The influence of the N-terminal region proximal to the core domain on the assembly and chaperone activity of αB-crystallin

Journal article

αB-Crystallin (HSPB5) is a small heat-shock protein that is composed of dimers that then assemble into a polydisperse ensemble of oligomers. Oligomerisation is mediated by heterologous interactions between the C-terminal tail of one dimer and the core "α-crystallin" domain of another and stabilised by interactions made by the N-terminal region. Comparatively little is known about the latter contribution, but previous studies have suggested that residues in the region 54-60 form contacts that stabilise the assembly. We have generated mutations in this region (P58A, S59A, S59K, R56S/S59R and an inversion of residues 54-60) to examine their impact on oligomerisation and chaperone activity in vitro. By using native mass spectrometry, we found that all the αB-crystallin mutants were assembly competent, populating similar oligomeric distributions to wild-type, ranging from 16-mers to 30-mers. However, circular dichroism spectroscopy, intrinsic tryptophan and bis-ANS fluorescence studies demonstrated that the secondary structure differs to wild type, the 54-60 inversion mutation having the greatest impact. All the mutants exhibited a dramatic decrease in exposed hydrophobicity. We also found that the mutants in general were equally active as the wild-type protein in inhibiting the amorphous aggregation of insulin and seeded amyloid fibrillation of α-synuclein in vitro, except for the 54-60 inversion mutant, which was significantly less effective at inhibiting insulin aggregation. Our data indicate that alterations in the part of the N-terminal region proximal to the core domain do not drastically affect the oligomerisation of αB-crystallin, reinforcing the robustness of αB-crystallin in functioning as a molecular chaperone.

Authors: Jovcevski, B; Andrew Aquilina, J; Benesch, J; Ecroyd, H

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Springer Verlag
• Journal: Cell Stress Chaperones
• Volume: 23
• Issue: 5
• Pages: 827–836
• Publication date: 2018-03-08
• Acceptance date: 2018-02-24
• DOI: 10.1007/s12192-018-0889-y
• EISSN: 1466-1268
• ISSN: 1355-8145
• Language: English
• Keywords: molecular chaperone; amyloid fibrils; proteostasis; HSPB5; small heat-shock protein; protein aggregation; αB-crystallin; native mass spectrometry