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. 2017 Feb;14(2):174-180.
doi: 10.1038/nmeth.4081. Epub 2016 Dec 5.

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

Björn Hellenkamp  1 , Philipp Wortmann  1 , Florian Kandzia  2 , Martin Zacharias  2 , Thorsten Hugel  1
Affiliations

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

Björn Hellenkamp et al. Nat Methods. 2017 Feb.

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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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Global domain arrangement of Hsp90’s open state.
(a) Corrected FRET efficiencies are calculated from photon bursts of single molecules using a confocal setup with alternating excitation and color- and polarization-sensitive detection. Photophysical correction parameters are verified within a global network. Each measurement consists of at least 1000 single molecules and is repeated at least three times. See main text and Supplementary Notes 2, 3 for determination and verification of precise distances from efficiencies. (b) The self-consistent distance network of Hsp90’s open state is visualized for the final model structure (left). It is used to optimally arrange domain structures (middle, blue/violet) considering the accessible volumes of the dyes (green). The initial global domain arrangement is refined considering changes of accessible volumes and local structural elements to obtain an optimal arrangement (right). (c) The arrangement procedure is repeated for different compilations of domains and subdomains. The full-length x-ray structure of yeast Hsp90 in the closed state (pdb: 2cg9) and x-ray structures of individual domains and homologues served as basic components. The final model structure fulfills the distances with an RMSD of 3.7 Å. The procedure is verified with subsets of the data (see Fig. 2b).
Figure 2
Figure 2. Analyzing networks of distances and fluorescence parameters.
(a) If the combined anisotropy rC is low enough, κ²=2/3 is normally justified (green distance, top). The threshold for the combined anisotropy rc=0.22 is found by determining the RMSD between experimental distances and the x-ray crystal structure of the closed state (bottom). (b) Subsets of distances were systematically generated from 50 experimental distances to arrange Hsp90’s middle domains. The RMSD from the x-ray structure, its variability and the number of possible solutions strongly decrease with an increasing number of redundant distances. The number of possible solutions was reduced to one if using 26 distances or more (dashed black line). We use a χ2 criterion (Online Methods) to rate the solutions. The error bars represent the standard deviations, each calculated from 5 random subsets. (c) Dyes may change their residual anisotropy due to a changing accessible volume (top, green) or due to local conformational changes (top, black). Correlating accessible volumes and residual anisotropies for various dye positions within the closed structure (blue squares) and the arranged open structure (red circles) allows us to find state dependent local conformational changes. The green lines denote positions at domain interfaces. Long black lines indicate a state-dependent local conformational change not located at interfaces. The dashed gray line denotes the average anti-correlation between accessible volume and residual anisotropy for all dye positions.
Figure 3
Figure 3. Network of time-correlated distance distributions.
(a) Efficiency histograms recorded under different conditions are shown for a photon binning time of 1 ms for the FRET pair highlighted in the top right. The fits indicate the efficiency distribution of Hsp90’s closed state (blue) or Hsp90’s open state (red) assuming Gaussian distributed distances. (b) Time-correlated distance distributions – i.e. fluctuations versus observation time – are quantified for the closed state (blue), the open state (red), the open state in the presence of 2mM ATP (gray) and the open state in the presence of 2mM ATP and 20 µM of the model client Δ131Δ (purple). See Supplementary Fig. 4 for additional data and Supplementary Note 5 for the calculation of distance fluctuations. (c) The distance fluctuations for an observation time of 1 ms are represented as average value and standard error over all distances of the indicated domain pair. The individual distance distributions are listed in Supplementary Table 1 for every FRET pair.
Figure 4
Figure 4. Time-correlated structural ensembles and dynamics of Hsp90.
(a) Dynamic ensembles of Hsp90 structures for the closed state (blue) and open state (red) for an observation time of 1 ms. The mean structures are shown in solid color and the less probable structures in transparent colors. (b) Global conformational dynamics and state-dependent inter-domain fluctuations of Hsp90 are illustrated for an observation time of 0.1 ms to emphasize the effect of the interaction with the client protein. The rigid closed state (left) is mainly stabilized by NN-contacts. The highly dynamic open state (middle) is characterized by globally stable but locally flexible domain interfaces. The CM-interface enables large but defined inter-monomer fluctuations on the millisecond timescale. The MN-interface enables a transient rotational motion of the N-terminal domain on the sub-millisecond timescale (see Supplementary Note 6). The CC-interface remains globally unchanged during the described conformational changes. The interconversion between the global states occurs on the seconds timescale. The model client Δ131Δ suppresses inter-monomer fluctuations on timescales shorter than 0.1 ms (right). The transition rates are indicated by the arrow thickness in a logarithmic scale.
Figure 5
Figure 5. Structural insights from unrestrained MD simulations.
(a) Root mean square fluctuations from MD simulations for Hsp90 indicating several flexible elements in the open state. (b, c) The number of contributions per residue to the buried surface area (i.e. the contact probability) was calculated within the closed state (blue frames) and the open state (red frames) for the CM-interface (b) and the MN-interface (c). Interfacial salt-bridges are highlighted as sticks.
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