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. 2004 Mar 30;101(13):4367-72.
doi: 10.1073/pnas.0306276101. Epub 2004 Mar 19.

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

Victor F Lundin  1 , Peter C Stirling, Juan Gomez-Reino, Jill C Mwenifumbo, Jennifer M Obst, José M Valpuesta, Michel R Leroux
Affiliations

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

Victor F Lundin et al. Proc Natl Acad Sci U S A. .

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 coiled-coil 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.

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Figures

Fig. 1.
Fig. 1.
Chaperone activity of intradomain swap (switch) mutant complexes. (A) Schematic representations of α and β switch mutants. For the wild-type subunits, the N- and C-terminal helices are colored white and gray, respectively; the interhelical a/d residues are represented as dark ovals and correspond to those shown in the PFD subunit alignments (Fig. 6). For the switch mutants, the numbering scheme and colors used correspond to the wild-type sequences and show where the crossover points occur. (B) Effect of PFD switch mutants on the aggregation of denatured lysozyme. Relative aggregation of 2 μM lysozyme (monitored at 360 nm) during 10 min in buffer alone or in the presence of wild-type or prefoldin variants (as shown on the right of each curve). PFD complexes were at 2 μM unless otherwise indicated.
Fig. 2.
Fig. 2.
Chaperone activity of chimeric complexes. (A) Schematic representations of chimeric PFD subunits. The exogenous coiled-coil region is colored black, and the numbers adjacent to the helical regions refer to the amino acid position at which the fusion has taken place. (B) Effect of chimeric PFD mutants on the aggregation of 2 μM denatured lysozyme. PFD complexes (as shown on the right of each curve) were at 2 μM unless otherwise indicated.
Fig. 3.
Fig. 3.
Hydrophobic a/d coiled-coil residues are required for chaperone activity. (A) Schematic representations of the a/d residue point mutants. Hydrophobic residues between the coiled coils are dark ovals; residues mutated to serine are colored white. (B) Effect of hydrophobic a/d point mutants on the aggregation of 2 μM denatured lysozyme. Each PFD complex, shown on the right, was tested at 2 μM. (C) Mutated hydrophobic residues shown on the PFD crystal structure. PFD is shown looking into the cavity (Left), and from inside, viewing the cavity surface (Right). Residues that were mutated are colored green. Not shown is one pair of hydrophobic residues (L3 and A113) in theβ subunit termini and one amino acid (D3) in the N terminus of the α subunit, which were not resolved in the crystal structure but may also form part of the coiled coil (4, 6).
Fig. 4.
Fig. 4.
Substrate binding occurs near the ends of flexible coiled coils. (A) Coelution of PFD and GFP on a superdex 200 size-exclusion column (Left). The green dashed line is PFD (PhPFD) plus denatured GFP monitored at 222 nm, the thick blue line is GFP alone monitored at its excitation maximum (396 nm), and the red line is PFD plus denatured GFP monitored at 396 nm. Coelution was also demonstrated by SDS/PAGE analysis of peak A (1.25–1.50 ml) with and without PhPFD and of peak B without PhPFD (1.65–1.80 ml) (Right). (B) Interaction of unfolded GFP with PFD. (1) Molecular surface of PFD crystal structure; negatively stained and averaged EM images of substrate-free prefoldin (2), and GFP-bound PFD (4). (3 Upper) Merged contour maps (blue, PFD alone; red, PFD + GFP). (Lower) The approximate tilt angle change (≈12° opening) of the substrate-bound PFD subunits (red) relative to that of PFD alone (the contour map is shaded blue). (C) The putative hinge domains connecting PFD coiled coils and theβ-barrel domain are shown with arrows. Gray dashed lines indicate the (approximate) 12° opening motion, and the β-barrel oligomerization domain regions are circled with narrow black dashed lines. (D) Effect of wild-type (Ph and Mt) and MtαHM3βHM4 PFD complexes on the aggregation of denatured conalbumin (75 kDa). Aggregation assays were performed as for denatured lysozyme except conalbumin was 0.75 μM and PFD or its variants (Right) were added at a 5:1 ratio over substrate (3.75 μM).
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