MANF antagonizes nucleotide exchange by the endoplasmic reticulum chaperone BiP

Despite its known role as a secreted neuroprotectant, much of the mesencephalic astrocyte-derived neurotrophic factor (MANF) is retained in the endoplasmic reticulum (ER) of producer cells. There, by unknown mechanisms, MANF plays a role in protein folding homeostasis in complex with the ER-localized Hsp70 chaperone BiP. Here we report that the SAF-A/B, Acinus, and PIAS (SAP) domain of MANF selectively associates with the nucleotide binding domain (NBD) of ADP-bound BiP. In crystal structures the SAP domain engages the cleft between NBD subdomains Ia and IIa, stabilizing the ADP-bound conformation and clashing with the interdomain linker that occupies this site in ATP-bound BiP. MANF inhibits both ADP release from BiP and ATP binding to BiP, and thereby client release. Cells lacking MANF have fewer ER stress-induced BiP-containing high molecular weight complexes. These findings suggest that MANF contributes to protein folding homeostasis as a nucleotide exchange inhibitor that stabilizes certain BiP-client complexes.


MANF antagonizes nucleotide exchange by the endoplasmic reticulum (ER) chaperone BiP
Yan and Rato et al.

Supplementary Information
Source data for panel "b" and uncropped images for panel "c" are provided as a Source Data file.

Supplementary Figure 2
Supplementary BLI traces for Fig. 2 a Bio-layer interferometry (BLI) signals of streptavidin biosensors loaded with biotinylated SAPLIP (the first phase shown) and sequentially incubated with the BiP SBD or NBD. Note the absence of a binding signal.
b Traces of time-dependent BLI signal from a representative experiment shown in Fig. 2e of biosensors loaded with biotinylated BiP NBD and exposed to solutions containing the indicated concentrations of MANF to record association, and then transferred into buffer for dissociation. Association signals at 300 seconds were used to create the binding curve to the right. The K1/2 max value was extracted by fitting the data to a saturated one site specific binding function in Prism 5. c As in "b" above but with SAP in solution.

Supplementary Figure 3
Crystal structure of BiP oligomers a Cartoon representation of BiP molecules in the crystal of apo BiP V461F (PDB 6HAB). Though a single BiP molecule was found in the asymmetric unit, in the crystal the substrate binding domain (SBD) of a symmetry-related molecule binds the interdomain linker of another molecule, forming "daisy-chain" oligomers. The nucleotide binding domain (NBD), SBD, and the interdomain linker of one BiP protomer are annotated.
b Shown is a representative autoradiograph (one of two experiments performed) of 32 P-labeled ATP and ADP separated by thin layer chromatography, the products of a single-turnover ATPase assay testing if MANF has a stimulatory effect on ATP hydrolysis by BiP. Pre-formed complexes between purified BiP protein and α-32 P-ATP were incubated in the absence of additional proteins (control) or in the presence of the indicated concentrations of MANF, its derivatives, or the J-domain of ERdJ6 as a positive control. ATPase activity was assessed by comparing the loss of ATP signal over time and the signals were quantified in the plot to the right. Of note, the ADP signal present at t = 0 arises from a combination of factors: non-enzymatic hydrolysis of the (unlabelled) γ phosphate during storage of the precursor 32 P α-labelled ATP, enzymatic hydrolysis during formation of the BiP-ATP complex and possibly hydrolysis that occurs during sample freezing and thawing. However, as this is a single turnover experiment and only preformed BiP-ATP complexes can hydrolyze ATP, BiP-ADP complexes are inert and the pre-experimental conversion of ATP to ADP is ignored and only the ATPase activity of BiP-ATP complexes during the experiment are taken into account.

Supplementary Figure 4
Supplementary data for Fig. 5 a Plot of koff for release of MABA-ADP from BiP NBD (as in Fig. 5b) against final concentration of MANF or SAP. The mean values and SD bars of three independent experiments are plotted. Single exponential best fit lines are shown.
b As in "a" but with intact BiP and ATP as competitor. Related to Fig. 5c. c Overlay of the NBD-MANF complex structure (as in Fig. 3a) and the Sse1p-Hsp70 complex (a nucleotide exchange factor bound to an Hsp70 NBD; PDB 3D2F). MANF (gold) binds to the opposite site of the NBD (blue surface) where the NEF Sse1p (yellow) binds.
Source data for panels "a" and "b" are provided as a Source Data file. Fig. 6) a Schema of the design of the SILAC experiment to quantify relative changes in abundance of BiP peptides incorporated into detergent insoluble high molecular weight (HMW) complexes in CHO-K1 S21 wildtype (wt) cells untreated and treated with tunicamycin (Tm; 2.5 μg/mL, 15 hours).

More BiP is recovered in high molecular weight complexes from tunicamycintreated cells (supplementary data for
b LC-MS spectra of a representative doubly-charged tryptic BiP peptide (VEIIANDQGNR 60 ) from the input (top) and HMW complexes (bottom) of experiments outlined in "a". The spectrum on the left is from lysate of untreated (Unt) cells cultured in light medium combined with lysate from cells cultured in heavy medium and exposed to tunicamycin (Tm), and the spectrum on the right is of BiP from untreated cells cultured in heavy medium combined with lysate from cells cultured in light medium and exposed to tunicamycin. Source data for panels "c" and "d" and uncropped image for panel "d" are provided as a Source Data file.

Preliminary gating for live cells was done based on FSC-A/SSC-A and for singlets based on FSC-W/SSC-A.
Supplementary Table 1 List of plasmids used, their lab names, description, first appearance in the figures and their corresponding label, and references.