Interplay of disordered and ordered regions of a human small heat shock protein yields an ensemble of ‘quasi-ordered’ states
- University of Washington, United States
Figures
Figure 1 with 1 supplement
sHSP domain architecture and sequence alignment.
(A) All sHSPs are defined by a conserved α-crystallin domain (ACD), that is flanked by N-terminal and C-terminal regions (NTR and CTR, respectively). Constructs used in this study are shown and …
Figure 1—figure supplement 1
Sequence alignment of HSPB1 orthologs, residues of interest, and known and predicted secondary structure.
HSPB1 sequences from human, mouse, frog, chicken, and fish show considerable conservation throughout most of the NTR, in contrast to other sHSP types as seen in the human HSPB5 sequence at the …
Figure 2 with 2 supplements
NMR analysis of NTR-ACD reveals changes in the ACD and increased heterogeneity when the NTR is present.
(A) Comparison of B1-ACD (black), NTR-ACD (blue), and a mixture of 15N-B1-ACD and unlabeled NTR-ACD (red) 1H-15N HSQC-TROSY spectra. (B) CSPs measured for NTR-ACD compared to B1-ACD. The following …
Figure 2—figure supplement 1
NMR spectra comparison of HSPB1dimer and NTR-ACD.
1H-15N HSQC-TROSY spectra of the two constructs, showing considerable overlap; strong peaks in the center of the spectrum correspond to disordered regions of the protein (NTR and CTR). Center peaks …
Figure 2—figure supplement 2
Perturbed residues in the mixed 15N-B1-ACD/NTR-ACD dimer spectrum.
Residues for which a distinct, new peak arises in the same position as the NTR-ACD spectrum are highlighted in red. These mainly cluster around the β4/β8 groove. Residues for which the peak is …
Figure 3
Summary of results from peptide-binding and PRE NMR experiments.
Regions of the ACD that are perturbed upon peptide binding or lose intensity in the presence of a spin label are highlighted on the HSPB1 primary structure (top) and NMR structure (PDB 2N3J, …
Figure 4 with 1 supplement
Perturbations of 15N-B1-ACD due to peptide binding.
(A) The distal peptide, consisting of HSPB1 residues 1–13, causes CSPs in the 15N-HSQC spectrum of B1-ACD (black). Peak shifts occur along a trajectory toward the peak positions of the same residues …
Figure 4—figure supplement 1
Effect of phosphorylation on peptide binding.
(A) CSPs in the aromatic peptide binding experiment with 15N-B1-ACD. A phosphorylated form of the aromatic peptide with phophoserine at position 15 does not cause strong CSPs (diamonds), indicating …
Figure 5
Paramagnetic relaxation enhancement NMR reveals contacts between NTR sites and residues in the ACD.
The spin label MTSL was conjugated at five positions in the NTR of 15N-NTR-ACD. Spectra were collected in the presence of the active spin label (paramagnetic) and after it had been quenched by …
Figure 6
Assignment of NTR residues reveal disorder and heterogeneity.
(A) 1H-15N HSQC-TROSY spectrum of NTR-ACD, highlighting strong peaks in the center of the spectrum that correspond to disordered regions of the protein (NTR and CTR). For part of the NTR, pairs of …
Figure 7 with 2 supplements
HDXMS analysis of WT oligomers and HSPB1dimer reveals changes in protection and heterogeneity in the NTR.
Representative peptides are indicated as horizontal bars. The midpoint of each peptide is represented by a gray circle showing the deuteration level of the peptide after 3 s (see Source Data one for …
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Figure 7—source data 1
HDXMS profiles for all forms of HSPB1.
The deconvoluted fractions of each population from bimodal distributions are shown in the first two columns for each mutant. The fractional deuterium uptake for each of these populations is shown in the next two columns, followed by a weighted average in the fifth column. All spectra were binomial fit using HX-Express (Guttman et al., 2013).
- https://doi.org/10.7554/eLife.50259.015
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Figure 7—source data 2
Statistics for HDXMS experiments on all mutants.
Statistics listed are for C137S forms of the following mutants of HSPB1. Sequence coverage takes into account the lack of observed deuterium uptake in the first two residues of each peptide. Back-exchange is calculated as the % deuterium uptake of the fully deuterated sample divided by the theoretical maximum deuteration for each peptide (89%), excluding prolines and the first two residues.
- https://doi.org/10.7554/eLife.50259.016
Figure 7—figure supplement 1
Example bimodal spectra for peptides 1–10, 29–41, 77–99, and 179–185 of C137S oligomers.
Multiple populations (bimodals) are observed in earlier timepoints for several regions of the protein. HX-Express (Guttman et al., 2013) was used to deconvolute the two populations into two binomial …
Figure 7—figure supplement 2
Comparison of changes in HDXMS between WT and HSPB1dimer across biological replicates at 3 s.
Identical constructs expressed, purified, and examined separately are shown in black and gray. The native C137-containing constructs with reducing agent are shown in red. Points in the upper half …
Figure 8 with 1 supplement
Modeling of NTR-ACD interactions.
(A) Cartoon showing the starting structure of the ACD with two copies of the distal motif (red arrows), one copy of the conserved motif (orange arrow), and two copies of β2 (blue arrows). The …
Figure 8—figure supplement 1
Models in which the conserved motif is connected to the distal motif oriented in the opposite direction.
While we believe this connectivity is less favored than the models shown in the main figure, we were able to generate physically realistic models. Depending on which copy of β2 the conserved motif …
Figure 9 with 1 supplement
Analysis of disease-associated HSPB1 NTR mutations G34R, G84R, and P39L.
(A) HDXMS analysis of each mutant. Representative peptides are indicated as horizontal bars. The midpoint of each peptide is represented by a gray circle showing the deuteration level of the peptide …
Figure 9—figure supplement 1
Circular dichroism spectra and subunit exchange kinetics of disease mutants.
(A) Far-UV CD spectra show overall similar secondary structure for WT, G34R, P39L, and G84R mutants, but P39L shows the strongest increase in ellipticity. The HSPB1dimer has a unique positive peak …
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.50259.021
