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. 2012 Feb 17;287(8):5661-72.
doi: 10.1074/jbc.M111.275057. Epub 2011 Dec 8.

Unique peptide substrate binding properties of 110-kDa heat-shock protein (Hsp110) determine its distinct chaperone activity

Xinping Xu  1 , Evans Boateng Sarbeng, Christina Vorvis, Divya Prasanna Kumar, Lei Zhou, Qinglian Liu
Affiliations

Unique peptide substrate binding properties of 110-kDa heat-shock protein (Hsp110) determine its distinct chaperone activity

Xinping Xu et al. J Biol Chem. .

Abstract

The molecular chaperone 70-kDa heat-shock proteins (Hsp70s) play essential roles in maintaining protein homeostasis. Hsp110, an Hsp70 homolog, is highly efficient in preventing protein aggregation but lacks the hallmark folding activity seen in Hsp70s. To understand the mechanistic differences between these two chaperones, we first characterized the distinct peptide substrate binding properties of Hsp110s. In contrast to Hsp70s, Hsp110s prefer aromatic residues in their substrates, and the substrate binding and release exhibit remarkably fast kinetics. Sequence and structure comparison revealed significant differences in the two peptide-binding loops: the length and properties are switched. When we swapped these two loops in an Hsp70, the peptide binding properties of this mutant Hsp70 were converted to Hsp110-like, and more impressively, it functionally behaved like an Hsp110. Thus, the peptide substrate binding properties implemented in the peptide-binding loops may determine the chaperone activity differences between Hsp70s and Hsp110s.

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Figures

FIGURE 1.
FIGURE 1.
Structural comparison of Hsp70s and Hsp110s. A, schematics of Hsp70 and Hsp110 sequences. NBD is in blue. SBD is divided into two subdomains: SBDα and SBDβ are in red and green, respectively. The interdomain linker is shown in purple, whereas the extreme C-terminal end is shown in white. B and C, ribbon diagrams of the Sse1-ATP structure (Protein Data Bank code 2QXL) and bHsc70 isolated NBD structure (Protein Data Bank code 1HPM), respectively, with the NBD aligned. The coloring is the same as in A. ATP in Sse1 and ADP in bHsc70 are shown as bonds. The associated ions, Mg2+ and K+, are shown as balls. D and E, ribbon diagrams of SBD structures of Sse1 (Protein Data Bank code 2QXL) and DnaK (Protein Data Bank code 1DKX), respectively, with the SBDβ aligned. The coloring is the same as in A with the peptide substrate NR in DnaK shown in cyan.
FIGURE 2.
FIGURE 2.
Hsp70s and Hsp110s have different peptide substrate preference. A, chaperone binding to four different peptides analyzed by fluorescence anisotropy assay. The peptides are TRP2 (red circle), p53 (blue diamond), p12 (green triangle), and NR (black circle). From top to bottom, the plots are DnaK, Ssa1, Sse1, Sse2, and hHsp110, respectively. B, chaperone binding to the mutated TRP2 peptides TRP2_FF (green triangle) and TRP2_W (black diamond) with TRP2 peptide (red filled circle) as a control. The sequence of the plots is the same as in A. mP, millipolarization level.
FIGURE 3.
FIGURE 3.
Binding of DnaK and Sse1 to mutant TRP2 peptides. A, DnaK. B, Sse1. The mutant TRP2 peptides are labeled between A and B: TRP2_W (black filled circle), TRP2_F (red filled square), TRP2_L (orange filled triangle), TRP2_V (green filled triangle), TRP2_A (blue filled diamond), TRP2_S (blue open circle), and TRP2_D (black open diamond). mP, millipolarization level.
FIGURE 4.
FIGURE 4.
Kinetics of Hsp110 peptide substrate binding and stability of Hsp110-peptide complexes. A, ATP reduces the peptide substrate binding affinity in Sse1. Left, DnaK protein binding to the NR peptide. Right, Sse1 binding to the TRP2 peptide. B and C, peptide binding kinetics in the absence and presence of ATP, respectively. Left, DnaK binding to the NR peptide. Right, Sse1 binding to the TRP2 peptide. The concentrations of DnaK and Sse1 used were 40 (red), 20 (orange), 10 (yellow), 5 (green), 2.5 (blue), and 1.25 μm (magenta). D, the stability of the Hsp70-peptide and Hsp110-peptide complexes were analyzed using native PAGE. The fluorescein-labeled peptides were visualized using a phosphorimaging system (left panel), and the chaperone proteins on the same gel were stained with Coomassie Blue (right panel). The positions of the free peptides and the chaperone-peptide complexes, respectively, are indicated in the left panel. The arrowheads in the right panel point to the monomeric forms of DnaK and Sse1, respectively. The oligomeric forms of DnaK ran above the monomeric form. mP, millipolarization level.
FIGURE 5.
FIGURE 5.
Peptide-binding loops determine peptide binding properties in Hsp70. A, peptide substrate binding to the isolated SBDs. The peptide binding of DnaK SBD (left) and Sse2 SBD (right) were measured with fluorescence anisotropy assays. The peptides are TRP2 (red circle), p53 (blue diamond), p12 (green triangle), and NR (black circle). B, sequence alignment of Loop1,2 (L1,2) and Loop3,4 (L3,4) regions from a number of Hsp110s and Hsp70s. The hydrophobic residues that form direct contacts with the bound NR peptide in the DnaK crystal structure are highlighted in red. C, ribbon diagrams of the SBDβ structures from Sse1 (left) and DnaK (right), respectively, with the β strands aligned. Loop1,2 (L1,2) and Loop3,4 (L3,4) are highlighted in red and purple, respectively. The NR peptide in DnaK is in cyan. D, the loop-swapping mutation in DnaK (DnaK_Loop) changed the peptide substrate binding properties. The binding of WT DnaK (black circle) and DnaK_Loop mutant protein (red diamond) to NR (left) and TRP2_181 (right) peptides was determined after binding reached equilibrium. E, swapping the peptide-binding loops or mutating the peptide binding cleft has little effect on the peptide binding of Sse1. The binding of the NR peptide (left) and the TRP2_181 peptide (right) to Sse1_Loop (red diamond) and Sse1_P (green triangle) mutants was determined and compared with that of the WT Sse1 (black circle). F, sequence alignment of peptide binding cleft residues that form hydrophobic interactions with the NR peptide in the structure of isolated DnaK SBD. mP, millipolarization level.
FIGURE 6.
FIGURE 6.
Influence of loop swapping on chaperone activity of DnaK. A, the DnaK_Loop mutant lost folding activity. After heat denaturation, refolding of denatured firefly luciferase by WT DnaK (black circle) and DnaK_Loop mutant (red diamond) was monitored over time. B, mutating peptide-binding loops in DnaK resulted in loss of in vivo function. Serial dilutions of fresh E. coli cultures were spotted on LB agar plates with empty vector and WT DnaK as negative and positive controls, respectively. 30 °C growth was used as culture control. C, the DnaK_Loop mutant gained holdase activity. Holdase activity was assayed by analyzing the aggregation of firefly luciferase (Lucif) in the presence of various chaperones, Sse1 (left), WT DnaK (middle), and DnaK_Loop (right), at a 1:4 (green) and 4:1 (red) ratio to luciferase. The final concentration of luciferase was kept at 200 nm for all the reactions. The aggregation of luciferase by itself (black) was used as a no chaperone control.
FIGURE 7.
FIGURE 7.
Model of Hsp70-Hsp110 chaperone machinery. A, model of the cooperative actions of Hsp70 and Hsp110 in protein folding. Hsp70s and Hsp110s prefer different segments in polypeptide substrates. Based on this study, we speculate that the segments preferred by Hsp70s (highlighted in green) are rich in aliphatic residues, whereas the segments preferred by Hsp110s (highlighted in red) are rich in aromatic residues. B, comparison of the chaperone cycles of Hsp70 and Hsp110. Peptide substrates are bound to and released from Hsp70 in the ATP-bound state due to fast kinetics; whereas, in the ADP-bound state, Hsp70 holds the bound peptide stably. For Hsp110, peptide substrates are bound and released constantly regardless of the nucleotide-bound state.
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