Oligomerization of a molecular chaperone modulates its activity
- Hokkaido University, Japan
- PRESTO, Japan Science and Technology Agency, Japan
- St. Jude Children's Research Hospital, United States
Figures
Figure 1 with 1 supplement
Dimerization of TF in solution.
(A) Structure of E. coli TF (PDB code: 1W26). PPD, SBD, and RBD are shown in green, pink, and blue, respectively. The residue boundaries for each one of the three domains are shown in parentheses. …
Figure 1—figure supplement 1
Dynamic monomer-dimer transition of TF.
(A) Observation of PRE for TF K46C attached with MTSL in the presence and absence of its substrate PhoA. Overlay of 1H-13C methyl HMQC spectra of [U-2H; Met-13CH3; Ile-δ1-13CH3; Leu,Val-13CH3/13CH3]-…
Figure 2 with 4 supplements
Structural basis for TF dimerization.
(A) The lowest-energy structure of the TF dimer is shown as space-filling model. TF forms a dimer in a head-to-tail orientation. RBD, SBD, and PPD are shown in blue, magenta, and green, …
Figure 2—figure supplement 1
NMR of dimeric TF.
(A) 1H–13C methyl HMQC spectrum of [U-2H; Ala-13CH3; Met-13CH3; Ile-δ1-13CH3; Leu,Val-13CH3/13CH3; Thr-13CH3]-labelled TF. (B) TF is enriched in hydrophobic amino acids, such as methyl-bearing (Ala, …
Figure 2—figure supplement 2
SEC-MALS of TF mutants.
SEC-MALS profiles of TFG348E/G352E (A), TFM374A/Y378A/V384A/F387A (B), TFM140E (C), TFΔPPD (D), TFΔRBD (E), TFV39E/I76E/I80A (TFmon) (F), and TFF44A/R45A/K46A (G) are shown. Proteins were injected …
Figure 2—figure supplement 3
Comparison with PRE-based docking models.
The structure of TF dimer superimposed with previously reported PRE-based docking models (Morgado et al., 2017); conformer 1 [PDB code: 5OWI] (A), and conformer 2 [PDB code: 5OWJ] (B). PPD, SBD, and …
Figure 2—figure supplement 4
Examples of the inter-molecular NOEs.
Representative strips from 13C-edited NOESY-HMQC and HMQC-NOESY-HMQC NMR experiments. The intermolecular NOE cross peaks are designated by a dashed-line red rectangle.
Figure 3 with 2 supplements
Conformational changes of TF upon dimerization.
(A) The structure of one subunit in the TF dimer (colored as in Figure 1A) and the crystal structure of monomeric TF (colored grey) [Protein Data Bank (PDB) code: 1W26] are superimposed for SBD. The …
Figure 3—figure supplement 1
The ribosome-binding loop in the TF dimer.
Close-up view of the ribosome-binding loop in the TF dimer (A) or TF in complex with the ribosome (PDB ID: 1W2B) (B). The amino acid residues of the ribosome-binding loop involved in the interaction …
Figure 3—figure supplement 2
Characterization of small substrate proteins in complex with TF.
(A) SEC-MALS of E. coli S7 in complex with TF indicating two S7 molecules bind to the monomer of TF. (B) SEC-MALS of E. coli reverse transcriptase (RT)-Ec86 255–320 in complex with TF indicating one …
Figure 4 with 2 supplements
Effect of TF dimerization on chaperone activities.
Aggregation of GAPDH in the absence or presence of TF and TFmon at 0.5 μM (A) and OmpA in the absence or presence of TF and TFmon at 4 μM (B). (C) Refolding of MBP in the absence or presence of TF …
Figure 4—figure supplement 1
Aggregation of GAPDH in the absence or presence of 1 µM TF and TFmon.
The aggregation of GAPDH was monitored by 90° light scattering at 620 nm.
Figure 4—figure supplement 2
Sequence hydrophobicity of the substrate proteins of TF (Roseman algorithm, window = 9).
(A) Hydrophobicity plot of PhoA as a function of its primary sequence. TF-binding sites determined by NMR (Saio et al., 2014) are highlighted in green. (B) Hydrophobicity plot of GAPDH (left panel) …
Figure 5 with 2 supplements
Effect of TF dimerization on binding kinetics.
(A) Association of unfolded PhoA with TF monitored by tryptophan fluorescence. (B) Fitting of the data for the association of PhoA with TF by a single exponential function (gray line) or the sum of …
Figure 5—figure supplement 1
ITC traces of the titration of unfolded proteins to TF.
ITC traces of the titration of PhoA220-310 (A) into TF (left panel), TFmon (middle panel), and TFΔRBD (right panel), or MBP198-265 (B) into TF (left panel) and TFΔRBD (right panel), Monomeric …
Figure 5—figure supplement 2
Substrate-binding sites in the dimer.
(A) Accessible surface area of the substrate-binding sites in monomeric and dimeric TF. (B) The residues identified by NMR to interact with unfolded MBP are colored blue on the surface of TF dimer. …
Figure 6
Chaperone activities of TF in the cell.
The ribosome is shown in light blue. The protein substrate is shown in orange, and TF is represented as spheres with the subunits colored as in Figure 2A. See text for details.
Author response image 1
NOE mapping for the interfaces for PPD-RBD and Arm1-RBD (left panel) and Arm2-RBD.
The intermolecular NOEs are indicated by red lines. TF domains are colored as in Figure 2.
Tables
Table 1
Structural and NMR statistics of TF dimer.
https://doi.org/10.7554/eLife.35731.004| Distance restraints* | |
|---|---|
| NOEs | |
| Short range (intraresidue and sequential) | 870 |
| Medium range (2 < | i-j | < 5) | 467 |
| Long range ( | i-j | > 5 ) | 1230 |
| Intermolecular | 54 |
| Hydrogen bonds | 374 |
| Dihedral angle restraints (cp and | 1358 |
| Violations (mean and SD)* | |
| Distance restraints (A) | 0.005 ± 0.025 |
| Dihedral angle restraints (°) | 0.02 ± |
| 0.23 | |
| Structural coordinates rmsd* | |
| RBD core (1-39, 51-112) | |
| Chain A | |
| Backbone atoms | 1.50 ± 0. A |
| All heavy atoms | 2.04 ± 0.29 A |
| Chain B | |
| Backbone atoms | 1.56 ± 0.41 A |
| All heavy atoms | 2.07 ± 0.38 A |
| PPD core (157-190,195-241) | |
| Chain A | |
| Backbone atoms | 0.87 ± 0.09 A |
| All heavy atoms | 1.38 ± 0.07 A |
| Chain B | |
| Backbone atoms | 0.82 ± 0.14 A |
| All heavy atoms | 1.30 ± 0.11 A |
| SBD core (115-149, 250-321, 329-428) | |
| Chain A | |
| Backbone atoms | 1.40 ± 0.21A |
| All heavy atoms | 2.17 ± 0.23 A |
| Chain B | |
| Backbone atoms | 1.34 ± 0.16A |
| All heavy atoms | 2.14 ± 0.20 A |
| Ramachandran plot* | |
| Most-favored regions | 85.4% |
| Additionally allowed regions | 14.3% |
| Generously allowed regions | 0.3% |
| Disallowed regions | 0.0% |
-
*The statistics apply to the 20 lowest-energy structures.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers |
|---|---|---|---|
| Strain, strain background (E. coli) | BL21 (DE3) | NIPPON GENE CO., LTD. | ECOS Competent E. coli BL21 (DE3) |
| Recombinant DNA reagent | PhoA | Saio et al. (2014),PMID: 24812405 | NCBIGene:945041 |
| Recombinant DNA reagent | OmpA | Tsirigotaki et al., (2018),PMID: 29606594 | NCBIGene:945571 |
| Recombinant DNA reagent | RT | Inouye et al., (1999),PMID: 10531319 | UniProtKB: P23070 |
| Recombinant DNA reagent | MBP | Huang et al. (2016), PMID: 27501151 | NCBIGene: 948538 |
| Recombinant DNA reagent | TF | Takara Bio inc. | pCold-TF (TKR 3365) |
| S7 | S7 | GenScript | Gene synthesis |
| Peptide, recombinant protein | GAPDH | Sigma-Aldrich | G-2267 |
| Software, algorithm | CYANA3.97 | Güntert (2004),PMID: 15318003 | RRID:SCR_014229 |
| Software, algorithm | CNS | Brunger (2007), PMID: 18007608 | RRID:SCR_014223 |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.35731.020
