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. 2015 Dec 1;23(12):2191-2203.
doi: 10.1016/j.str.2015.10.012. Epub 2015 Nov 19.

Close and Allosteric Opening of the Polypeptide-Binding Site in a Human Hsp70 Chaperone BiP

Jiao Yang  1 , Melesse Nune  1 , Yinong Zong  1 , Lei Zhou  1 , Qinglian Liu  2
Affiliations

Close and Allosteric Opening of the Polypeptide-Binding Site in a Human Hsp70 Chaperone BiP

Jiao Yang et al. Structure. .

Abstract

Binding immunoglobulin protein (BiP), an essential and ubiquitous Hsp70 chaperone in the ER, plays a key role in protein folding and quality control. BiP contains two functional domains: a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD). NBD binds and hydrolyzes ATP; the substrates for SBD are extended polypeptides. ATP binding allosterically accelerates polypeptide binding and release. Although crucial to the chaperone activity, the molecular mechanisms of polypeptide binding and allosteric coupling of BiP are poorly understood. Here, we present crystal structures of an intact human BiP in the ATP-bound state, the first intact eukaryotic Hsp70 structure, and isolated BiP-SBD with a peptide substrate bound representing the ADP-bound state. These structures and our biochemical analysis demonstrate that BiP has a unique NBD-SBD interface that is highly conserved only in eukaryotic Hsp70s found in the cytosol and ER to fortify its ATP-bound state and promote the opening of its polypeptide-binding pocket.

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Figures

Figure 1
Figure 1. Constructs for crystallization of a full-length BiP and its isolated SBD
a, BiP binds NR peptide through its SBD and in an ATP-sensitive manner. Fluorescence polarization assay with serial dilutions of BiP proteins was used to measure NR binding. DnaK was used as a positive control. b, Peptide NR binding affinities for DnaK and BiP proteins. Dissociation constants (Kd) were calculated based on the results in a. Standard errors were calculated from at least 6 assays on more than two protein purifications. c, Schematics of BiP domain structure and the construct for crystallization of a full-length BiP. The coloring of domains is: NBD (blue), Linker (purple), SBDβ (green), and SBDα (red). The signal sequence (first 24 residues) and the last 20 residues are not colored. The residue numbers marking domains are labeled on the top. d, Schematics of the BiP-SBD constructs for crystallization. Domain coloring is the same as in c. The Tev linker and linked NR peptide are shown as an orange line and in cyan, respectively. e, Neither BiP SBD-Tev–NR nor SBD-L3,4′-Tev-NR showed appreciable binding to NR peptide. WT BiP SBD was used as a positive control. Binding assay was carried out as in a. f, BiP-T229A mutant binds NR peptide in an ATP-sensitive manner like that of WT BiP, and L3,4′ modification drastically compromised the NR peptide binding. WT BiP was used as a positive control. Binding assay was carried out as in a.
Figure 2
Figure 2. Structural analysis of isolated BiP-SBD structures
a, Ribbon diagrams of the BiP SBD-Tev–NR structure. Domain coloring is the same as Fig. 1c with the TEV linker and NR peptide shown in blue and cyan, respectively. b, Comparison of the BiP SBD-Tev–NR structure with the DnaK SBD structure (PDB code: 1DKZ). The coloring of BiP SBD-Tev–NR is the same as in a. The DnaK SBD structure is shown in orange with the bound NR peptide in purple. The structures were superimposed based on Cα atoms of SBDβ. c, Comparison of the bound NR peptide in the BiP SBD-Tev–NR (top) and DnaK SBD (bottom; PDB code:1DKZ) structures. The two structures were superimposed as in b. The side-chains of NR peptides are highlighted in stick presentation, and the carbon atoms of the NR peptide are colored in grey and orange for the BiP SBD-Tev–NR and DnaK SBD structures, respectively. d, Y570 and R492 form novel hydrophobic contacts with L4 of bound NR peptide in the BiP-SBD structure. The BiP-SBD structure is shown in ribbon representation and colored as in a. L4 (NR peptide), V429, R492 and Y570 are highlighted as sticks. e, Ribbon diagram of the BiP SBD-L3,4′-Tev-NR structure. Domain coloring is the same as in Fig. 1c with the Tev linker and NR peptide in blue and cyan, respectively. f, Superposition of BiP SBD-L3,4′-Tev-NR (purple) with BiP SBD-Tev–NR (domain coloring is the same as in a) based on Cα atoms of SBDβ. g, Superposition of the NR peptides in BiP SBD-L3,4′-Tev-NR (purple) and BiP SBD-Tev–NR (cyan). The two structures were superimposed as in f. The side-chains are shown in stick representation with carbon atoms shown in orange for SBD-L3,4′-Tev-NR and grey for SBD-Tev–NR. h, Comparison of NR-contacting residues (in stick representation) between BiP SBD-Tev–NR (orange) and SBD-L3,4′-Tev-NR (green). The two structures were superimposed based on Cα atoms of SBDβ.
Figure 3
Figure 3. Overall structure of BiP-ATP
a, Ribbon diagram of human BiP-ATP structure. Domain coloring is the same as in Fig. 1c. The bound ATP is in stick presentation, and its associating Zn ion is shown as a grey ball. Left, the classic front-face view of NBD; right, view orthogonal to that of the left panel. b, Comparison of human BiP-ATP structure with our previously published DnaK-ATP (PDB code: 4JNE) structure. The domain coloring of BiP-ATP is the same as a. The domain coloring for DnaK-ATP: NBD (cyan), Linker (orange), SBDβ (yellow) and SBDα (grey). The superposition was based on Cα atoms. The view point is the same as the right panel of a. c, Superposition of SBDβ domains from the BiP-ATP (green) and DnaK-ATP (yellow) structures. The loops are labeled. The superposition was based on Cα atoms. d, A unique hydrogen bond was formed between D483 and D529 in the BiP-ATP structure. D483, D529, T527, N528 and Q530 are shown in stick presentation. Hydrogen bonds are shown as dotted lines.
Figure 4
Figure 4. The unique NBD-SBDα interface and NBD-Lα,β contact in BiP-ATP
a–c, Ribbon diagrams of NBD-SBDα interfaces in the BiP-ATP (a), DnaK-ATP (PDB code: 4JNE) (b), and Sse1-ATP (PDB code: 2QXL) (c) structures. NBDs are in blue and SBDαs are in red. Residues forming the two clusters of contacts are shown in stick presentation. Residues labeled in green are highly conserved between BiP-ATP and DnaK-ATP; residues labeled in orange are conserved between BiP-ATP and Sse1-ATP. d, Sequence alignment among Hsp70s. Secondary structure assignments are labeled on the top with cylinder for helix and arrow for strand. R532 and F548 are highlighted in red and green, respectively. h, human; d, Drosophila melanogaster; b, bovine; v, Virgibacillus halodenitrificans. DnaK is from E.coli. Kar2, Ssa1 and Ssc1 are from saccharomyces cerevisiae. e, The unique contact of NBD-Lα,β in the BiP-ATP structure. R532 forms two hydrogen bonds with D178 on NBD (blue). SBDβ and SBDα are in green and red, respectively. f, Fluorescence anisotropy assay of NR peptide binding affinity for BiP R532E mutant. WT BiP was used as a control. Assays were carried out as in Fig. 1 in the presence of ATP (+ATP) or ADP (+ADP). g, BiP R532E has a defect in releasing NR peptide upon addition of ATP. BiP proteins were incubated with F-NR peptide in the presence of ADP. After binding reached equilibrium, ATP was added (indicated by an arrow), and the release of F-NR was monitored over time.
Figure 5
Figure 5. Comparisons of the BiP-ATP structure with the isolated domain structures
a, Comparison of the NBD from the BiP-ATP structure with the isolated BiP NBD structure in complex with ADP (3IUC). Subdomain coloring for NBD of BiP-ATP is: Lobe I (blue), and Lobe II (red); for isolated BiP NBD structure: Lobe I (brown), and Lobe II (green). Left panel, the classic front-face view; right panel, top view of the left panel. NBDs are in backbone worm representation and superimposed on the basis of Lobe I Cα positions. b, Superposition of the BiP-ATP structure to the BiP SBD-Tev–NR structure based on the Cα positions of SBDβ. Domain coloring for BiP-ATP is the same as Fig. 3a. BiP SBD-Tev–NR is colored in orange with the NR peptide highlighted in cyan. c, Superposition of the BiP-ATP structure to the BiP SBD-L3,4′-Tev-NR structure based on the Cα positions of SBDβ. Domain coloring for BiP-ATP is the same as Fig. 3a. BiP SBD-L3,4′-Tev-NR is colored in purple with the NR peptide highlighted in cyan. d,e, Close-up view of b and c, respectively. Only SBDβ domains are shown. The Cα atoms of R492 are shown as blue spheres. f–h, Comparisons of polypeptide-binding site conformations. The polypeptide-binding site for BiP-ATP (f), superposition of f with BiP SBD-Tev–NR structure (g), and superposition of f with BiP SBD-L3,4′-Tev-NR structure (h) are shown in backbone worms representations. The superposition is based on Cαs in L1,2 and L4,5. Residues that form van der Waals contacts with NR peptide in BiP SBD-Tev–NR and SBD-L3,4′-Tev-NR are highlighted in stick representation.
Figure 6
Figure 6. The importance of two conserved glycine residues on L5,6 of Hsp70s
a, Peptide binding affinity determined by fluorescence polarization assay. Assays were carried out in the presence of ADP (+ADP) or ATP (+ATP). b, Fluorescence anisotropy assay of peptide substrate binding kinetics. The binding reactions of F-NR peptide were carried out in the presence of either ADP (+ADP) or ATP (+ATP), and the measurements were started right after mixing F-NR with the indicated protein. c–g, NR peptide and Hsp40 stimulation of BiP and DnaK in a single turn-over ATPase assay. Fold of stimulation was calculated by setting the intrinsic ATPase activity as 1. c, NR peptide failed to stimulate the ATPase activity of the BiP-PP mutant. d, f, Neither ERdj3 (d) nor DnaJ (f) showed appreciable stimulation on the ATPase activity of the BiP-PP mutant. e, g, The DnaK-PP protein manifested significant stimulation by both DnaJ (e) and ERdj3 (g).
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