Allostery in Hsp70 chaperones is transduced by subdomain rotations

Abstract

Hsp70s (heat shock protein 70 kDa) are central to protein folding, refolding, and trafficking in organisms ranging from archaea to Homo sapiens under both normal and stressed cellular conditions. Hsp70s are comprised of a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD). The nucleotide binding site in the NBD and the substrate binding site in the SBD are allosterically linked: ADP binding promotes substrate binding, while ATP binding promotes substrate release. Hsp70s have been linked to inhibition of apoptosis (i.e., cancer) and diseases associated with protein misfolding such as Alzheimer's, Parkinson's, and Huntington's. It has long been a goal to characterize the nature of allosteric coupling in these proteins. However, earlier studies of the isolated NBD could not show any difference in overall conformation between the ATP state and the ADP state. Hence the question: How is the state of the nucleotide communicated between NBD and SBD? Here we report a solution NMR study of the 44-kDa NBD of Hsp70 from Thermus thermophilus in the ADP and AMPPNP states. Using the solution NMR methods of residual dipolar coupling analysis, we determine that significant rotations occur for different subdomains of the NBD upon exchange of nucleotide. These rotations modulate access to the nucleotide binding cleft in the absence of a nucleotide exchange factor. Moreover, the rotations cause a change in the accessibility of a hydrophobic surface cleft remote from the nucleotide binding site, which previously has been identified as essential to allosteric communication between NBD and SBD. We propose that it is this change in the NBD surface cleft that constitutes the allosteric signal that can be recognized by the SBD.

Figure 1

Stereograph of the superposition of five X-ray crystallography structures for bovine Hsc70 NBD (4–380). The proteins were crystallized in the following states: wt-APO (2QW9.pdb, chain A), wt-ADP.PO₄ (3HSC.pdb and 2QWL.pdb, chain A), wt-ADP.VO₄ (2QWM.pdb, chain A) and K71M-ATP (1KAX.pdb). The proteins were overlaid on the secondary structure elements of sub domain IA (Hsc70-count residues 1–39, 116–188, 361–381; blue) to accentuate potential changes in sub domains IB (residues 40–115; green), IIA (residues 189–228, 307–360; purple) and IIB (residues 229–306; cyan). The N-terminus is in red, the C-terminus of this domain is shown in yellow. The nucleotide, PO₄³⁻, Mg²⁺ and two Na⁺ present in 3HSC.pdb are shown in space fill. The figure was prepared with MacPyMOL.

Figure 2

A: ¹⁵N-¹H chemical shift differences between the ATP and ADP.Pi conformation of Hsc-70-NBD. Orange: significant shift, green: no shift, grey: not known. Residues R171 (R167 in DnaK E. coli) and I181 (L177 in DnaK E. coli) are rendered as sticks. B: ¹⁵N-¹H chemical shift differences (Δδ) between the AMPPNP and ADP.Pi conformation of TTh-NBD. Red: Δδ> 2σ_Δδ; Orange: σ_Δδ< Δδ < 2 σ_Δδ; yellow: 0.5 σ_Δδ < Δδ < σ_Δδ; green: Δδ < 0.5 σ_Δδ; grey: not known, where Δδ=(Δδ_H² + Δδ_N^2)1/2. ADP is in light blue, PO₄³⁻ in dark blue. Residues R164 (R167 in DnaK E. coli) and L174 (L177 in DnaK E. coli) are rendered as sticks. See also Figure 3. The figure was prepared with MacPyMOL.

Figure 2

A: ¹⁵N-¹H chemical shift differences between the ATP and ADP.Pi conformation of Hsc-70-NBD. Orange: significant shift, green: no shift, grey: not known. Residues R171 (R167 in DnaK E. coli) and I181 (L177 in DnaK E. coli) are rendered as sticks. B: ¹⁵N-¹H chemical shift differences (Δδ) between the AMPPNP and ADP.Pi conformation of TTh-NBD. Red: Δδ> 2σ_Δδ; Orange: σ_Δδ< Δδ < 2 σ_Δδ; yellow: 0.5 σ_Δδ < Δδ < σ_Δδ; green: Δδ < 0.5 σ_Δδ; grey: not known, where Δδ=(Δδ_H² + Δδ_N^2)1/2. ADP is in light blue, PO₄³⁻ in dark blue. Residues R164 (R167 in DnaK E. coli) and L174 (L177 in DnaK E. coli) are rendered as sticks. See also Figure 3. The figure was prepared with MacPyMOL.

Figure 3

Globe graphs (Sanson-Flamsteed plots) showing the orientations and experimental uncertainties of the S_zz principal alignment axes (around 60 °West and 5 °South) of the different subdomains of NBD of DnaK-Tth as derived from the NMR RDC measurements using REDCAT. A and B: RDC input: conserved residues and experimentally reproducible A (left) AMP-PNP state; B (right) ADP.Pi state. Color code: IA (1–37, 109–180, 357–377; blue); IB (residues 38–108; green); IIA (residues 181–219, 307–356; purple) and IIB (residues 220–304; cyan). The reference structure for the NBD of DnaK-Tth was modeled on the NBD of DnaK E. coli (1DKG.pdb) and bovine Hsc70.ADP.Pi (3HSC.pdb). Scale: Horizontal: 20 ° per gridline, vertical, 10 ° per gridline. (The S_zz principal alignment axes also appear at the other side of the globe (120 °East, and 5 °North). The S_xx axes appear as smears around 150 ° West, 45 ° North and 30 °East, 45 °South.

Figure 3

Globe graphs (Sanson-Flamsteed plots) showing the orientations and experimental uncertainties of the S_zz principal alignment axes (around 60 °West and 5 °South) of the different subdomains of NBD of DnaK-Tth as derived from the NMR RDC measurements using REDCAT. A and B: RDC input: conserved residues and experimentally reproducible A (left) AMP-PNP state; B (right) ADP.Pi state. Color code: IA (1–37, 109–180, 357–377; blue); IB (residues 38–108; green); IIA (residues 181–219, 307–356; purple) and IIB (residues 220–304; cyan). The reference structure for the NBD of DnaK-Tth was modeled on the NBD of DnaK E. coli (1DKG.pdb) and bovine Hsc70.ADP.Pi (3HSC.pdb). Scale: Horizontal: 20 ° per gridline, vertical, 10 ° per gridline. (The S_zz principal alignment axes also appear at the other side of the globe (120 °East, and 5 °North). The S_xx axes appear as smears around 150 ° West, 45 ° North and 30 °East, 45 °South.

Figure 4

Model of the ADP state of the NBD of DnaK T.Th (RED) superposed on domain IA of the model for the AMPPNP state (BLUE) calculated using all RDCs

Figure 5

The average of the Tth-NBD self-validation ensemble for AMPPNP (blue) and Tth-NBD ADP (red) compared to the self-validation ensemble of Tth-NBD ADP (salmon)

Figure 6

Comparison of structures. The figures were made by superposing the CA-positions of the secondary structure elements of domains IA. A: Hsc70 bovine (3HSC.pdb, green) and NBD-TTh with AMPPNP (blue) B: Hsc70 bovine and NBD-TTh with ADP (red) C: Hsc70 bovine and Hsc70 bovine complexed with BAG (1HX1.pdb, orange; BAG is not shown) The figure was prepared with MacPyMOL.

Figure 6

Comparison of structures. The figures were made by superposing the CA-positions of the secondary structure elements of domains IA. A: Hsc70 bovine (3HSC.pdb, green) and NBD-TTh with AMPPNP (blue) B: Hsc70 bovine and NBD-TTh with ADP (red) C: Hsc70 bovine and Hsc70 bovine complexed with BAG (1HX1.pdb, orange; BAG is not shown) The figure was prepared with MacPyMOL.

Figure 6

Comparison of structures. The figures were made by superposing the CA-positions of the secondary structure elements of domains IA. A: Hsc70 bovine (3HSC.pdb, green) and NBD-TTh with AMPPNP (blue) B: Hsc70 bovine and NBD-TTh with ADP (red) C: Hsc70 bovine and Hsc70 bovine complexed with BAG (1HX1.pdb, orange; BAG is not shown) The figure was prepared with MacPyMOL.

Figure 7

Comparison of structures. The figures were made by superposing the CA-positions of the secondary structure elements of domains IA. A: Hsc70-bovine complexed with BAG (1HX1.pdb; orange; BAG not shown) and Hsp110. (1QXL.pdb; NBD of one monomer shown, in brown). B: DnaK-TTh-NBD-model in the AMPPNP state (blue) and DnaK-TTh-NBD-model in the ADP state (red). The figure was prepared with MacPyMOL.

Figure 7

Comparison of structures. The figures were made by superposing the CA-positions of the secondary structure elements of domains IA. A: Hsc70-bovine complexed with BAG (1HX1.pdb; orange; BAG not shown) and Hsp110. (1QXL.pdb; NBD of one monomer shown, in brown). B: DnaK-TTh-NBD-model in the AMPPNP state (blue) and DnaK-TTh-NBD-model in the ADP state (red). The figure was prepared with MacPyMOL.

Figure 8

View of the IA / IIA interface of DnaK-TTh. This view is a 90 degree rotation around the horizontal axis as compared to the other figures. It shows the “bottom” as compared to these other figures. A: in the ADP state. B: in the AMPPNP state Color coding: In domain IA (left) hydrophobics, light-green; positive, light-blue; negative, purple; polar, grey In domain IIA (right) hydrophobics, green; positive, blue; negative, red; polar, white The C-terminus (residue 372 in this construct) is rendered in cyan. Domain IB (at left top) is in grey; domain IIB (at right top) is in black.

Figure 8

View of the IA / IIA interface of DnaK-TTh. This view is a 90 degree rotation around the horizontal axis as compared to the other figures. It shows the “bottom” as compared to these other figures. A: in the ADP state. B: in the AMPPNP state Color coding: In domain IA (left) hydrophobics, light-green; positive, light-blue; negative, purple; polar, grey In domain IIA (right) hydrophobics, green; positive, blue; negative, red; polar, white The C-terminus (residue 372 in this construct) is rendered in cyan. Domain IB (at left top) is in grey; domain IIB (at right top) is in black.