Small heat-shock proteins (sHSPs) are ubiquitously expressed molecular chaperones that protect the proteome against proteostasis failure. However, their structure-function relationship remains elusive. Human sHSPs populate a polydisperse ensemble of oligomers that readily exchange subunits (1). This polydispersity renders sHSPs refractory to biophysical characterization by X-ray diffraction and cryo-electron microscopy, however the central, highly conserved α-crystallin domain (ACD) can be crystallized. The ACD adopts a β-sandwich fold and forms antiparallel dimers via an extended β-strand, creating a shared β-sheet between the two subunits (2). sHSP oligomers are widely regarded to act as storage forms of these chaperone-active, exchangeable dimers. Indeed, ACD dimers have been shown to be chaperone-active (2). However, the exchange of sHSP monomers from oligomers has been observed, and the chaperone function(s) of the monomers are unknown. We and others have begun to characterize the ACD monomer via NMR (3,4) and sedimentation velocity analytical ultracentrifugation.
We investigated the interaction of the ACD with apolipoprotein C-II and α-synuclein amyloid fibrils using NMR. Significant resonance broadening was observed in the presence of amyloid fibrils, indicative of an ACD-fibril interaction. Substantially more signal broadening was observed in the presence of short, fragmented fibrils relative to an equal mass of long fibrils, demonstrating that the ACD interacts preferentially with fibril ends. Furthermore, resonances corresponding to the ACD monomer were broadened to a greater extent than those corresponding to the dimer. This indicates that the primary fibril-interacting species is monomeric and implicates the intra-dimer interface as an interactive site. These findings are supported by our mutagenesis studies in which disulfide cross-linked ACD dimers were less effective at inhibiting amyloid fibril elongation and naturally occurring fibril end-to-end joining than non-covalent dimers. Our results provide further evidence for the dynamic nature of sHSP assemblies and the importance of this dynamism for substrate binding and chaperone function.
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