HSPB5 disease-associated mutations have long-range effects on structure and dynamics through networks of quasi-ordered interactions
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Evaluation Summary:
The human small heat shock protein (sHSP) HSPB5 is an ATP-independent molecular chaperone involved in maintaining protein homeostasis. This manuscript reports on dynamic interactions between the disordered N-terminal region (NTR) and the structured alpha-crystallin domain (ACD) in HSPB5 oligomers. The authors show that two mutations, associated with early cataract and myopathy development, disrupt the interaction of the ACD core with the unfolded NTRs and generate a much more dynamic and hyperactive version of the chaperone. These findings will be of interest to colleagues studying molecular chaperones and their implications for disease in humans.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Small heat shock proteins (sHSPs) are chaperones whose importance in protein homeostasis is exemplified by dozens of missense mutations associated with tissue-specific disease states. Despite decades of studies, the structure, dynamics, and mechanism of chaperone activity remain unclear. Here we show that the human sHSP HSPB5 distinguishes native lens protein γD-crystallin from damaged γD-crystallin even though the mutant/damaged client is folded. The disordered N-terminal region of HSPB5 (NTR) is essential for its chaperone activity, whereas the structured domain (ACD) has no intrinsic activity. Nevertheless, two sHSP mutational hotspots associated with disease, D109 and R120, are located in the ACD. Our studies on wild-type HSPB5 oligomers reveal that distinct regions within the NTR interact with specific grooves presented on the ACD dimer and/or with other NTR sub-regions and that the number of binding partners is greater than the number of binding sites, leading to a large, but finite number of potential combinations of interactions at any given time. The ACD mutations result in increased dynamics and accessibility of the disordered NTR and enhanced chaperone activity in vitro. Our findings reveal that HSPB5 quasi-order is delicately balanced and that perturbations arising from mutations within the structured core cause alterations that contribute to misbalance in eye lens protein homeostasis that lead to cataract formation.
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Evaluation Summary:
The human small heat shock protein (sHSP) HSPB5 is an ATP-independent molecular chaperone involved in maintaining protein homeostasis. This manuscript reports on dynamic interactions between the disordered N-terminal region (NTR) and the structured alpha-crystallin domain (ACD) in HSPB5 oligomers. The authors show that two mutations, associated with early cataract and myopathy development, disrupt the interaction of the ACD core with the unfolded NTRs and generate a much more dynamic and hyperactive version of the chaperone. These findings will be of interest to colleagues studying molecular chaperones and their implications for disease in humans.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the …
Evaluation Summary:
The human small heat shock protein (sHSP) HSPB5 is an ATP-independent molecular chaperone involved in maintaining protein homeostasis. This manuscript reports on dynamic interactions between the disordered N-terminal region (NTR) and the structured alpha-crystallin domain (ACD) in HSPB5 oligomers. The authors show that two mutations, associated with early cataract and myopathy development, disrupt the interaction of the ACD core with the unfolded NTRs and generate a much more dynamic and hyperactive version of the chaperone. These findings will be of interest to colleagues studying molecular chaperones and their implications for disease in humans.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In this manuscript by Woods et al, the authors study the small heat shock protein HSPB5, specifically focusing on two cataract-associated mutations. They show that the mutations, which are located in the ACD core of the protein, disrupt the interaction of the core with the unfolded N-termini and generate a much more dynamic version of the protein. A surprising feature of the mutants is that they enhance in vitro chaperone activity directed against damaged GammaD-crystallin.
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Reviewer #2 (Public Review):
Combining data from site-specific crosslinking, hydrogen-deuterium exchange mass spectrometry (HDX-MS), and NMR, the authors demonstrate a high variability in the binding of NTR regions to the folded α-crystallin domain (ACD) of HSPB5. The observed behavior is comparable to an ensemble of "quasi-ordered" states as claimed for HSPB1 (a further human sHsp) in a previous study by the Klevit lab (Clouser et al., 2019). The concept of "quasi-ordered" states, formed by varying interactions between specific contact points was introduced by the authors to explain the observed dynamic ensembles of oligomers.
The structural analysis of the HSPB5 wild-type protein and disease-related mutations together with functional data on the chaperone activity of the variants allow the authors to conclude that perturbations in the …
Reviewer #2 (Public Review):
Combining data from site-specific crosslinking, hydrogen-deuterium exchange mass spectrometry (HDX-MS), and NMR, the authors demonstrate a high variability in the binding of NTR regions to the folded α-crystallin domain (ACD) of HSPB5. The observed behavior is comparable to an ensemble of "quasi-ordered" states as claimed for HSPB1 (a further human sHsp) in a previous study by the Klevit lab (Clouser et al., 2019). The concept of "quasi-ordered" states, formed by varying interactions between specific contact points was introduced by the authors to explain the observed dynamic ensembles of oligomers.
The structural analysis of the HSPB5 wild-type protein and disease-related mutations together with functional data on the chaperone activity of the variants allow the authors to conclude that perturbations in the quasi-order are directly correlated to alterations in chaperone activity. In their investigations, the authors focus on two disease-related hotspot mutations, D109 and R120, which have been implicated in cataract formation in the eye lens. The authors conclude that misbalancing the quasi-order by the respective point mutations in HSPB5 is causative for cataracts.
The study by Woods et al. provides new insight into the dynamic interactions within the HSPB5 oligomers. The authors show which sub-regions of the NTR interact with respective sites in the ACD. Together with the description of changes in the interaction pattern and function induced by the mutations, the findings advance our understanding of the structural basis for the concept of quasi-ordered ensembles of sHSPS.
The comprehensive set of experiments is well designed and thoroughly conducted. The manuscript is well written and the results are clearly presented and discussed.
However, some concerns exist. The authors analyze oligomeric, dimeric ACD only, and crosslinked dimer setups. In this context, the validity of conclusions on NTR-NTR-interactions within the oligomers needs to be explained in more detail. The quasi-ordered state model of the authors also leaves the impression that all potential interactions are similar in the strength of the interaction. A qualitative (or, if possible, quantitative) comparison of the different interactions would strengthen the concept.
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Reviewer #3 (Public Review):
The manuscript by Woods et al. describes a highly interesting study on signalling between the alpha-crystallin domain (ACD) and the disordered N-terminal domain (NTD) in the small heat shock protein HSPB5 (alphaB-crystallin). The authors show that distinct regions in the NTD interact with specific grooves in the ACD. The data are supported with aggregation assays, SEC, HDX, NMR, and X-linking MS experiments. This is a very timely and valuable contribution that will be well received by the community.
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