Molecular clamp mechanism of substrate binding by hydrophobic coiled-coil residues of the archaeal chaperone prefoldin

Abstract

Prefoldin (PFD) is a jellyfish-shaped molecular chaperone that has been proposed to play a general role in de novo protein folding in archaea and is known to assist the biogenesis of actins, tubulins, and potentially other proteins in eukaryotes. Using point mutants, chimeras, and intradomain swap variants, we show that the six coiledcoil tentacles of archaeal PFD act in concert to bind and stabilize nonnative proteins near the opening of the cavity they form. Importantly, the interaction between chaperone and substrate depends on the mostly buried interhelical hydrophobic residues of the coiled coils. We also show by electron microscopy that the tentacles can undergo an en bloc movement to accommodate an unfolded substrate. Our data reveal how archael PFD uses its unique architecture and intrinsic coiled-coil properties to interact with nonnative polypeptides.C oiled coils consist of two or more parallel or antiparallel amphipathic ␣-helices that twist around one another to form supercoils (1). The primary sequences of the helices display a heptad repeat (abcdefg), where apolar residues are found preferentially in the first (a) and fourth (d) positions. Although the knobs-into-holes packing of the hydrophobic residues is the predominant stabilizing force for a coiled coil, inter-and intrahelical ionic interactions can act to further stabilize or destabilize its supersecondary structure (1). Coiled coils are found in several molecular chaperones, a diverse family of proteins whose collective cellular role is to ensure the quality control (e.g., folding, assembly, and transport) of nonnative proteins (2, 3). Archaeal prefoldin (PFD) is a chaperone that contains six canonical antiparallel coiled coils whose N-and C-terminal helices project outward from a double ␤-barrel oligomerization domain; the overall shape of the hexameric protein complex, assembled from two PFD␣ and four PFD␤ subunits (␣ 2 ␤ 4 ), resembles a jellyfish with six tentacles (4). In solution, its tentacles are likely to be fully solvated and independently mobile (4). A lower-resolution electron microscope image of recombinant human PFD, which consists of six different proteins (two ␣ class and four ␤ class subunits), reveals that it possesses the same overall structure (5).Like other chaperones, archaeal PFD can selectively interact with and stabilize nonnative (unfolded) polypeptides that expose hydrophobic surfaces in vitro, helping to prevent their aggregation (4, 6, 7). Preliminary studies have shown that deletion of the distal coiled-coil regions in either the ␣ or ␤ subunit abrogates chaperone activity in vitro, implying that PFD grasps its substrates in a multivalent manner (4). Similarly, the eukaryotic PFD-actin complex recently visualized by EM shows that nonnative actin, one of its substrates, makes multiple contacts with the distal regions of the tentacles (5).In the crowded cellular environment, eukaryotic PFD is likely to transiently stabilize ribosome-bound nascent polypeptides (8) before shuttling them to a chaperonin (an ATP-depe...

New insights into the biogenesis of nuclear RNA polymerases?This paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

“…2 for a schematic representation of the prefoldin-like module components). Once assembled, the β hairpins form the core of the complex, two 8-stranded β barrels, while the coiled-coil helixes resemble tentacle-like appendages that first mediate substrate binding (Siegert et al 2000;Lundin et al 2004). The PFD complex binds unfolded, newly synthesized poly-peptides and delivers them to the CCT chaperone for proper folding.…”

The cavity-chaperone Skp protects its substrate from aggregation but allows independent folding of substrate domains

“…The Skp structure was unexpectedly similar to that of prefoldin, a cytosolic molecular chaperone present in archea and eukarya (8)(9)(10). Although Skp is a trimer (8,9,11) and prefoldin is a hexamer (12,13), both proteins share jellyfish architectures with a central cavity ( Fig. 1 A and B).…”

“…1 A and B). In prefoldin, this cavity has been shown to bind substrate proteins (10,13). Prefoldin thus belongs to a family of chaperones sometimes referred to as ''holdases.''…”

Functional Interaction between Phosducin-like Protein 2 and Cytosolic Chaperonin Is Essential for Cytoskeletal Protein Function and Cell Cycle Progression

“…CCT cooperates with another chaperone called Prefoldin (PFD) in the folding of actins and tubulins (Geissler et al, 1998;Vainberg et al, 1998). PFD uses six long coiled-coil "tentacles" to stabilize substrate proteins at the opening of its jellyfish-shaped cavity before their delivery to the open CCT cavity (Vainberg et al, 1998;Siegers et al, 1999;Siegert et al, 2000;Lundin et al, 2004). Together, PFD and CCT compose a folding pathway for cytoskeletal proteins, which, along with PhLPs, control the folding of actin and tubulin (Siegers et al, 1999;Lacefield and Solomon, 2003;Stirling et al, 2006).…”