Allosteric fine-tuning of the conformational equilibrium poises the chaperone BiP for post-translational regulation

  1. Lukasz Wieteska
  2. Saeid Shahidi
  3. Anastasia Zhuravleva  Is a corresponding author
  1. University of Leeds, United Kingdom
6 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Two main functional conformations of BiP.

Ribbon representation of the structure of the ATP-bound BiP (PDB 5e84 [(Yang et al., 2015]), referred as the domain-docked conformation, and ADP-bound, which was modeled from the structures of the …

https://doi.org/10.7554/eLife.29430.002
Figure 1—figure supplement 1
Sequence alignment of eukaryotic and bacterial Hsp70 proteins.

The aligned sequences of the βSBD, including human HSP70s: BiP (GRP78 or HSPA5), HSP70 (HSPA1A or HSP72), HSPA2, HSC70 (HSPA8 or HSP73), HSPA9 (GRP75, Mortalin-2 or MTHSP70), and HSPA6; Hsp70s from …

https://doi.org/10.7554/eLife.29430.003
Figure 2 with 1 supplement
NMR fingerprints of two main functional conformations of BiP.

The isoleucine region of methyl-TROSY spectra of ADP-bound (left) and ATP-bound (right) BiP* (the full-length ATPase deficient T229G BiP construct, in black) overlaid with the spectra of …

https://doi.org/10.7554/eLife.29430.005
Figure 2—figure supplement 1
NMR fingerprints of the main steps of the BiP allosteric cycle.

The isoleucine region of methyl-TROSY spectra of BiP* (the full-length ATPase deficient T229G BiP construct, in black) overlaid with the spectra of corresponding nucleotide-bound state of NBD* (the …

https://doi.org/10.7554/eLife.29430.006
ATP-induced linker binding to the BiP NBD.

(A) Ribbon representation of the structures of two BiP NBD conformations: linker-bound and linker-unbound. The ATP-bound NBD structure (grey, linker bound; the linker is shown in yellow) is taken …

https://doi.org/10.7554/eLife.29430.007
Figure 4 with 5 supplements
Allosteric regulation of the BiP conformational ensemble.

(A) Ribbon representation of the domain-docked conformations (PDB 5e84 [Yang et al., 2015]). Four ‘soft’ mutations are shown as red spheres. The AMPylation site (Thr 518) is shown in green. (B) …

https://doi.org/10.7554/eLife.29430.008
Figure 4—source data 1

NMR analysis of populations for the domain-docked and -undocked conformations.

The percentage of the population of the domain-docked conformation was calculated from methyl peak intensities of three non-overlapping peak doublets (P1, P2 and P3), each containing peaks for the domain-docked (D) and -undocked (U) conformations, using the following equation: pD=IDID+IU×100%, where ID and IU are the intensities of peaks corresponding to the domain-docked and -undocked conformations, respectively. Errors were set as standard deviations (SDs) from the means for three doublets or uncertainties from the errors in peak intensities, whatever is larger. BiPT229G (ATP-bound): pD = 53 ± 6.6%.

https://doi.org/10.7554/eLife.29430.014
Figure 4—figure supplement 1
The isoleucine regions of methyl-TROSY spectra of ATP- and ADP-bound full-length (FL) BiP* (A, in black), overlaid with the spectra of a corresponding nucleotide-bound state of NBD*(1-413) (B, in red).

Peaks in the spectra of FL BiP* were assigned to the domain-undocked (U) conformation when they were overlapped with peaks in the spectrum of the isolated NBD*(1-413) in the corresponding nucleotide …

https://doi.org/10.7554/eLife.29430.009
Figure 4—figure supplement 2
The isoleucine regions of methyl-TROSY spectra of ATP- and ADP-bound states of the V461F variant of full-length (FL) BiP* (A, in black), overlaid with the spectra of a corresponding nucleotide-bound state of NBD*(1-413) (B, in red).

Peaks in the spectra of the FL protein were assigned to the domain-undocked (U) conformation when they were overlapped with peaks in the spectrum of the isolated NBD*(1-413) in the corresponding …

https://doi.org/10.7554/eLife.29430.010
Figure 4—figure supplement 3
The isoleucine regions of methyl-TROSY spectra of ATP- and ADP-bound states of the I437V variant of full-length (FL) BiP* (A, in black), overlaid with the spectra of a corresponding nucleotide-bound state of NBD*(1-413) (B, in red).

Peaks in the spectra of the FL protein were assigned to the domain-undocked (U) conformation when they were overlapped with peaks in the spectrum of the isolated NBD*(1-413) in the corresponding …

https://doi.org/10.7554/eLife.29430.011
Figure 4—figure supplement 4
The isoleucine regions of methyl-TROSY spectra of ATP- and ADP-bound states of the I526V variant of full-length (FL) BiP* (A, in black), overlaid with the spectra of a corresponding nucleotide-bound state of NBD*(1-413) (B, in red).

Peaks in the spectra of the FL protein were assigned to the domain-undocked (U) conformation when they were overlapped with peaks in the spectrum of the isolated NBD*(1-413) in the corresponding …

https://doi.org/10.7554/eLife.29430.012
Figure 4—figure supplement 5
The isoleucine regions of methyl-TROSY spectra of ATP- and ADP-bound states of the I538V variant of full-length (FL) BiP* (A, in black), overlaid with the spectra of a corresponding nucleotide-bound state of NBD*(1-413) (B, in red).

Peaks in the spectra of the FL protein were assigned to the domain-undocked (U) conformation when they were overlapped with peaks in the spectrum of the isolated NBD*(1-413) in the corresponding …

https://doi.org/10.7554/eLife.29430.013
Figure 5 with 7 supplements
Allosteric regulation of the BiP by AMPylation.

(A) Populations of the domain-docked conformation for AMPylated and non-AMPylated BiP* and its V461F variant in the presence of ATP and ADP, calculated using methyl peak intensities of the …

https://doi.org/10.7554/eLife.29430.015
Figure 5—source data 1

NMR analysis of populations for the domain-docked and -undocked conformations for AMPylated BiP.

The percentage of populations of the domain-docked conformation were calculated from methyl peak intensities of three non-overlapping peak doublets (P1, P2 and P3), each containing peaks for the domain-docked (D) and -undocked (U) conformations, using the following equation: pD=IDID+IU×100%, where ID and IU are the intensities of peaks corresponding to the domain-docked and -undocked conformations, respectively. Errors were set as standard deviations (SDs) from the means for three doublets or uncertainties from the errors in peak intensities, whatever is larger.

https://doi.org/10.7554/eLife.29430.023
Figure 5—source data 2

Analysis of the thermodynamic linkage between domain docking and nucleotide binding.

(Top) Thermodynamic parameters of ATP and ADP binding obtained from ITC experiments for FL BiP* and its non-AMPylated and AMPylated V461F variant; the measurements were repeated three times and the standard deviations for all parameters were less than 10%. (Bottom) Ratio of populations of the domain-docked (pD) and undocked (pU) conformations obtained from the analysis of NMR peak intensities (Figure 4—source data 1 and Figure 5—source data 1). (Grey) The experimental free energy of nucleotide binding and domain docking plotted in Figure 5B. The free energy of binding was calculated as ΔG(binding)/RT= Ln(Kd), where Kd is ADP or ATP binding constant obtained by ITC, R is the ideal gas constant, and T is the temperature in K; the free energy of domain docking was calculated from populations of the domain-undocked and -docked conformations obtained from the NMR analysis using the following equation: ΔG(docking)/RT= Ln(pD/pu).

https://doi.org/10.7554/eLife.29430.024
Figure 5—figure supplement 1
The isoleucine regions of methyl-TROSY spectra of ATP- and ADP-bound states of AMPylated full-length (FL) BiP* (A, in black), overlaid with the spectra of a corresponding nucleotide-bound state of NBD*(1-413) (B, in red).

Peaks in the spectra of the FL protein were assigned to the domain-undocked (U) conformation when they were overlapped with peaks in the spectrum of the isolated NBD*(1-413) in the corresponding …

https://doi.org/10.7554/eLife.29430.016
Figure 5—figure supplement 2
The isoleucine regions of methyl-TROSY spectra of ATP- and ADP-bound states of AMPylated full-length (FL) V461F BiP* (A, in black), overlaid with the spectra of a corresponding nucleotide-bound state of NBD*(1-413) (B, in red).

Peaks in the spectra of the FL protein were assigned to the domain-undocked (U) conformation when they were overlapped with peaks in the spectrum of the isolated NBD*(1-413) in the corresponding …

https://doi.org/10.7554/eLife.29430.017
Figure 5—figure supplement 3
The methyl TROSY spectrum of ATP-bound AMPylated BiP*-V461F (black) overlaid with the spectrum of ADP-bound AMPylated BiP*-V461F (red).
https://doi.org/10.7554/eLife.29430.018
Figure 5—figure supplement 4
The amide TROSY spectrum of ATP-bound AMPylated BiP*-V461F (black) overlaid with the spectrum of ADP-bound AMPylated BiP*-V461F (red).

Blowups of the representative regions of the TROSY spectra highlighting local chemical shift perturbations between ADP- and ATP-bound states.

https://doi.org/10.7554/eLife.29430.019
Figure 5—figure supplement 5
Representative panels showing nucleotide (ATP or ADP) binding to BiP* and isolated NBD*(1-413) and NBD*(1-417) measured by ITC. 

Experiments were performed at 25°C using 20–80 μM protein in the cell and 0.1–0.4 mM ADP (or ATP) injected from the syringe. The top panels show raw data and the bottom panels show integrated …

https://doi.org/10.7554/eLife.29430.020
Figure 5—figure supplement 6
Representative panels showing nucleotide (ATP or ADP) binding to non-AMPylated and AMPylated V461F BiP* measured by ITC. 

Experiments were performed at 25°C using 30 μM protein in the cell and 0.25–0.35 mM ADP (or ATP) injected from the syringe. Top panels show raw data, and bottom panels show integrated normalized data.

https://doi.org/10.7554/eLife.29430.021
Figure 5—figure supplement 7
LC-MS data for molecular mass determination of unmodified (panels A-B) and AMPylated (panels C-D) BiP. 

Molecular mass difference of ~330 Da as reported before (Preissler et al., 2015) corresponds to modification by AMPlation. An additional peak at ±180 Da corresponds to α-N-Glucosylation of the …

https://doi.org/10.7554/eLife.29430.022
The unique features of the BiP conformational landscape and its posttranslational regulation: Schematic illustration of three key ligand-bound states (ATP-, ATP- and substrate-, and ADP-bound) of DnaK and BiP. For both chaperones the conformational landscapes comprises three structurally and functionally distinct conformations: domain-docked (D), intermediate domain-undocked linker-bound (I), and domain-undocked linker-unbound (U).

Blue and red arrows illustrate how local perturbations of the SBD affect the BiP conformational landscape: the I437V and I538V substitutions favor domain undocking (blue arrows, Figure 4), while the …

https://doi.org/10.7554/eLife.29430.025

Tables

Table 1
Thermodynamics of nucleotide binding for BiP and DnaK.

ITC experiments for full-length (FL) BiP* and its two NBD constructs with and without the interdomain linker, NBD(1-413) and NBD(1-417), were repeated three times and the standard deviations for all …

https://doi.org/10.7554/eLife.29430.004
ANPKd (µM)∆G (kcal/mol)∆H (kcal/mol)-T∆S (kcal/mol)
DnaK*
FL
ATP0.16−9.39.0−18.3
ADP0.25−9.0−3.2−5.8
DnaK*
NBD
ATP0.61−8.9−4.1−3.7
ADP0.17−9.2−3.3−6.0
BiP*
FL
ATP0.80−8.311.4−19.7
ADP5.73−7.1−7.40.3
BiP*
NBD(1-413)
ATP7.41−7.012.3−19.3
ADP5.27−7.2−7.40.2
BiP*
NBD(1-417)
ATP1.15−8.114.7−22.3
ADP3.94−7.4−6.41.0
  1. *Thermodynamic parameters for ATPase deficient DnaK (DnaK*) and its NBD construct without the interdomain linker were taken from Taneva et al (Taneva et al., 2010).

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