Characterization of the Grp94/OS-9 chaperone-lectin complex

Abstract

Grp94 is a macromolecular chaperone belonging to the hsp90 family and is the most abundant glycoprotein in the endoplasmic reticulum (ER) of mammals. In addition to its essential role in protein folding, Grp94 was proposed to participate in the ER-associated degradation quality control pathway by interacting with the lectin OS-9, a sensor for terminally misfolded proteins. To understand how OS-9 interacts with ER chaperone proteins, we mapped its interaction with Grp94. Glycosylation of the full-length Grp94 protein was essential for OS-9 binding, although deletion of the Grp94 N-terminal domain relieved this requirement suggesting that the effect was allosteric rather than direct. Although yeast OS-9 is composed of a well-established N-terminal mannose recognition homology lectin domain and a C-terminal dimerization domain, we find that the C-terminal domain of OS-9 in higher eukaryotes contains "mammalian-specific insets" that are specifically recognized by the middle and C-terminal domains of Grp94. Additionally, the Grp94 binding domain in OS-9 was found to be intrinsically disordered. The biochemical analysis of the interacting regions provides insight into the manner by which the two associate and it additionally hints at a plausible biological role for the Grp94/OS-9 complex.

Keywords: ER-associated degradation; Grp94; Hsp90; chaperone; intrinsically disordered protein.

Figure 1. OS-9 and Grp94 protein constructs used in these studies

(a) OS-9 constructs from rat or yeast, as indicated. Grey hashed boxes in the rat OS-9 sequence are insertions found only in the mammalian protein. Corresponding regions found in the yeast protein are indicated by a dashed line. (b) Grp94 constructs used in this study. For the constructs indicated, the “charged linker” (CL) was replaced with a four glycine linker (4×Gly). ss = signal sequence, MRH = mannose recognition homology domain, L = proline-rich linker, CTE = mammalian C-terminal extension, N = N-terminal domain, M = middle domain, and C = Cterminal domain.

Figure 2. Grp94 binds to the C-terminal domain of OS-9

(a) SDS gel of bacterially co-expressed OS-9 and His-Grp94 following Ni-NTA affinity purification. The bands for OS-9 and His-Grp94 overlap. Left panel, coomassie stained, right panel, OS-9 Western blot. S = supernatant, E = elute. (b) SDS gel of bacterially co-expressed OS-9 ΔMRH (230–666) and His-Grp94-NMC (73–754). Left panel, coomassie stained, right panel, OS-9 Western blot. (c) Ni-NTA affinity pull-down with OS-9 ΔMRH (267–666) and His-Grp94 NMC expressed in Sf21 insect cells (with post-translational modifications, PTMs). (d) as in C except using His-Grp94 expressed in E. coli (without PTMs). (e) as in C except using His-Grp94-MC expressed in E. coli. (f) as in (c) except using His-Grp94-NMC + 1 mM AMP-PNP. (g) as in (c) except using His-Grp94-NMCΔ41 expressed in E. coli. (h) Ni-NTA affinity pull-downs using purified OS-9 ΔMRH and His-tagged GRP94-NMC expressed in Sf21 insect cells. Pulldowns were carried out in the absence (APO) or presence of 1 mM ADP or AMP-PNP. Grp94 doublet band reflects differential phosphorylation and glycosylation states characteristic of Grp94 overexpression in insect cells. (i) Band intensities from Grp94/OS-9 ΔMRH pull-downs (as shown in C) were quantified by densitometry and the fraction of OS-9 co-eluting with Grp94, determined from the average of three independent experiments, was plotted as a function of ligand.

Figure 3. Grp94-MC binds to OS-9 ΔMRH

(a) ITC titration of 352 uM Grp94-MC dimer titrated into 48 uM OS-9 ΔMRH hexamer. The endothermic heat of dilution was determined from the saturated baseline and subtracted from the exothermic binding reaction. (b) ITC of 341 uM Grp94-N titrated into 48 uM OS-9 ΔMRH hexamer. (c) Top, S200 gel filtration of purified Grp94-MC titrated into buffer and recovered from the ITC cell; Bottom, S200 gel filtration of the mixture of Grp94-MC and OS-9 ΔMRH recovered from the ITC titration in (a). (d) Top, Coomassie stained SDS gel of the peak fractions in panel (c), top. Bottom, Coomassie stained SDS gel of the peak fractions in panel (c), bottom. (e) As in (d) above except for Grp94-N and OS-9 ΔMRH.

Figure 4. GRP94 MC binds a C-terminal extension of mammalian OS-9

(a) Coomassie stained SDS gel of Ni-NTA affinity pull-down of His-Grp94-MC with OS-9 ΔMRH from rat (267–666) or yeast (266–527). S = supernatant, W = wash, E = elute. (b) Coomassie stained SDS gel (8–25% top and middle, 12.5% bottom) of Ni-NTA affinity pulldowns using His-GRP94-MC and rat OS-9 truncation constructs. (c) Band intensities from Grp94-OS-9 pull-downs (shown in (c)) were quantified by densitometry and the fraction of OS-9 co-eluting with His-Grp94 was plotted for each OS-9 construct shown in (a). (d) ITC titration of 1.6 mM OS-9 CTE hexamer into 83 µM GRP94-MC dimer. Heat of dilution was determined from the saturated baseline and the binding isotherm was fit to a one-site model.

Figure 5. GRP94 Middle and C-terminal domains bind OS-9 with similar affinities

(a) ITC titration of 1.59 mM OS-9 CTE hexamer into 81 uM Grp94-M. (b) Coomassie stained SDS gel of the recovered ITC titration in (a) following gel filtration on S200. The top panel is purified Grp94-M, and the bottom panel is the post-ITC mixture of Grp94-M and OS-9 CTE. (c) ITC titration of 1.57 mM OS-9 CTE hexamer into 77 uM Grp94-C dimer. (d) Coomassie stained SDS gel of the ITC titration in (c) following gel filtration on an S200 column. (e) ITC titration of 1.4 mM OS-9 CTE hexamer into 79 uM Grp94-N. (f) Coomassie stained SDS gel of the ITC titration in (e) following S200 gel filtration.

Figure 6. Grp94 binds the CTD of OS-9 in vivo

EK293 cells were transfected with S-tagged rat OS-9 constructs encoding the MRH domain (residues 31–229), the ΔMRH domain (residues 267–666), the full length (residues 31–666), or C-terminal truncation constructs 486 (residues 31–486) or 589 (residues 31–589). Cell lysates were immunoprecitpitated using S-protein agarose beads and western blotted as indicated.

Figure 7. The OS-9 C-terminal domain is intrinsically unstable

(a) Circular dichroism spectra of OS-9 ΔMRH (267–666) collected at 50 µg/ml in phosphate buffer. (b) Change in ellipticity at 222 nm as a function of temperature. (c and d) As in (a and b) except for OS-9 CTE (589–666). (e) Limited trypsin digest of 2mg/ml OS-9 ΔMRH, OS-9 CTE, α-casein, or GRP94-M (337–629) at trypsin concentrations of 0: 0 µg/ml, 1: 0.5 µg/ml, 2: 5 µg/ml, or 3: 10 µg/ml. Asterisk marks α-casein aggregate.