Short hydrophobic loop motifs in BRICHOS domains determine chaperone activity against amorphous protein aggregation but not against amyloid formation

Abstract

ATP-independent molecular chaperones are important for maintaining cellular fitness but the molecular determinants for preventing aggregation of partly unfolded protein substrates remain unclear, particularly regarding assembly state and basis for substrate recognition. The BRICHOS domain can perform small heat shock (sHSP)-like chaperone functions to widely different degrees depending on its assembly state and sequence. Here, we observed three hydrophobic sequence motifs in chaperone-active domains, and found that they get surface-exposed when the BRICHOS domain assembles into larger oligomers. Studies of loop-swap variants and site-specific mutants further revealed that the biological hydrophobicities of the three short motifs linearly correlate with the efficiency to prevent amorphous protein aggregation. At the same time, they do not at all correlate with the ability to prevent ordered amyloid fibril formation. The linear correlations also accurately predict activities of chimeras containing short hydrophobic sequence motifs from a sHSP that is unrelated to BRICHOS. Our data indicate that short, exposed hydrophobic motifs brought together by oligomerisation are sufficient and necessary for efficient chaperone activity against amorphous protein aggregation.

Molecular basis for different substrate‐binding sites and chaperone functions of the BRICHOS domain

“…The approximate locations of the loops in the Bri2 BRICHOS oligomer structure show that the hydrophobic motifs, that is, Y65‐P67, Y70‐I72, and M76‐V78, located in the loop regions, get exposed and come together during oligomerization. This arrangement subsequently generates binding sites for amorphous protein aggregates, supported by that the hydrophobicity of these three motifs correlates well with Bri2 BRICHOS capacity against amorphous protein aggregation, but not the anti‐amyloid capacity (Chen et al, 2023 ).…”

“…BRICHOS domains, including Bri2 BRICHOS, inhibit both fibrillar and amorphous aggregation to varying degrees (Chen et al, 2017 ; Chen et al, 2023 ; Leppert et al, 2023 ; Poska et al, 2016 ; Poska et al, 2020 ), but the molecular basis for how different BRICHOS domains recognize and affect different substrates remains unclear. As highly accurate protein structure predictions can now be achieved by AlphaFold2 (Jumper et al, 2021 ) and RoseTTAFold (Baek et al, 2021 ; Jumper et al, 2021 ), the BRICHOS domain from human Bri2 was modeled using both of these procedures (Figure 1a and Figure S1a ).…”

“…The polydispersity is consistent with the additional density of the map, shown by contouring at lower levels, which could house up to 12 additional symmetry related subunits (Figure S7 ). In the core, the loops between α‐helix 1 and 2 extend from the outside of the assembly toward the center and are involved in intersubunit interactions (Figure 5c,d ), providing a molecular explanation for loop manipulations, such as weakening hydrophobic interactions, which results in a reduction in the average sizes of Bri2 BRICHOS oligomers (Chen et al, 2023 ). The distribution of intermolecular disulfides in the oligomer is shown in Figure 5e–g , supporting the formation of disulfide bonds between subunits in Bri2 BRICHOS oligomers (Chen et al, 2017 ; Leppert et al, 2022 ).…”

“…Some chaperones are active against both fibrillar and amorphous protein aggregation (Wentink et al, 2019 ), for example, αB‐crystallin and DNAJB (Mainz et al, 2015 ; McMahon et al, 2021 ). BRICHOS domains collectively show activity against both fibrillar and amorphous aggregations, but the level of activity as well as the specificity for the different types of substrates depend on the BRICHOS family as well as the assembly state (Chen et al, 2017 ; Chen et al, 2023 ; Leppert et al, 2023 ; Poska et al, 2016 ; Poska et al, 2020 ).…”

A new kid in the folding funnel: Molecular chaperone activities of the BRICHOS domain

“…While all BRICHOS domains studied to date show antiamyloid activities only the BRICHOS domains from Bri2 and Bri3 prevent amorphous, non-fibrillar protein aggregation by forming transient complexes with misfolded clients (Chen et al, 2017(Chen et al, , 2022Poska et al, 2016Poska et al, , 2020. This feature is linked to the exposure of substrate binding sites made up of short hydrophobic motifs that are brought together via disulfide-dependent oligomerization processes (Chen, Leppert, et al, 2023). The mechanism of substrate binding of BRICHOS oligomers is appealingly similar to ATP-independent molecular chaperones, like sHSP26 and sHSP42 from baker's yeast, which bind non-selectively to misfolded substrates (Haslbeck & Vierling, 2015;Poska et al, 2016).…”