β-Glucocerebrosidase Modulators Promote Dimerization of β-Glucocerebrosidase and Reveal an Allosteric Binding Site

Abstract

β-Glucocerebrosidase (GCase) mutations cause Gaucher's disease and are a high risk factor in Parkinson's disease. The implementation of a small molecule modulator is a strategy to restore proper folding and lysosome delivery of degradation-prone mutant GCase. Here, we present a potent quinazoline modulator, JZ-4109, which stabilizes wild-type and N370S mutant GCase and increases GCase abundance in patient-derived fibroblast cells. We then developed a covalent modification strategy using a lysine targeted inactivator (JZ-5029) for in vitro mechanistic studies. By using native top-down mass spectrometry, we located two potentially covalently modified lysines. We obtained the first crystal structure, at 2.2 Å resolution, of a GCase with a noniminosugar modulator covalently bound, and were able to identify the exact lysine residue modified (Lys346) and reveal an allosteric binding site. GCase dimerization was induced by our modulator binding, which was observed by native mass spectrometry, its crystal structure, and size exclusion chromatography with a multiangle light scattering detector. Finally, the dimer form was confirmed by negative staining transmission electron microscopy studies. Our newly discovered allosteric site and observed GCase dimerization provide a new mechanistic insight into GCase and its noniminosugar modulators and facilitate the rational design of novel GCase modulators for Gaucher's disease and Parkinson's disease.

Figure 1

Structure and activity of GCase modulators. (a) Chemical structures of noniminosugar pharmacological chaperones. (b) Dose-response curves for GCase modulator JZ-4109 and JZ-5029, and IFG. (c) Thermal melting curves of GCase with different concentrations of modulator JZ-4109 (increase in T_m relative to a DMSO control shown). (d) Western blot of fibroblast cell lysate after 3 days of compound treatments with DMSO as a control. (e) Normalization of GCase signal to GAPDH signal for each sample calculated from Western blots. (f) GCase activity assay for cell lysate with 4MU-β-Glc substrate after 3 days of treatment. (g) Time-dependent enzyme activity decrease with the treatment of inactivator JZ-5029. Each data point was collected in triplicate, and error bars represent the mean values ± standard deviation (SD). The data shown are representative of three independent experiments.

Figure 2

Cellular assays for modulator JZ-4109 treatment. (a) Endo H and PNGase F assays for wild-type fibroblast after compound treatment. Modulator JZ-4109 (2 µM), IFG (2 µM), n = 3. (b) Lysosomal enrichment assay for HEK cells after JZ-4109 (2 µM) and IFG (2 µM) treatment. (c, d) statistical analysis for GCase in the lysosomal fraction (C, GCase only; D, GCase normalized with Lamp1). Single tail test, n = 3. (e) Western blot and Endo H assay of N370S fibroblast cells for GCase after JZ-4109 (0.2 and 2 µM) and IFG (0.2 and 2 µM) treatment, n = 3. (f) Normalized (to DMSO control) GCase activity in N370S fibroblast cells after JZ-4109 and IFG treatment. n = 3.

Figure 3

Multitiered native mass spectrometry analysis of JZ-5029-modified GCase. (a) Intact native mass spectrum of the GCase dimer bound to quinazoline modulator JZ-5029 with deconvoluted neutral masses in the inset. Multiple peaks correspond to variable glycosylation. (b) Top: spectrum of the 15+ monomer species directly measured from solution; Bottom: spectrum of the 15+ monomer resulting from gas-phase isolation of the dimer and ejection of the monomer using collisional activation. (c) Graphical fragment ion map with blue flags indicating ions fragmented from the monomer that have been matched to within 10 ppm of those predicted from the GCase sequence. Only residues 276–497 are shown; no N-terminal fragments were observed. The orange markers correspond to the two potential modulator binding sites (+500.2576 Da).

Figure 4

Crystal structure of JZ-5029-modified GCase. (a) Crystallographic unit cell of JZ-5029-modified GCase. (b) Location of the JZ-5029 binding site in the GCase dimer interface. (c) Interaction diagram of JZ-5029 (in cyan, the modified Lys346 is also presented) with GCase (Chain A, yellow; Chain B, pink). (d) Loop 1, loop 2, and loop 3 conformation changes in JZ-5029-modified GCase (cyan) compared to apo-GCase (mauve, pdb: 1OGS), and IFG bound GCase (yellow, pdb: 2NSX).

Figure 5

Surface representation and residue conformation of loop 1 and loop 2 in GCase. Surface representation of (a) JZ-5029-modified GCase (cyan), (b) apo-GCase (purple, pdb: 1OGS), (c) IFG-bound GCase (yellow, pdb: 2NSX). The residue movement of loop 1 and loop 2 of (d) JZ-5029-modified GCase (cyan), (e) apo-GCase (purple, pdb: 1OGS), (f) IFG-bound GCase (yellow, pdb: 2NSX).

Figure 6

GCase dimer conformation and SEC-MALS chromatogram. (a) Comparison of JZ-5029-modified GCase dimer (pink) conformation to apo-GCase dimer (cyan, pdb: 1OGS); aligned by chain B: (b) Left, compound JZ-5029 (tan) modified GCase dimer surface representation with exposed active site (indicated in red); Right, predicted apo-GCase dimer surface representation (cyan, pdb: 1OGS). (c) SEC-MALS chromatogram of wild-type GCase (black) and a mixture of JZ-4109 (4.12, 8.25, 16.5, and 33.0 µM) and GCase (16.5 µM) with the calculated molar masses (g/mol) dotted on the chromatogram. (d) SEC-MALS chromatogram of wild-type GCase (black) and JZ-5029-modified (red) GCase with the calculated molar masses (g/mol) dotted on the chromatogram. (e) SEC-MALS chromatogram of wild-type GCase (black) and a mixture of IFG (8.25, 16.5, 33.0, and 165 µM) and GCase (16.5 µM) with the calculated molar masses (g/mol) dotted on the chromatogram

Figure 7

TEM results for JZ-5029-modified GCase dimer. (a) Representative negative staining TEM micrograph of the JZ-5029-modified GCase dimer. (b) Representative reference free class averages of JZ-5029-modified GCase dimer. (c) Docking of JZ-5029-modified GCase dimer (blue, Chain A; red, Chain B; spheres, JZ-5029) into 3D TEM model (purple mesh)