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PreprintCited by 0Heat shock proteins researchProtein Structure and DynamicsBacterial Genetics and Biotechnology

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The nucleotide exchange factor, GrpE, modulates substrate affinity by interaction of its N-terminal tails with the DnaK substrate-binding domain

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Maqtedar, Akshitha, Rossi, Maria-Agustina, Clerico, Eugenia M., Williams, Robert V., Gierasch, Lila M.·2025·Journal of Biological Chemistry
DOI 10.1101/2025.10.21.683677PMC PMC12915176

Preprint — published as 10.1016/j.jbc.2026.111176 · Paper page

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Abstract

The 70-kDa heat shock proteins (Hsp70s) assist in protein folding through allosteric communication between their nucleotide-binding domains (NBDs) and substrate-binding domains (SBDs), which are connected by an interdomain linker. Their nucleotide-dependent allosteric cycle is modulated by ligand binding and co-chaperones, including nucleotide exchange factors (NEFs). GrpE, the NEF for the E. coli Hsp70, DnaK, has been proposed to have a dual effect on the chaperone, facilitating the exchange of ADP for ATP in the NBD in a temperature-dependent fashion and promoting substrate release from the SBD. We recently reported NMR-based evidence that GrpE binding to DnaK has a direct structural effect on the SBD. Here, we expanded on these findings and obtained new evidence for a model in which the disordered N-terminal tails of GrpE facilitate peptide dissociation from the nucleotide-free DnaK/GrpE complex by transiently binding to the canonical substrate-binding site in the SBD. This GrpE/SBD interaction, while weak, is favored by the high local concentration of the tails around the SBD after complex formation. Moreover, we identified the DnaK binding motif in GrpE’s N-terminal disordered tails as 17 IIM 19 , which is conserved across many bacterial species. Excitingly, our data further suggest a mechanism for the temperature-dependence of GrpE’s modulation of DnaK’s refolding activity: as the temperature increases, unfolding of GrpE’s coiled-coil weakens its contacts with the SBD, reducing N-terminal tail binding, and thus increasing DnaK affinity to substrates.

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Figures

Figure 1 AlphaFold2-predicted Escherichia coli DnaK–GrpE complex reveals conserved interaction modes. The AlphaFold2-predicted structure of the E. coli DnaK–GrpE complex is shown in the center, and a close-up view of the interaction interface between DnaK SBD and GrpE coiled-coil domain is illustrated on the right and left. GrpE is highlighted in white, and in the Hsp70, the NBD is in red, the SBD is in green, and the interdomain linker is in blue. The left panel shows a zoomed view highlighting contacting residues between the GrpE coiled-coil (light gray) and DnaK SBD (green), whereas the right panel shows the same view with conserved GrpE residues indicated in red. DnaK, 70-kDa heat shock protein of prokaryote; GrpE, nucleotide exchange factor of prokaryote; Hsp70, 70-kDa heat shock protein; NBD, nucleotide-binding domain; SBD, substrate-binding domain.
Figure 1 AlphaFold2-predicted Escherichia coli DnaK–GrpE complex reveals conserved interaction modes. The AlphaFold2-predicted structure of the E. coli DnaK–GrpE complex is shown in the center, and a close-up view of the interaction interface between DnaK SBD and GrpE coiled-coil domain is illustrated on the right and left. GrpE is highlighted in white, and in the Hsp70, the NBD is in red, the SBD is in green, and the interdomain linker is in blue. The left panel shows a zoomed view highlighting contacting residues between the GrpE coiled-coil (light gray) and DnaK SBD (green), whereas the right panel shows the same view with conserved GrpE residues indicated in red. DnaK, 70-kDa heat shock protein of prokaryote; GrpE, nucleotide exchange factor of prokaryote; Hsp70, 70-kDa heat shock protein; NBD, nucleotide-binding domain; SBD, substrate-binding domain.CC BY · PubMed Central
Figure 2 DnaK and GrpE homologs exhibit sequence conservation in the interaction interfaces of the DnaK–GrpE complex. A, sequence alignment of DnaK from Mycobacterium tuberculosis, human (mortalin, HSPA9), Escherichia coli, and Geobacillus kaustophilus. Residues in the interdomain linker sequence are highlighted in blue. SBD loop 1 residues are shown in green (E. coli K414–P419, G. kaustophilus R382–P388, H. sapiens mortalin HSPA9 R460–P465, and M. tuberculosis R388–P393). The residues that contact the GrpE coiled-coil domain are highlighted in red: Asn (E. coli N415, G. kaustophilus N383, mortalin HSPA9 N461, and M. tuberculosis N389), Thr (E. coli T417, G. kaustophilus T385, mortalin HSPA9 T463, and M. tuberculosis N391), and Asp (E. coli D477, G. kaustophilus D446, mortalin HSPA9 D523, and M. tuberculosis D451). B, sequence alignment of GrpE homologs where conserved residues are highlighted (hydrophobic in green, polar in light blue, negatively charged in red, positively charged in blue, and aromatic in yellow). The (1) L63–R75 region in the coiled-coil domain that interacts with DnaK is highlighted with a red box; the (2) I91–D110 region that contacts DnaK NBD subdomain IB; and the (3) L148–I157 region that interacts with DnaK NBD subdomain IIB are highlighted with black boxes. The numbering in this caption corresponds to the E. coli GrpE. C, sequence alignment for the N-terminal disordered tails of GrpE homologs. The only sequence similarity region in the N-terminal tails is highlighted (hydrophobic residues in green, negatively charged ones in red). DnaK, 70-kDa heat shock protein of prokaryote; GrpE, nucleotide exchange factor of prokaryote; NBD, nucleotide-binding domain; SBD, substrate-binding domain.
Figure 2 DnaK and GrpE homologs exhibit sequence conservation in the interaction interfaces of the DnaK–GrpE complex. A, sequence alignment of DnaK from Mycobacterium tuberculosis, human (mortalin, HSPA9), Escherichia coli, and Geobacillus kaustophilus. Residues in the interdomain linker sequence are highlighted in blue. SBD loop 1 residues are shown in green (E. coli K414–P419, G. kaustophilus R382–P388, H. sapiens mortalin HSPA9 R460–P465, and M. tuberculosis R388–P393). The residues that contact the GrpE coiled-coil domain are highlighted in red: Asn (E. coli N415, G. kaustophilus N383, mortalin HSPA9 N461, and M. tuberculosis N389), Thr (E. coli T417, G. kaustophilus T385, mortalin HSPA9 T463, and M. tuberculosis N391), and Asp (E. coli D477, G. kaustophilus D446, mortalin HSPA9 D523, and M. tuberculosis D451). B, sequence alignment of GrpE homologs where conserved residues are highlighted (hydrophobic in green, polar in light blue, negatively charged in red, positively charged in blue, and aromatic in yellow). The (1) L63–R75 region in the coiled-coil domain that interacts with DnaK is highlighted with a red box; the (2) I91–D110 region that contacts DnaK NBD subdomain IB; and the (3) L148–I157 region that interacts with DnaK NBD subdomain IIB are highlighted with black boxes. The numbering in this caption corresponds to the E. coli GrpE. C, sequence alignment for the N-terminal disordered tails of GrpE homologs. The only sequence similarity region in the N-terminal tails is highlighted (hydrophobic residues in green, negatively charged ones in red). DnaK, 70-kDa heat shock protein of prokaryote; GrpE, nucleotide exchange factor of prokaryote; NBD, nucleotide-binding domain; SBD, substrate-binding domain.CC BY · PubMed Central
Figure 3 The binding of GrpE reduces the affinity of the DnaK SBD for a model peptide. A, apparent affinity of DnaK for the model peptide FITC-p5 in the absence or presence of 1 μM GrpE, GrpE33–197, and GrpE ASA at 22 °C. B, apparent affinity of DnaK for the model peptide FITC-p5 when the concentration of GrpE and GrpE33–197 was varied at 22 °C. DnaK, 70-kDa heat shock protein of prokaryote; GrpE, nucleotide exchange factor of prokaryote; SBD, substrate-binding domain.
Figure 3 The binding of GrpE reduces the affinity of the DnaK SBD for a model peptide. A, apparent affinity of DnaK for the model peptide FITC-p5 in the absence or presence of 1 μM GrpE, GrpE33–197, and GrpE ASA at 22 °C. B, apparent affinity of DnaK for the model peptide FITC-p5 when the concentration of GrpE and GrpE33–197 was varied at 22 °C. DnaK, 70-kDa heat shock protein of prokaryote; GrpE, nucleotide exchange factor of prokaryote; SBD, substrate-binding domain.CC BY · PubMed Central
Figure 4 DnaK SBD reporter NMR signals, I401 and I438, indicate which residues bind to the central pocket when complexed with GrpE and its variants. Ile resonances of the substrate-binding pocket on the HMQC at 25 °C of (left) 13C-methyl-labeled ILV-DnaK–GrpE complex (black) and isolated apo-DnaK (red), (middle) 13C-methyl-labeled ILV-DnaK–GrpE33–197 complex (blue) and isolated apo-DnaK (red), and (right)13C-methyl-labeled ILV-DnaK–GrpE complex (black), 13C-methyl-labeled ILV-DnaK–GrpE33–197 complex (blue), and 13C-methyl-labeled ILV-DnaK–GrpE ASA complex (orange). An arrow marks the I438 peak in the ILV-DnaK–GrpE complex spectrum. The residue marked with an asterisk in the apo-DnaK spectrum corresponds to residue I338. DnaK, 70-kDa heat shock protein of prokaryote; GrpE, nucleotide exchange factor of prokaryote; HMQC, heteronuclear multiple quantum correlation; SBD, substrate-binding domain.
Figure 4 DnaK SBD reporter NMR signals, I401 and I438, indicate which residues bind to the central pocket when complexed with GrpE and its variants. Ile resonances of the substrate-binding pocket on the HMQC at 25 °C of (left) 13C-methyl-labeled ILV-DnaK–GrpE complex (black) and isolated apo-DnaK (red), (middle) 13C-methyl-labeled ILV-DnaK–GrpE33–197 complex (blue) and isolated apo-DnaK (red), and (right)13C-methyl-labeled ILV-DnaK–GrpE complex (black), 13C-methyl-labeled ILV-DnaK–GrpE33–197 complex (blue), and 13C-methyl-labeled ILV-DnaK–GrpE ASA complex (orange). An arrow marks the I438 peak in the ILV-DnaK–GrpE complex spectrum. The residue marked with an asterisk in the apo-DnaK spectrum corresponds to residue I338. DnaK, 70-kDa heat shock protein of prokaryote; GrpE, nucleotide exchange factor of prokaryote; HMQC, heteronuclear multiple quantum correlation; SBD, substrate-binding domain.CC BY · PubMed Central
Figure 5 15N-GrpE NMR analysis supports transient binding of the GrpE N-terminal tails to the DnaK SBD. A, the 1H,15N-HSQC spectrum of 15N-GrpE (left panel) shows backbone amide resonances corresponding to disordered GrpE N-terminal tail residues. Resonance assignments are indicated, whereas unassigned peaks are labeled as ∗∗. An overlay (right) comparing equivalent spectra collected for the GrpE–DnaK complex (blue) and GrpE–DnaK–NBD (red) shows that the largest differences in peak position between the two complexes are observed for I17, I18, and E23 (indicated with arrows), whereas T8 and H22 experience more broadening in the GrpE–DnaK spectrum (indicated with asterisks). B, CSPs were measured for GrpE N-terminal amide signals in complex with DnaK (blue) or a truncated NBD (red) relative to those of isolated 15N-GrpE. B, top, bar plot comparing CSPs (Δδ) for 15N-GrpE in complex with full-length DnaK (blue) or the DnaK NBD1–392 (red) relative to 15N-GrpE alone. Missing CSPs correspond to proline residues (P9, P14, and P30) or shifts that could not be measured because of resonance overlap (A27, A34). Bottom,15N-{1H}-NOE values for GrpE (black), GrpE–DnaK (blue), and GrpE–DnaK–Ash2 peptide (green) complexes. Several NOEs are not indicated as there is a proline residue in the sequence (P9, P14, and P30), or there was resonance overlap resonance (A27 and A34), or there was insufficient intensity in the saturated spectrum (H22). CSP, chemical shift perturbation; DnaK, 70-kDa heat shock protein of prokaryote; GrpE, nucleotide exchange factor of prokaryote; HSQC, heteronuclear single quantum coherence; SBD, substrate-binding domain.
Figure 5 15N-GrpE NMR analysis supports transient binding of the GrpE N-terminal tails to the DnaK SBD. A, the 1H,15N-HSQC spectrum of 15N-GrpE (left panel) shows backbone amide resonances corresponding to disordered GrpE N-terminal tail residues. Resonance assignments are indicated, whereas unassigned peaks are labeled as ∗∗. An overlay (right) comparing equivalent spectra collected for the GrpE–DnaK complex (blue) and GrpE–DnaK–NBD (red) shows that the largest differences in peak position between the two complexes are observed for I17, I18, and E23 (indicated with arrows), whereas T8 and H22 experience more broadening in the GrpE–DnaK spectrum (indicated with asterisks). B, CSPs were measured for GrpE N-terminal amide signals in complex with DnaK (blue) or a truncated NBD (red) relative to those of isolated 15N-GrpE. B, top, bar plot comparing CSPs (Δδ) for 15N-GrpE in complex with full-length DnaK (blue) or the DnaK NBD1–392 (red) relative to 15N-GrpE alone. Missing CSPs correspond to proline residues (P9, P14, and P30) or shifts that could not be measured because of resonance overlap (A27, A34). Bottom,15N-{1H}-NOE values for GrpE (black), GrpE–DnaK (blue), and GrpE–DnaK–Ash2 peptide (green) complexes. Several NOEs are not indicated as there is a proline residue in the sequence (P9, P14, and P30), or there was resonance overlap resonance (A27 and A34), or there was insufficient intensity in the saturated spectrum (H22). CSP, chemical shift perturbation; DnaK, 70-kDa heat shock protein of prokaryote; GrpE, nucleotide exchange factor of prokaryote; HSQC, heteronuclear single quantum coherence; SBD, substrate-binding domain.CC BY · PubMed Central
Figure 6 GrpE modulates peptide release from apo-DnaK less at elevated temperatures. A, apparent affinity of DnaK for the model peptide FITC-p5 in the absence or presence of 1 μM GrpE and GrpE33–197 at 38 °C. B, Ile resonances of the substrate binding pocket on the HMQC of (left) 13C-methyl-labeled ILV-DnaK–GrpE complex (black) at 25 °C, 13C-methyl-labeled ILV-DnaK–GrpE complex (green) at 38 °C, and isolated apo-DnaK (red) at 25 °C and (right) 13C-methyl-labeled ILV-DnaK–GrpE33–197 complex (blue) at 25 °C, 13C-methyl-labeled ILV-DnaK–GrpE33–197 complex (pink) at 38 °C, and isolated apo-DnaK (red) at 25 °C. The residue marked with an asterisk in the apo-DnaK spectrum corresponds to residue I338. DnaK, 70-kDa heat shock protein of prokaryote; GrpE, nucleotide exchange factor of prokaryote; HMQC, heteronuclear multiple quantum correlation.
Figure 6 GrpE modulates peptide release from apo-DnaK less at elevated temperatures. A, apparent affinity of DnaK for the model peptide FITC-p5 in the absence or presence of 1 μM GrpE and GrpE33–197 at 38 °C. B, Ile resonances of the substrate binding pocket on the HMQC of (left) 13C-methyl-labeled ILV-DnaK–GrpE complex (black) at 25 °C, 13C-methyl-labeled ILV-DnaK–GrpE complex (green) at 38 °C, and isolated apo-DnaK (red) at 25 °C and (right) 13C-methyl-labeled ILV-DnaK–GrpE33–197 complex (blue) at 25 °C, 13C-methyl-labeled ILV-DnaK–GrpE33–197 complex (pink) at 38 °C, and isolated apo-DnaK (red) at 25 °C. The residue marked with an asterisk in the apo-DnaK spectrum corresponds to residue I338. DnaK, 70-kDa heat shock protein of prokaryote; GrpE, nucleotide exchange factor of prokaryote; HMQC, heteronuclear multiple quantum correlation.CC BY · PubMed Central

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Cites

Research works printed but not linked: 12

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Metadata

Authors
Maqtedar, A., Rossi, M.-A., Clerico, E. M., Williams, R. V., & Gierasch, L. M.
Affiliations
University of Massachusetts Amherst (US)
Corresponding author
Gierasch, Lila M.
ORCID
Venue
Journal of Biological Chemistry
Publisher
openRxiv
Published
2025-10-21
Volume
302
Issue
3
Pages
111176-111176
ISSN
0021-9258 (ISSN-L)
DOI
10.1101/2025.10.21.683677
PMC
PMC12915176
OpenAlex
W7125431941
Cited by
0
References
23
FWCI
0.00
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (submittedVersion, cc-by-nc-nd)
Funders
National Institutes of Health (GM18161, F32-GM156016)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Heat shock proteins research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
GrpE, DnaK, HSP70, nucleotide exchange factor, substrate-binding domain, substrate release, allosteric communication, temperature dependence, protein folding, Escherichia coli

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