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. 2021 Oct 12;118(41):e2016962118.
doi: 10.1073/pnas.2016962118.

Selective promiscuity in the binding of E. coli Hsp70 to an unfolded protein

Eugenia M Clerico  1 , Alexandra K Pozhidaeva  1 , Rachel M Jansen  1 , Can Özden  1   2 , Joseph M Tilitsky  3 , Lila M Gierasch  4   3
Affiliations

Selective promiscuity in the binding of E. coli Hsp70 to an unfolded protein

Eugenia M Clerico et al. Proc Natl Acad Sci U S A. .

Abstract

Heat shock protein 70 (Hsp70) chaperones bind many different sequences and discriminate between incompletely folded and folded clients. Most research into the origins of this "selective promiscuity" has relied on short peptides as substrates to dissect the binding, but much less is known about how Hsp70s bind full-length client proteins. Here, we connect detailed structural analyses of complexes between the Escherichia coli Hsp70 (DnaK) substrate-binding domain (SBD) and peptides encompassing five potential binding sites in the precursor to E. coli alkaline phosphatase (proPhoA) with SBD binding to full-length unfolded proPhoA. Analysis of SBD complexes with proPhoA peptides by a combination of X-ray crystallography, methyl-transverse relaxation optimized spectroscopy (methyl-TROSY), and paramagnetic relaxation enhancement (PRE) NMR and chemical cross-linking experiments provided detailed descriptions of their binding modes. Importantly, many sequences populate multiple SBD binding modes, including both the canonical N to C orientation and a C to N orientation. The favored peptide binding mode optimizes substrate residue side-chain compatibility with the SBD binding pockets independent of backbone orientation. Relating these results to the binding of the SBD to full-length proPhoA, we observe that multiple chaperones may bind to the protein substrate, and the binding sites, well separated in the proPhoA sequence, behave independently. The hierarchy of chaperone binding to sites on the protein was generally consistent with the apparent binding affinities observed for the peptides corresponding to these sites. Functionally, these results reveal that Hsp70s "read" sequences without regard to the backbone direction and that both binding orientations must be considered in current predictive algorithms.

Keywords: DnaK; Hsp70 molecular chaperone; NMR; crystallography; substrate binding.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
How the DnaK SBD binds a model peptide. (A) The crystal structure of the SBD of DnaK (gray) bound to the model peptide NR (NRLLLTG; maroon; PDB ID code 1DKZ) showing the peptide bound to a cleft on the β-subdomain covered by the α-helical lid (11). (B) Top view of the SBD substrate binding cleft showing the mode of binding of NR to the five pockets created by the topography of this domain. Note in particular the deep 0th pocket here occupied by L4. The SBD Ile401 (red) and Ile438 (green) are shown as spheres. Residues 507 to 603 and residues 404 to 429 are not shown to better visualize the bound peptide. (Structures here and elsewhere are depicted using PyMol [Schrodinger LCC].) NBD, nucleotide binding domain.
Fig. 2.
Fig. 2.
Multiple sequences in proPhoAS4 are bound by the DnaK SBD. The Ile401 and Ile438 region of 1H-13C-HMQC spectra of ILV-13CH3–labeled DnaK SBD in the presence of unlabeled, unfolded proPhoAS4 at low (Left) and high (Right) chaperone to substrate ratios showing several resonances arising from proPhoAS4 binding.
Fig. 3.
Fig. 3.
Potential DnaK binding sites on proPhoA and designed peptide models. (A) Schematic of the proPhoA sequence with strong DnaK binding sites identified from a proPhoA peptide array (9) labeled as A, B, C, D, and E and colored in red, orange, green, blue, and magenta, respectively (this color code is used in all figures). (B) Model peptides and their SBD apparent binding affinities (KD) (SI Appendix, Fig. S4). (Underlined residues are not the part of proPhoA sequence.)
Fig. 4.
Fig. 4.
Peptides containing proPhoA sites C and D bind to the DnaK SBD in opposite orientations. (A and B) Crystal structures (top view) of the SBD (gray) in complex with proPhoA peptide C (A), which binds in an N to C orientation (green), and Dsh (B), which binds in a C to N orientation (blue). Residues 507 to 603 are not shown. The side chains of SBD residues contacting the peptide are in red sticks, and hydrogen bonds between the peptide backbone and the SBD are in yellow. (C) Schematic showing that the bound peptide interacts with the β3-strand of the SBD (magenta) in a parallel or antiparallel strand–strand arrangement for the N to C [Left; peptide in maroon, PDB ID code 1DKZ (11)] or the C to N binding mode [Right; peptide in blue, PDB ID code 4EZY (12)]. (D) The Ile401 and Ile438 region of 1H-13C-HMQC spectra of ILV-13CH3–labeled DnaK SBD in the presence of unlabeled peptides C (green) and D (blue). (Peptides and SBD are at 40 μM.) (E) PRE NMR of SBD complexes with CysC and CysD peptides. SBD residues significantly broadened in the presence of spin-labeled peptides are mapped on the SBD structure (in green, CysC; in blue, CysD). Residues for which no data are available are shown in white. (F) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of SBDCys425 and SBDCys458 (cysteine residues in magenta and yellow spheres in the structures in Left) cross-linked to CysC and CysD peptides.
Fig. 5.
Fig. 5.
Peptides containing proPhoA sites E and B both populate more than one binding mode, one N to C, and another minor state, C to N. (A) Crystal structure of the DnaK SBD (gray) complexed with proPhoA peptide E (magenta). (B) The Ile401 and Ile438 region of 1H-13C-HMQC spectra of ILV-13CH3 ILV-13CH3-DnaK SBD in the presence of unlabeled peptide E (magenta). Two binding modes are populated; the minor C to N binding mode is denoted E’ (SI Appendix, Fig. S8). (C) The Ile401 and Ile438 region of 1H-13C-HMQC spectra of ILV-13CH3 DnaK SBD in the presence of unlabeled peptides B (orange), BshN (blue), and BshC (black). The major resonances for peptide B represent an N to C binding mode with I50 in the central pocket; the minor resonances can be assigned to C to N binding mode with I46 in the 0th pocket (SI Appendix, Fig. S9).
Fig. 6.
Fig. 6.
The peptide containing proPhoA site A spans an SBD dimer in crystals and in solution binds 1:1 to the SBD, populating more than one binding mode. (A) Crystal structure of the DnaK SBD in complex with peptide A. Two SBDs (green and gray) are aligned at the twofold axis, and peptide A (red sticks) extends through both SBDs with two sequences in the binding clefts, one in an N to C and one in a C to N backbone direction. (B) The Ile401 and Ile438 region of 1H-13C-HMQC spectra of ILV-13CH3–labeled DnaK SBD in the presence of unlabeled peptides A (red), AshN (blue), and AshC (black). The binding modes were assigned as 1) N to C with a leucine in the central pocket and 2) C to N with P(−10) in the central pocket. (C) Competition binding assays for peptides A, AshN, and AshC to the SBD bound to a fluorescein isothiocyanate (FITC)-labeled peptide (SI Appendix, SI Materials and Methods and Fig. S10).
Fig. 7.
Fig. 7.
From peptide to protein, the DnaK SBD binds multiple sites on proPhoA with differing affinities, largely consistent with studies of peptides, and one previously unidentified high-affinity site. (A) Titration of unlabeled proPhoAS4 with increasing amounts of ILV-13CH3 DnaK SBD. The Ile401 and Ile438 resonances of the SBD in complex with proPhoAS4 are shown in blue, and resonances for complexes with the proPhoA peptides A to E are color coded as in Fig. 3. At a 1:1 ratio of SBD to proPhoAS4, the resonances for the protein complex coincide with those for peptide A (red), and an additional resonance is seen (labeled U) that does not correspond to any of the peptides tested. At a 3:1 ratio of SBD to proPhoAS4, additional resonances appear that overlay on those for the SBD complexes with peptides C and D and major species for complexes with peptides B and E. Finally, at a 4:1 ratio of SBD to proPhoAS4, all the resonances seen for the peptides appear. (B) SDS-PAGE of SBDCys425 cross-linked to proPhoAS4 using Sulfo-GMBS. Addition of the cross-linker to SBD or proPhoAS4 alone does not result in higher–molecular mass species (lanes 1 and 2). At a 1:1 ratio of SBD to proPhoAS4, a 1:1 complex is observed (lane 3), while at a 5:1 ratio of SBD to proPhoAS4, complexes with multiple SBD bound to one proPhoA molecule are seen. (Controls are shown in SI Appendix, Fig. S13.) (C) Summary of DnaK SBD binding sites on proPhoAS4, their occupancy of SBD binding pockets, and their orientation. The prime symbol indicates an alternative minor binding mode. (D) Cartoon depicting DnaK binding to proPhoA. Site A, which has the highest binding affinity to DnaK, is occupied by the chaperone at the lowest chaperone to substrate ratios and remains bound as the chaperone to substrate ratio increases. Other binding sites on proPhoA are occupied based largely on the affinities they manifest as peptides but in a dynamic manner, with several or all of them occupied by chaperone simultaneously.
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References

    1. Balchin D., Hayer-Hartl M., Hartl F. U., In vivo aspects of protein folding and quality control. Science 353, aac4354 (2016). - PubMed
    1. Rosenzweig R., Nillegoda N. B., Mayer M. P., Bukau B., The Hsp70 chaperone network. Nat. Rev. Mol. Cell Biol. 20, 665–680 (2019). - PubMed
    1. Clerico E. M., et al. ., Hsp70 molecular chaperones: Multifunctional allosteric holding and unfolding machines. Biochem. J. 476, 1653–1677 (2019). - PMC - PubMed
    1. Mayer M. P., Intra-molecular pathways of allosteric control in Hsp70s. Philos. Trans. R. Soc. Lond. B Biol. Sci. 373, 20170183 (2018). - PMC - PubMed
    1. Mayer M. P., Gierasch L. M., Recent advances in the structural and mechanistic aspects of Hsp70 molecular chaperones. J. Biol. Chem. 294, 2085–2097 (2019). - PMC - PubMed

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