The dynamic dimer structure of the chaperone Trigger Factor

Abstract

The chaperone Trigger Factor (TF) from Escherichia coli forms a dimer at cellular concentrations. While the monomer structure of TF is well known, the spatial arrangement of this dimeric chaperone storage form has remained unclear. Here, we determine its structure by a combination of high-resolution NMR spectroscopy and biophysical methods. TF forms a symmetric head-to-tail dimer, where the ribosome binding domain is in contact with the substrate binding domain, while the peptidyl-prolyl isomerase domain contributes only slightly to the dimer affinity. The dimer structure is highly dynamic, with the two ribosome binding domains populating a conformational ensemble in the center. These dynamics result from intermolecular in trans interactions of the TF client-binding site with the ribosome binding domain, which is conformationally frustrated in the absence of the ribosome. The avidity in the dimer structure explains how the dimeric state of TF can be monomerized also by weakly interacting clients.

Fig. 1

Domain organization of full-length TF and secondary structure elements in solution. a On the ribbon representation of a published TF crystal structure (PDB 1W26), the three domains ribosome-binding domain (RBD), substrate-binding domain (SBD), and peptidyl-prolyl-cis/trans isomerase domain (PPD) are colored in red, blue, and yellow, respectively. b Domain constructs of E. coli TF used in this work. Six constructs of TF domains are shown with amino acid numbering corresponding to full-length TF. The names define a color code used throughout this work. c Secondary ¹³C chemical shifts plotted against the amino acid residue number of TF, as determined from triple-resonance experiments in the domain constructs SBD–PPD (green), RBD (red), and SBD (blue). A 1–2–1 weighting function for residues (i−1)–i–(i + 1) has been applied to the raw data to reduce noise and highlight regular secondary structure elements. Secondary structure elements were calculated for the crystal structure (PDB 1W26, gray) with DSSP and for the NMR data with CSI 3.0 and are indicated on top. The red arrows and boxes highlight structural elements detected only in solution

Fig. 2

Localization of pairwise interaction sites on individual TF domains. a NMR titration of unlabeled SBD–PPD to 100 μM [U-¹⁵N] RBD in sample buffer (20 mM K-phosphate pH 6.5, 100 mM KCl, 0.5 mM EDTA) at 25 °C and 700 MHz. b NMR titration of unlabeled RBD to 250 μM [U-²H,¹⁵N] SBD–PPD in sample buffer at 25 °C and 700 MHz. c Chemical shift perturbation of the amide moieties observed in the titrations: [U-¹⁵N] RBD + SBD–PPD (top left), [U-¹⁵N] RBD + SBD (bottom left), [U-²H,¹⁵N] SBD–PPD + RBD (top right), and [U-²H,¹⁵N] SBD + RBD (bottom right). Light-shaded bars represent peaks undergoing line-broadening. Dashed lines are plotted at defined thresholds (mean value of the chemical shift perturbations plus one time and plus two times the standard deviation corrected to zero). d Significant chemical shift perturbations plotted in TF crystal structure (PDB 1W26). The affected residues are plotted with color gradient from light to dark for peaks with chemical shift changes above the threshold and that broaden beyond detection, respectively

Fig. 3

Determination of the lifetime of the TF dimer. a Experimental scheme. At the onset of the experiment (t = 0), separately produced samples of [U-¹⁵N,²H]-labeled (green) and randomly spin-labeled (brown) TF are mixed in equal ratio in sample buffer (left). The NMR signal intensity is then monitored in real time t during the equilibration to the end point (right). NMR signals of protomers bound to a spin-labeled protomer are reduced in intensity by the intermolecular PRE, symbolized by light green color. b Intensity of ¹⁵N-filtered NMR signals, I _{Δ, rel}, following the experimental scheme in a. The lifetime of the TF dimer is obtained by non-linear least-square fits (lines) to the data (dots). See Supplementary Note 1 for mathematical details. The experiment was performed at five temperatures in the range 15–35 °C, as indicated

Fig. 4

Domain contacts in the full-length TF dimer. Result of PRE experiments with a paramagnetic spin label attached to one of the positions S30, V49, S61, S72, A223, and E326 in full-length TF measured in sample buffer (20 mM K-phosphate pH 6.5, 100 mM KCl, 0.5 mM EDTA) at 25 °C and 700 MHz. The peak volume ratio between oxidized and reduced samples from 2D [¹⁵N,¹H]-TROSY is plotted against the residue number. For visualization purposes, a value of 0.15 is shown for the peaks that were broadened beyond detection in the paramagnetic sample. Data are shown only for non-overlapping resonances. The black line outlines the PRE effect observed for each mutant. The orange-shaded regions correspond to intermolecular PRE and the green shaded to intramolecular effects, as expected from the monomeric crystal structure (PDB 1W26). The colored bars on top show the sequence domain organization as in Fig. 1

Fig. 5

Structure of the TF dimer in solution. a Flowchart for structural model determination of TF dimer. Structural models are indicated in orange boxes. Experimental data contributions are indicated in green boxes. Software packages are identified in purple boxes. b Lowest energy structures from the two clusters obtained with HADDOCK docking based on chemical shift perturbation data. Both monomers are represented in surface view, and one of them is semi-transparent to show the backbone. c XPLOR-NIH results represented as (I) ensemble of the 10 lowest energy structures with the flexible residue segments in gray, (II) lowest energy structures with both monomers represented in surface view, one of them depicted semi-transparent to show the backbone, and (III) lowest energy structures in surface representation with experimental PRE distances represented, intermolecular (A–B, top) and intramolecular (A–A, bottom)

Fig. 6

Equilibrium and frustration of the TF dimer in solution. a TF dimer is highly dynamic and is in equilibrium in solution with its monomeric form and with its ribosome-bound form. The ribosome is represented in gray. b Frustration analysis of TF. Local frustration for TF crystal structure was calculated with the online tool Protein Frustratometer 2 (PDB 1W26) and is plotted on TF crystal structure (left) and on the dimer structural models (right). Minimally frustrated interactions are depicted as green lines, highly frustrated interactions as red lines