Promiscuous binding by Hsp70 results in conformational heterogeneity and fuzzy chaperone-substrate ensembles
- The University of Toronto, Canada
- University of Toronto, Canada
- Weizmann Institute of Science, Israel
- Hospital for Sick Children, Program in Molecular Structure and Function, Canada
Figures
Figure 1
Architecture of DnaK and hTRF1 molecules.
(A) Cartoon representation of DnaK/ADP (PDB ID: 2KHO [Bertelsen et al., 2009]) highlighting the nucleotide binding (green, NBD) and substrate binding (SBD) domains, the latter of which is further …
Figure 2 with 2 supplements
DnaK binds substrate via a multiplicity of interactions.
The Ile region of 1H-13C HMQC spectra of ILVM-13CH3 hTRF1 in the absence (A) and in the presence (B) of two-fold excess U-2H DnaK/ADP. The single Ile 29 peak in the unbound state (A) disperses into …
Figure 2—figure supplement 1
Verifying binding with a PFG diffusion experiment.
Relative diffusion coefficients of DnaK-bound (hTRF1 Ile residues A,B,D, Figure 2B) and native hTRF1 measured using a sample of 0.3 mM ILVM-13CH3 labeled hTRF1 and 0.5 mM U-2H DnaK/ADP and a 2D 1H-13…
Figure 2—figure supplement 2
Multiple conformers are observed upon DnaK binding to hTRF1 peptides.
(A) Superposition of 1H-13C HMQC spectra of IM-13CH3 DnaK/ADP bound to hTRF1 (black) and hTRF123-38 peptide containing the high affinity DnaK binding region (red, left) or hTRF134-49 peptide that …
Figure 3
Linking multiple resonances and hence conformers of DnaK and hTRF1.
(A) Zoomed region of the crystal structure of a DnaK-NRLLLTG peptide complex (PDB ID: 1DKZ [Zhu et al., 1996]) highlighting interactions between sidechains of the peptide (magenta) bound to the …
Figure 4 with 3 supplements
Assigning residues of hTRF1 at the central position of the DnaK binding cleft.
Selected regions of 1H-13C HMQC spectra of wild-type ILVM-13CH3 hTRF1 bound to U-2H DnaK/ADP (black), overlaid with ILVM-13CH3 labeled L31I (red), L30I (orange), V41I (cyan) and V18I (green) hTRF1 …
Figure 4—figure supplement 1
Assigning states 'a' and 'b' to L31 and L30 respectively.
Superposition of selected regions of 1H-13C HMQC spectra of ILVM-13CH3 hTRF1 bound to U-2H DnaK/ADP (red) and (A) U-13C L30 hTRF123-38 or (B) U-13C L31 hTRF123-38 bound to U-2H DnaK/ADP (green). …
Figure 4—figure supplement 2
Assigning states 'c' and 'd' to Val residues of hTRF1 present at the 0 position of the DnaK binding cleft.
Overlay of selected regions from 1H-13C HMQC spectra of LV-13CH3 hTRF1 (blue) and V-13CH3 hTRF1 (red) bound to DnaK/ADP showing that peaks d and c derive from Val residues.
Figure 4—figure supplement 3
Additional unassigned DnaK-hTRF1 conformations.
Overlay of HMQC spectra of unlabeled hTRF134-49 bound to IM-13CH3 DnaK/ADP (blue) and Iγ-proR-LV-13CH3 labeled hTRF1 bound to IM-13CH3 labeled DnaK/ADP (red). Unassigned Ile 401 and Ile 438 peaks of …
Figure 5 with 2 supplements
Quantifying the stoichiometry of hTRF1 binding to DnaK.
Peak intensities from an NMR titration focusing on the four hTRF1 residues Leu 30, Leu 31, Val 18 and Val 41 that occupy the central position of the DnaK binding cavity in different DnaK-bound …
Figure 5—figure supplement 1
Simulating multiple binding equilibria for quantifying stoichiometry.
Simulated profiles reporting the concentrations of different components formed during the course of a titration as a function of the ratio of [KT]/ [NT]. For the specific case analyzed here, [KT] = …
Figure 5—figure supplement 2
Quantifying the stoichiometry of hTRF1 binding to DnaK.
Experimental peak intensity profiles measured as a function of the ratio of [DnaK]T/[hTRF1]T, where [DnaK]T and [hTRF1]T are the concentrations of all DnaK and hTRF1 species in solution, …
Figure 6
Both human Hsc70 and E.coli DnaK interact with hTRF1 at multiple sites.
Overlay of a 1H-13C HMQC spectrum of 200 μM ILVM-13CH3 hTRF1 containing a two-fold excess of U-2H Hsc70/ADP (red) with the spectrum of (A) free ILVM-13CH3 hTRF1 (black) and (B) ILVM-13CH3 hTRF1 …
Figure 7 with 1 supplement
Model of a substrate with three Hsp70 binding sites (indicated in different colors) forming three distinct Hsp70 bound sub-ensembles.
By increasing the number of interaction sites a larger region of conformational space can be explored from which folding of the substrate occurs upon release from the chaperone. Proteins stuck in …
Figure 7—figure supplement 1
Folding of hTRF1 without and with DnaK/DnaJ/GrpE (K/J/E).
Simulated curves showing the time-dependent formation of native hTRF1 starting from the unfolded state in the absence (pink) and presence (blue) of the E.coli Hsp70 chaperone system (K/J/E). …
Tables
Table 1
Percentages of Trans and Gauche- Ile 401 and Ile 438 Conformations*.
| a (Leu 31) | b (Leu 30) | c (Val 41) | d (Val 18) | |||||
|---|---|---|---|---|---|---|---|---|
| Ile 401 | Ile 438 | Ile 401 | Ile 438 | Ile 401 | Ile 438 | Ile 401 | Ile 438 | |
| Trans | 0.86 | 0.28 | 0.99 | 0.54 | 0.66 | 0.00 | 0.58 | 0.00 |
| Gauche- | 0.14 | 0.72 | 0.01 | 0.46 | 0.34 | 1.00 | 0.42 | 1.00 |
-
*Fractional Trans and Gauche- conformations calculated from Ile 13Cδ1 chemical shifts according to Hansen et al. (2010) for conformers 'a'-'d'. The identities of hTRF1 residues at the 0 position are noted.
