence to Thorsten Hugel.

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Integrated supplementary information

Supplementary Figure 1 Flow chart for dynamic domain arrangement.

To generate dynamic ensembles of protein structures the given steps should be followed. For details see main text and Online Methods.

Supplementary Figure 2 Comparison of Hsp90’s global states.

(a) Superposition of the average open structure of yeast Hsp90 (red) with the closed x-ray structure of yeast Hsp90 (blue) at the C-domains. (b) The -2σ-structure of yeast Hsp90 (reddish) and the x-ray structure of ADP-bound Grp94 (pdb: 2o1v, yellow) match with an RMSD of 4.5 Å. The conformations between M- and N-domain match with an RMSD of 3 Å. (c) Superposition of the 1σ-structure of yeast Hsp90 (reddish) and the open conformation of the bacterial homologue HtpG (pdb: 2ioq, orange).

Supplementary Figure 3 Domain interface dynamics from unrestrained MD simulations.

(a) Root mean square deviations (RMSD) from start structure vs. simulation time for the full-length structure (black) and the structure without N-domains (gray) for the closed (left) and open state (right). (b) Normalized buried surface area per atom (surface illustration) and number of contributions per residue to the buried surface area (cartoon illustration) for the CM-interface. The values are color-coded for the M-domain (top) and C-domain (bottom) for the closed (blue) and open state (red) (see also Online Methods). (c) Distances between charged residues that are located at the CM-interface are plotted vs. simulation time to test for salt bridges. Several charged residues capture relative distances of 2.9 Å, which is characteristic for ionic or hydrogen bonds1,2. These bonds are stable for long time periods of several 10 ns and seem to be protected from water. Some salt bridges interchange in an anti-correlated pattern. A second plateau at 4.5-5 Å could be explained by hydrogen bonds interacting with an enclosed water molecule. The interacting charged residues are well conserved. All amino acids that are well conserved among Hsp90 eukaryotes (conservation score > 80%) are color-coded in red (cartoon plot on the right). A correlation between conservation degree and interface contacts can be seen by comparing this plot with Figure 5 in the main text.

1 Xu, D., Tsai, C.-J. & Nussinov, R. Hydrogen bonds and salt bridges across protein-protein interfaces. Protein engineering 10, 999-1012 (1997). 2 Musafia, B., Buchner, V. & Arad, D. Complex salt bridges in proteins: statistical analysis of structure and function. J Mol Biol 254, 761-770 (1995).

Supplementary Figure 4 Time-correlated distance distributions between different Hsp90 domains.

Time-correlated distance distributions – i.e. distance fluctuations versus observation time – are quantified for the closed state (blue) and the open state (red). Shown are the results for two different FRET pairs between the M-domains (a-b), for one FRET pair between M- and N-domain within a monomer (c), and for one FRET pair between C- and M-domain (d).

Supplementary Figure 5 The effect of cysteine point mutations, labeling and heating on the ATPase activity of Hsp90.

Protein activity was checked by a regenerating ATPase assay1. ATPase rates were measured at 30°C in 40 mM Hepes, 150 mM KCl, 10 mM MgCl2, pH 7.5. ATPase background could be detected by specific inhibition of Hsp90 with radicicol (Sigma) and was subtracted.

The ATPase activity of various constructs is compared to the wild type (WT) activity. The point mutation 51C does show a similar activity, but loses more than 70% of activity upon dye attachment (Atto550-Mal). In addition, this construct (51C, Atto550-Mal) did not close completely after addition of AMP-PNP (as monitored by smFRET). Therefore, this data was excluded from the FRET-network.

For the 517C point mutation the situation is very different. Here the ATPase activity does not significantly change upon the addition of dyes (Atto550-Mal or Atto647N-Mal). Even the exchange a 1:1 mix of Hsp90 517C-Atto550 and -Atto647N, which was heated 20 minutes at 47°C, did not significantly affect the ATPase activity. In addition, these constructs did completely close after the addition of AMP-PNP. This data was included for the FRET-network. Some constructs showed an increase in ATPase, but complete closing, they were also included for the FRET-network.

ATPase activity was measured with a regenerating assay with 1 μM protein under saturating ATP concentration (2 mM) at 37°C. The error bars represent the standard deviations from three independent measurements.

1 Hessling, M., Richter, K. & Buchner, J. Dissection of the ATP-induced conformational cycle of the molecular chaperone Hsp90. Nat Struct Mol Biol 16, 287-293 (2009).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Notes 1–6 (PDF 1941 kb)

Supplementary Table 1

Mean distance 〈R〉 and standard deviation σR of Gaussian distributed distances extracted from the measured efficiency histograms with the distance distribution analysis are listed for each FRET pair for the closed and open state of Hsp90, together with the deviation from the model (RM-〈R〉), the combined anisotropy rC and the selection criteria for every rejected distance. (XLSX 42 kb)

Supplementary Protocol (PDF 981 kb)

Supplementary Software

Multidomain Arrangement Software and MD Restraining Support. (ZIP 2568 kb)

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Hellenkamp, B., Wortmann, P., Kandzia, F. et al. Multidomain structure and correlated dynamics determined by self-consistent FRET networks. Nat Methods 14, 174–180 (2017). https://doi.org/10.1038/nmeth.4081

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