Oligomeric assembly is required for chaperone activity of the filamentous γ‐prefoldin

Abstract

Prefoldins (PFDs) are molecular chaperones with a distinctive jellyfishshape that have a general role in de novo protein folding in Archaea and in the biogenesis of cytoskeleton proteins in eukaryotes. In general, PFDs are hetero-hexameric protein assemblies consisting of two a and four b subunits. However, a PFD variant called gamma-prefoldin (cPFD), isolated from the hyperthermophilic archaeon Methanocaldococcus jannaschii, exhibits a unique filamentous structure that is composed of hundreds of monomeric subunits. In this study, we investigated the relationship between the morphology of the cPFD filament and its ability to prevent protein aggregation. A chaperone assay demonstrated that cPFD must be in a filamentous assembly for functional activity and the distal regions of the coiled-coils are required for binding of non-native proteins. Molecular dynamic simulations were used to model the interactions between in silico thermally denatured protein substrates and the coiled-coils of a cPFD filament. During molecular dynamic simulations at 300 and 353 K, each coiled-coil was highly flexible, enabling it to widen the central cavity of the filament to potentially capture various non-native proteins. Docking molecular dynamic simulations of cPFD filaments with unfolded citrate synthase or insulin showed a size-dependence between the substrate and the number of interacting coiled-coils. To confirm this observation, we generated filaments containing specific numbers of subunits, and showed that between six and eight cPFD subunits are required for chaperone activity to prevent citrate synthase from thermal aggregation. These results provide insights into structure-function relationships of oligomeric chaperones and illuminate the potential role of cPFD in its native environment.

Geometrical assembly of ultrastable protein templates for nanomaterials

“…The connectors were assembled with γPFD in a controlled manner by first denaturing the proteins in 8 M guanidinium-HCl and combining the proteins in various molar ratios, followed by rapid dilution and refolding in an aqueous buffer. The propensity of the connectors to attach to γPFD subunits and extend filaments was confirmed using a protein–protein interaction pull-down assay 11 ( Supplementary Fig. 4 ).…”

“…Importantly, the interface between each γPFD subunit is pliant and amenable to structural modification 9 ; the β-sheet oligomerization domains and the coiled-coils of the subunits are physically separate and can be modified independently for expanded function 10 ; and the assembled filament is extremely stable to thermal and solvent-induced denaturation 8 . The inherent molecular chaperone function of the filament 11 may also serve to stabilize and re-activate enzymes in denaturing conditions such as during production of composite materials and enzyme–nanomaterial conjugates. Furthermore, progress has been made to gain control over γPFD filament elongation through the creation of a capping protein called TERM (thermophilic extension resistant mutant) to control filament length 9 .…”

Enhanced Enzyme Activity through Scaffolding on Customizable Self‐Assembling Protein Filaments

“…We have shown previously that γ‐PFD can interact with a broad range of non‐native proteins to capture and stabilize them using its coiled coil domains, and that filamentous assembly is required for such activity . Thus, it is possible that γ‐PFD–SpyTag interacts with the attached enzymes in a similar manner to provide favorable conditions for their catalysis.…”

Chaperone‐like activity of the N‐terminal region of a human small heat shock protein and chaperone‐functionalized nanoparticles

“…One structural aspect of sHsps that likely contributes to their chaperone activity is their dynamic oligomerization. Many previous studies have suggested that both the oligomerization dynamics in addition to sequences of the chaperone that act as “interacting sites” contribute to the activity of sHsps . Since oligomerization implies potential multivalent protein architectures, we were interested in developing a chemical system to mimic potential oligomeric states that sHsps can assume.…”

“…Many previous studies have suggested that both the oligomerization dynamics in addition to sequences of the chaperone that act as "interacting sites" contribute to the activity of sHsps. 20,61,62 Since oligomerization implies potential multivalent protein architectures, we were interested in developing a chemical system to mimic potential oligomeric states that sHsps can assume. Our rationale is that this would allow for evaluation of multivalent effects on chaperone activity of sHsps in the absence of the dynamic properties of the native system, which currently leads to indeterminate results.…”