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 …
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 …
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 …
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 …
(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 …
(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) …
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: , 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%.
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 …
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 …
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 …
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 …
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 …
(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 …
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: , 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.
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 , 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: .
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 …
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 …
Blowups of the representative regions of the TROSY spectra highlighting local chemical shift perturbations between ADP- and ATP-bound states.
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 …
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.
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 …
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 …
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 …
ANP | Kd (µM) | ∆G (kcal/mol) | ∆H (kcal/mol) | -T∆S (kcal/mol) | |
---|---|---|---|---|---|
DnaK* FL | ATP | 0.16 | −9.3 | 9.0 | −18.3 |
ADP | 0.25 | −9.0 | −3.2 | −5.8 | |
DnaK* NBD | ATP | 0.61 | −8.9 | −4.1 | −3.7 |
ADP | 0.17 | −9.2 | −3.3 | −6.0 | |
BiP* FL | ATP | 0.80 | −8.3 | 11.4 | −19.7 |
ADP | 5.73 | −7.1 | −7.4 | 0.3 | |
BiP* NBD(1-413) | ATP | 7.41 | −7.0 | 12.3 | −19.3 |
ADP | 5.27 | −7.2 | −7.4 | 0.2 | |
BiP* NBD(1-417) | ATP | 1.15 | −8.1 | 14.7 | −22.3 |
ADP | 3.94 | −7.4 | −6.4 | 1.0 |
*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).