Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP

Abstract

The 70-kilodalton (kDa) heat-shock proteins (Hsp70s) are ubiquitous molecular chaperones essential for cellular protein folding and proteostasis. Each Hsp70 has two functional domains: a nucleotide-binding domain (NBD), which binds and hydrolyzes ATP, and a substrate-binding domain (SBD), which binds extended polypeptides. NBD and SBD interact little when in the presence of ADP; however, ATP binding allosterically couples the polypeptide- and ATP-binding sites. ATP binding promotes polypeptide release; polypeptide rebinding stimulates ATP hydrolysis. This allosteric coupling is poorly understood. Here we present the crystal structure of an intact ATP-bound Hsp70 from Escherichia coli at 1.96-Å resolution. The ATP-bound NBD adopts a unique conformation, forming extensive interfaces with an SBD that has changed radically, having its α-helical lid displaced and the polypeptide-binding channel of its β-subdomain restructured. These conformational changes, together with our biochemical assays, provide a structural explanation for allosteric coupling in Hsp70 activity.

Figure 1. Characteristics of DnaK locked in an ATP-bound state

a, Schematic of Hsp70 domain structure. Structured subdomains are given color assignments and positions of key elements are identified. b, Ribbon representation of the DnaK SBD- structure. Domain coloring is as in a. stands for loop. The Linker segment from a symmetry mate (in purple) binds to the polypeptide-binding site between and . c, Superposition of SBDβ from the DnaK SBD- structure (colored as in b) with that of the WT SBD structure (orange; PDB 1DKZ). The peptide substrate NR in the WT SBD structure is colored in cyan. d, Tryptophan-fluorescence emission spectra for assaying the ATP-induced allosteric coupling of DnaK.

Figure 2. Overall structure of ATP-bound DnaK (DnaK-ATP)

Domain coloring in the ribbon diagrams is as in Fig. 1a throughout. a, Ribbon diagrams of the DnaK dimer. Left: View with dyad axis (orange rod) vertical. Domains are labeled for protomer A; protomer B is on the right. Right: Orthogonal view from below, along the dyad axis (orange dot). b, Ribbon diagram of a DnaK protomer in the canonical, front-face NBD view (left) and in an orthogonal side view (right). The NBD lobes are labeled as I and II. The bound ATP molecule is in stick representation. c, Comparison with a DnaK-ADP structure. Left: ribbon diagram of the DnaK-ADP structure in the front-face NBD view (PDB 2KHO). Right: Side view of DnaK-ATP as in Fig. 2b right panel, but in backbone worm representation and superimposed onto DnaK-ADP based on NBD Lobe-I Cαs. Domain coloring is NBD from DnaK-ATP (blue), SBD from DnaK-ATP (red), NBD from DnaK-ADP (cyan) and SBD from DnaK-ADP (green). d, Comparison with the Sse1-ATP structure. Left: ribbon diagram of the Sse1-ATP structure in the front-face NBD view (PDB 2QXL). Right: Side view of DnaK-ATP as in Fig. 2b right panel, but in backbone worm representation and superimposed onto Sse1-ATP based on NBD Lobe-I Cαs. Domain coloring is NBD from DnaK-ATP (blue), SBD from DnaK-ATP (red), NBD from Sse1-ATP (cyan) and SBD from Sse1-ATP (green).

Figure 3. Unique conformations of NBD and SBD in DnaK-ATP

a, Comparison of NBD conformations in DnaK-ATP and human Hsp70-ADP (PDB 1S3X). NBDs are superimposed based on Cαs in lobe-II. Coloring has in DnaK-ATP Lobe-I (red) and Lobe-II (blue); human Hsp70-ADP Lobe-I (grey) and Lobe-II (green). The left panel shows the canonical front-face view, and the right panel shows the view from above the left panel with IB and IIB removed to show the IA-IIA changes. b, Comparison of DnaK-ATP and Sse1-ATP. Superpositions and coloring are as in a, except for replacement of human Hsp70-ADP by Sse1-ATP. Conformational differences between the two ATP-bound structures are slight. c, Comparison of SBDβ conformations. The left and middle panels are ribbon diagram of SBDβs from the DnaK-ATP and the isolated DnaK-SBD (PDB 1DKZ) structures, respectively. SBDβs are superimposed based on Cαs in and . Glycine residues Gly461 and Gly468 on loop are highlighted as purple and black balls, respectively. The Cα atoms of Arg467 are highlighted as orange balls. The peptide substrate NR is in cyan. The right panel is the superposition of the left and middle panels. d, Comparison of the polypeptide-binding sites. The left and middle panels are the details of the polypeptide-binding sites in the DnaK-ATP and isolated DnaK-SBD structures, respectively. The two structures are superimposed based on Cαs in and . Residues in van der Waals contacts with the three leucine residues of the NR peptide in the isolated DnaK-SBD structure are shown in sticks. The backbone of the NR peptide is in cyan, and the side chains of the three leucine residues in the NR peptide are highlighted in orange. The NR peptide in the left panel is from the isolated DnaK-SBD structure. The right panel is the superposition of the left and middle panels. The side chains of the NR peptide are not shown.

Figure 4. Two highly conserved glycine residues in

a, Comparison of phi and psi conformational angles for Gly461 and Gly468 in DnaK-ATP and isolated DnaK-SBD structures. b, Growth tests for glycine mutants in Hsp70-deficient E. coli and yeast backgrounds. Serial dilutions of fresh cultures were spotted onto plates and incubated at 37°C. Functional DnaK and Ssa1 are required for growth at 37°C. c, Refolding activity of WT and G461P G468P (PP) mutant DnaK. Refolding of the heat-denatured luciferase was started by adding the DnaK chaperone system (DnaK, DnaJ and GrpE) for the corresponding WT or PP DnaK. d, Fluorescence anisotropy assay of peptide substrate binding affinity. Serial dilutions of DnaK proteins were incubated with F-NR, a model peptide substrate. Fluorescence anisotropy measurements were made after binding reached equilibrium. e, Peptide substrate binding kinetics determined by fluorescence anisotropy assay. Immediately after mixing DnaK proteins with the F-NR peptide, peptide binding as a function of time was measured kinetics by fluorescence anisotropy.

Figure 5. Sequence conservation at domain interfaces in DnaK-ATP

The top panels are sequence conservation at the surface of NBD (a) and SBD (b). Conservation and surface mapping was calculated by ConSurf (http://consurf.tau.ac.il/). The conservation key is shown above b. The dotted yellow circle in b locates the polypeptide-binding site. The bottom panels are mappings of domain interfaces onto the surfaces of NBD (a) and SBD (b). In the bottom panel of a, the surface of NBD is blue, and the imprints of contacts are colored by domain: SBDα (red), SBDβ (green), Linker (purple). In the bottom panel of b, The surfaces of Linker, SBDβ and SBDα are purple, green, and red, respectively, and the imprints of contacts from NBD are colored by the sub-domain receiving that contact: SBDβ (blue), SBDα (marine), and Linker (purple, as it is buried entirely into the interface with NBD).

Figure 6. Characteristics of domain interfaces in DnaK-ATP

a, Identification of NBD-contacting loops from SBDβ (upper left; domain coloring as in Fig. 1a), and associated details of the individual contacts as labeled. Residues targeted for mutagenesis are featured in the details. Residues that were tested in the Sse1-ATP structure paper are labeled in black. b, Growth tests on interfacial mutant variants. Serial dilutions of fresh cultures, transformed with the respective mutant genes, were spotted onto plates and grown at 37°C. c, ATP-induced tryptophan-fluorescence shifts. Shifts in the maximum of Trp102 fluorescence are shown in the absence of peptide substrates (black bars) and after addition of 30μM substrate peptide NR (red bars). For each protein, the blue shift was averaged from five independent measurements from at least two purifications. Error bars are standard errors (n=5).

Figure 7. Schematic model for allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP

Hsp70 domain coloring is the same as in Fig. 1a. Polypeptide substrates are highlighted in black.