The endoplasmic reticulum (ER) chaperones BiP and Grp94 selectively associate when BiP is in the ADP conformation

Abstract

Hsp70 and Hsp90 chaperones are critical for protein quality control in the cytosol, whereas organelle-specific Hsp70/Hsp90 paralogs provide similar protection for mitochondria and the endoplasmic reticulum (ER). Cytosolic Hsp70/Hsp90 can operate sequentially with Hsp90 selectively associating with Hsp70 after Hsp70 is bound to a client protein. This observation has long suggested that Hsp90 could have a preference for interacting with clients at their later stages of folding. However, recent work has shown that cytosolic Hsp70/Hsp90 can directly interact even in the absence of a client, which opens up an alternative possibility that the ordered interactions of Hsp70/Hsp90 with clients could be a consequence of regulated changes in the direct interactions between Hsp70 and Hsp90. However, it is unknown how such regulation could occur mechanistically. Here, we find that the ER Hsp70/Hsp90 (BiP/Grp94) can form a direct complex in the absence of a client. Importantly, the direct interaction between BiP and Grp94 is nucleotide-specific, with BiP and Grp94 having higher affinity under ADP conditions and lower affinity under ATP conditions. We show that this nucleotide-specific association between BiP and Grp94 is largely due to the conformation of BiP. When BiP is in the ATP conformation its substrate-binding domain blocks Grp94; in contrast, Grp94 can readily associate with the ADP conformation of BiP, which represents the client-bound state of BiP. Our observations provide a mechanism for the sequential involvement of BiP and Grp94 in client folding where the conformation of BiP provides the signal for the subsequent recruitment of Grp94.

Keywords: BiP; Grp94; allostery; chaperone; fluorescence anisotropy; fluorescence resonance energy transfer (FRET); heat shock protein 90 (Hsp90); nuclear magnetic resonance (NMR); nucleotide; structural dynamics.

Figure 1.

Overview of Grp94 and BiP conformations. Mutation sites that disrupt binding between HtpG and DnaK are shown in red spheres. A, these sites are exposed on the Grp94 open conformation (ADP; Protein Data Bank (PDB) code 2O1V) and the closed conformation (ATP; PDB code 5ULS). B, in contrast, these sites are mostly exposed in the BiP ADP conformation (modeled from PDB code 2KHO) and blocked in the ATP conformation (PDB code 5E84).

Figure 2.

A, example of concentration-dependent BiP oligomerization. The BiP monomer elutes at 6.8 min. B, BiP can be maintained in a predominantly monomeric state at concentrations below 100 nm under all nucleotide conditions. Error bars are the S.E. of the mean for at least three measurements. C, design of a previously established BiP FRET pair (25). D, the BiP fluorescence spectrum shows anticorrelated changes at donor and acceptor emission under ADP and ATP conditions. mAU, milliabsorbance units; cps, counts per second.

Figure 3.

Fluorescence depolarization binding measurements show strong binding between BiP and Grp94 under ADP conditions and weak binding under ATP conditions. Error bars are the S.E. of the mean for at least three measurements. Buffer conditions were 25 mm Tris, pH 7.5, 50 mm KCl, 1 mm MgCl₂, 1 mm ADP or ATP, and 1 mg/ml BSA at 37 °C. mP, millipolarization units.

Figure 4.

A, direct binding between the BiP NBD and Grp94 MD shown by chemical shift perturbations ([¹⁵N]Grp94 MD alone, black, 150 μm, 32 scans; [¹⁵N]Grp94 MD with BiP NBD, red, 200 μm, 32 scans). B, the BiP NBD and full-length BiP have comparable affinities for Grp94 under ADP conditions. C, the Grp94 MD is sufficient for binding the BiP NBD, but full affinity requires both the MD and NTD. D, mutants that disrupt binding between the bacterial Hsp70 and Hsp90 system also disrupt binding between BiP and Grp94. Error bars are the S.E. of the mean for at least three measurements. Buffer conditions for binding experiments are the same as in Fig. 3. mP, millipolarization units.

Figure 5.

BiP activates the ATPase of Grp94 ATPase. The ATPase-deficient construct of BiP (T229G) shows that the increase in activity is due to Grp94. The binding-deficient mutants (BiP E243A and Grp94 K467A) show no activation, yielding roughly additive ATPase results. Error bars are the S.E. of the mean for at least three measurements. Buffer conditions were 25 mm Tris, pH 7.5, 50 mm KCl, 1 mm MgCl₂, 1 mm ATP, and 1 mg/ml BSA at 37 °C.

Figure 6.

A, weak binding to Grp94 under ATP conditions is only observed in BiP constructs that have the SBD. Buffer conditions are the same as in Fig. 3. B, a docking model shows that the BiP SBD clashes with the Grp94 MD when BiP is in the ATP conformation. Grp94 is shown as a surface, and BiP is shown as a ribbon. Domains are colored separately for Grp94 (CTD, blue; MD, green; NTD, orange) and BiP (NBD, blue; SBD-β, gray; SBD-α, orange). C, docking model suggests a salt bridge between Grp94 Lys-467 and BiP Glu-243 with Arg-466 at the periphery. Docking models were built using ClusPro 2.0 (39) with no constraints.

Figure 7.

A, Grp94 increases BiP FRET efficiency, indicative of a higher population of the BiP ADP conformation. The binding-defective mutant Grp94 K467A loses its capability to shift the BiP conformation. Solid lines are fits using Equation 1. B, kinetic FRET measurements show BiP transitioning from the ATP conformation to the ADP conformation (green circles). A similar experiment performed with Grp94 shows similar slow changes in FRET (red circles). C, binding kinetics between BiP and Grp94 is slow when BiP starts in the ATP conformation and transitions to the ADP conformation (red circles). In contrast, binding between BiP and Grp94 is fast when BiP starts in the ADP conformation (blue circles). Solid lines in B and C are single-exponential fits. D, kinetic model of conformation-specific binding between BiP and Grp94. Buffer conditions are the same as in Fig. 3. Error bars are the S.E. of the mean for at least three measurements. mP, millipolarization units.