A conserved histidine modulates HSPB5 structure to trigger chaperone activity in response to stress-related acidosis
- University of Washington, United States
- University of Michigan, United States
Figures
Figure 1
Solution structure ensemble of HSPB5-ACD at pH 7.5 is an anti-parallel dimer.
(A) Backbone traces of ten ACD structures determined by RosettaOligomer are aligned over all residues (RMSD of 1.4 Å). Subunits of the dimer are shown in blue and gray. (B) Cartoon representation of …
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Figure 1—source data 1
Comparison of the NMR solution structure and published structures.
Backbone root mean square deviations (rmsd) between the solution structure and published structures and the correlation between experimentally measured residual dipolar coupling (RDC) values and those calculated for each published structure (PALES Correlation Coefficient) are given. Values in parentheses are calculated without including loop regions between the β-strands. Protomer–protomer and dimer–dimer rmsd between 2KLR and solution structure ensembles are the averages between all pairwise combinations of all structures in each ensemble. Rmsd values between the solution structure ensemble and the crystal structures are the average of all pairwise rmsd between the ten structures in the ensemble and the crystal structure. 2KLR: solid-state NMR structure; 2WJ7:crystal structure at pH 9.0; 3L1G:crystal structure at pH 4.6; 4M5S: crystal structure at pH 6.0.
- https://doi.org/10.7554/eLife.07304.004
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Figure 1—source data 2
Multiple sequence alignment of the ten human sHSPs.
ClustalOmega was used to align the sequences. Structural features of HSPB5-ACD are highlighted as follows: ACD is denoted by the blue line; His-104 is identified by the blue arrow; Loop 5/6 is shown in red box; dimer interface is shown in green box. All ACD histidine residues are shown in bold font.
- https://doi.org/10.7554/eLife.07304.005
Figure 2
15N-CPMG relaxation dispersion experiments detect dimer-to-monomer exchange.
(A) Relaxation dispersion measurements reveal a two-state transition. Representative relaxation dispersion curves of 15N transverse relaxation rate (R2,eff) plotted as a function of field strength, …
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Figure 2—source data 1
Parameters from relaxation dispersion experiments performed on 0.7 mM HSPB5-ACD at 22°C and pH 7.5.
- https://doi.org/10.7554/eLife.07304.009
Figure 3
HSPB5-ACD undergoes a conformational transition between pH 7.5 and 6.5.
(A) 1H-15N HSQC spectra acquired on a 200 µM sample of HSPB5-ACD at pH 7.5 and 6.5 (22°C) reveal two states. Full spectra collected at pH 7.5 (black) and 6.5 (red) are overlaid (left). At pH 7.5, …
Figure 4 with 1 supplement
His-104 plays a key role in the dimer-monomer transition.
(A) The electrostatic surface of HSPB5-ACD at pH 7.5 (calculated using experimentally determined histidine pKR values) reveals patches of positive (blue) and negative (red) charges that cross the …
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Figure 4—source data 1
The pKR values and tautomeric states of HSPB5-ACD histidine side-chain imidazole rings (22°C) are listed.
n.d.: not determined because resonances broaden below pH 7.5 and become undetectable.
- https://doi.org/10.7554/eLife.07304.013
Figure 4—figure supplement 1
His-104 is at the center of a dynamic network of charged and H-bonding interactions.
Top panel: ten individual members of the solution ensemble are shown. One protomer of the dimer is in cyan; the other protomer is in gray. His-104 is shown as space-filling spheres; carbonyl groups …
Figure 5 with 1 supplement
Destabilizing the ACD dimer interface via low pH or His-104 mutation triggers large expansion of HSPB5 oligomers.
(A) SEC-MALS analysis showing protein elution profile (refractive index, right Y-axis) with average Mw (horizontal trace under peak corresponds to left Y-axis) for WT-HSPB5 at pH 7.5 (blue) and pH …
Figure 5—figure supplement 1
EM micrograph images, 2D classification, and particle diameter estimation for WT and H104K HSPB5.
(A) Representative micrograph images of WT-HSPB5 at pH 7.5 and 6.5 and H104K-HSPB5 at pH 7.5 negatively stained with 0.75% uranyl formate (scale bar equals 20 nm). Example individual particle …
Figure 6
His-104 mutants of HSPB5 are effective at delaying the onset of aggregation of a model client protein.
(Top panel) Aggregation of DTT-denatured bovine αLactalbumin at 42°C in the absence (green) and presence of WT-HSPB5 (blue) or HSPB5 mutants H104K (red) and H104Q (orange). Light scattering at 360 …
Figure 7 with 1 supplement
H104K- and H104Q-HSPB5 oligomers reorganize into small, long-lived client-bound complexes in the presence of αLac model client protein.
(A–C) SEC-MALS analysis and corresponding Mw of WT (A), H104K (B) and H104Q (C), HSPB5 oligomers (40 μM in subunit concentration) incubated with αLac (120 μM) in the absence (blue) and presence …
Figure 7—figure supplement 1
EM micrograph images and 2D classification of HSPB5 incubated with αLac.
(A) Representative micrograph images and single particles of WT-HSPB5 and H104K-HSPB5 incubated with or without αLac substrate and DTT prior to fractionation by SEC-MALS showing the shift in …
Tables
Table 1
NMR data and refinement statistics for HSPB5-ACD structures
| NMR distance and dihedral constraints | HSPB5-ACD |
|---|---|
| Distance constraints | |
| Total NOE | 838 |
| Intraresidue | 310 |
| Inter-residue | |
| Sequential (|i − j| = 1) | 255 |
| Medium-range (|i − j| < 4) | 85 |
| Long-range (|i − j| > 5) | 188 |
| Inter-molecular | 36 |
| Total dihedral angle restraints | |
| φ (TALOS) | 72 |
| ψ (TALOS) | 72 |
| Residual Dipolar Couplings (RDCs) | |
| 1H-15N RDCs | 81 |
| Structure statistics | |
| Violations (mean ± s.d.) | |
| Distance constraints (Å) | 0.48 ± 0.45 |
| Dihedral angle constraints (°) | 14.4 ± 14.7 |
| Average pairwise r.m.s deviation (Å)* | |
| Heavy | 2.46 ± 0.97 |
| Backbone | 1.48 ± 0.6 |
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*
Average pairwise r.m.s.d. was calculated among ten refined structures.
Table 2
Dimer-to-monomer dissociation constants for HSPB5-ACD determined by ITC*
| Temperature | pH | Kd (mM) | ΔH (cal/mol) |
|---|---|---|---|
| 25°C | 7.5 | 0.002 ± 0.002 | 8772 ± 3960 |
| 25°C | 6.5 | 0.030 ± 0.016 | 3242 ± 432 |
| 37°C | 7.5 | 0.036 ± 0.002 | 9328 ± 527 |
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*
See ‘Methods and materials’ for experimental details and data analysis.
