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

Abstract

Small heat-shock proteins (sHSPs) are a widely expressed family of ATP-independent molecular chaperones that are among the first responders to cellular stress. Mechanisms by which sHSPs delay aggregation of client proteins remain undefined. sHSPs have high intrinsic disorder content of up to ~60% and assemble into large, polydisperse homo- and hetero-oligomers, making them challenging structural and biochemical targets. Two sHSPs, HSPB4 and HSPB5, are present at millimolar concentrations in eye lens, where they are responsible for maintaining lens transparency over the lifetime of an organism. Together, HSPB4 and HSPB5 compose the hetero-oligomeric chaperone known as α-crystallin. To identify the determinants of sHSP function, we compared the effectiveness of HSPB4 and HSPB5 homo-oligomers and HSPB4/HSPB5 hetero-oligomers in delaying the aggregation of the lens protein γD-crystallin. In chimeric versions of HSPB4 and HSPB5, chaperone activity tracked with the identity of the 60-residue disordered N-terminal regions (NTR). A short 10-residue stretch in the middle of the NTR (“Critical sequence”) contains three residues that are responsible for high HSPB5 chaperone activity toward γD-crystallin. These residues affect structure and dynamics throughout the NTR. Abundant interactions involving the NTR Critical sequence reveal it to be a hub for a network of interactions within oligomers. We propose a model whereby the NTR critical sequence influences local structure and NTR dynamics that modulate accessibility of the NTR, which in turn modulates chaperone activity.

Dynamic fibrillar assembly of αB-crystallin induced by perturbation of the conserved NT-IXI motif resolved by cryo-EM

“…2f and Extended Data Fig. 6 ) 36,51,52 . We fitted this NTD region into the cryo-EM density, corresponding to αB-crystallin residues LFDQFFGE (residues 23-30) ( Fig.…”

The major inducible small heat shock protein HSP20-3 in the tardigrade Ramazzottius varieornatus forms filament-like structures and is an active chaperone

“…2 A, black rectangle) and its “critical-sequence” ( Fig. 2 A, purple box) identified in CRYAB 39 . This region affects the structure and dynamics of the NTD with consequences for CRYAB chaperone activity ( 39 ; Fig.…”

“… 68 . For CRYAB ssNMR studies reported an α-helix spanning residues 23–32 in CRYAB immediately preceding its critical-sequence 38 , 39 . The α-helix in the NTD of CRYAB will not fit the ACD central groove, implying that this part of the NTD will likely have to alter its conformation in the dimer 39 .…”

“…The predicted sequence for HSP20–8 is just 95 residues and contains an incomplete sHSP domain that would be expected to disrupt the IPI-β4/β8 surface interaction that is key to the oligomerisation of CRYAB (HSPB5; 33 , 34 ), but these features are present in the other seven sHSPs in R. varieornatus . The N- and C-terminal unstructured domains of sHSPs also regulate the interaction with client proteins as well as sHSP subunit oligomerisation 35 , 36 , 37 , 38 , 39 .…”

Roles of Nucleic Acids in Protein Folding, Aggregation, and Disease

“…Holdases include many disparate proteins, such as the small heat shock proteins, , the α-crystallins, − Hsp33, − and HdeA, , among others. − Holdases are thought to act in a more mechanistically simple manner by acting as a first line of defense to prevent widespread protein aggregation, later transferring their clients to the foldases for protein refolding. Although not requiring ATP, holdases can still be mechanistically complicated, using conditional disorder or heterogeneous oligomerization as regulatory mechanisms. , Some holdases do allow protein folding while bound, such as Spy. , …”