HSPB5 disease-associated mutations have long-range effects on structure and dynamics through networks of quasi-ordered interactions

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.

Disordered region encodes α-crystallin chaperone activity toward lens client γD-crystallin

“…A range of deuterium uptake was observed for homo-oligomeric assemblies of HSPB4 and HSPB5, with similar patterns of exchange to other reports ( SI Appendix , Figs. S12 and S13 ; HSPB1, 20 ; HSPB4, 15 ; and HSPB5 at pH 7.5, 11 ). CTR-derived peptides exchange very rapidly, ACD-derived peptides exchange slowly reflecting the stable β-sandwich structure, and peptides from the NTR have intermediate exchange rates.…”

“…HSPB4 and HSPB5 are the two most highly related of the 10 human sHSPs, with highest sequence similarity within the structurally conserved ACD and highest sequence divergence within the disordered NTR and CTR. Based on previous studies that revealed that the NTR of HSPB5 is required for its chaperone activity against γD-W130E ( 11 ), chimeric constructs were created in which the NTRs of HSPB4 and HSPB5 were swapped(“N4-B5” has the NTR of HSPB4 swapped onto HSPB5, “N5-B4” is the alternate chimera; SI Appendix , Fig. S3 ).…”

“…To test the generality of the effect of the Critical sequence on chaperone activity, we installed the Critical sequence swaps H31G/L33F/S35Y into the HSPB5 cataract- and myopathy-associated mutant R120G (“R120G-GFY”; 18 ). This mutant is an enhanced chaperone of γD-W130E and has strongly decreased protection from hydrogen/deuterium exchange (HDX) in the NTR and ACD ( 11 ). The Critical sequence substitutions substantially decrease R120G chaperone activity ( Fig.…”

“…However, client interactions involving highly activated HSPB5 species including known disease/cataract-associated mutants such as R120G ( Fig. 1 A ) result in long-lived co-complexes that eventually co-aggregate ( 11 ). Such observations indicate that there are negative consequences associated with constitutive high HSPB5 activity, suggesting there may be pressure to modulate client binding in lens.…”

“…The involvement of the NTR in chaperone activity is supported by its sequence characteristics: hydrophobic- and aromatic-rich segments interspersed by proline residues. We showed that the NTR of HSPB5 is required for productive interactions with lens client protein γD-crystallin ( 11 ). Among human sHSPs, the NTRs of HSPB4 and HSPB5 are most similar (67.6% similarity vs. 49.3% for next most similar sHSP pair; SI Appendix , Fig.…”