Structural features of membrane-bound glucocerebrosidase and α-synuclein probed by neutron reflectometry and fluorescence spectroscopy

Abstract

Mutations in glucocerebrosidase (GCase), the enzyme deficient in Gaucher disease, are a common genetic risk factor for the development of Parkinson disease and related disorders, implicating the role of this lysosomal hydrolase in the disease etiology. A specific physical interaction exists between the Parkinson disease-related protein α-synuclein (α-syn) and GCase both in solution and on the lipid membrane, resulting in efficient enzyme inhibition. Here, neutron reflectometry was employed as a first direct structural characterization of GCase and α-syn·GCase complex on a sparsely-tethered lipid bilayer, revealing the orientation of the membrane-bound GCase. GCase binds to and partially inserts into the bilayer with its active site most likely lying just above the membrane-water interface. The interaction was further characterized by intrinsic Trp fluorescence, circular dichroism, and surface plasmon resonance spectroscopy. Both Trp fluorescence and neutron reflectometry results suggest a rearrangement of loops surrounding the catalytic site, where they extend into the hydrocarbon chain region of the outer leaflet. Taking advantage of contrasting neutron scattering length densities, the use of deuterated α-syn versus protiated GCase showed a large change in the membrane-bound structure of α-syn in the complex. We propose a model of α-syn·GCase on the membrane, providing structural insights into inhibition of GCase by α-syn. The interaction displaces GCase away from the membrane, possibly impeding substrate access and perturbing the active site. GCase greatly alters membrane-bound α-syn, moving helical residues away from the bilayer, which could impact the degradation of α-syn in the lysosome where these two proteins interact.

Keywords: Gaucher Disease; Membrane Bilayer; Parkinson Disease; Protein-Lipid Interaction; Surface Plasmon Resonance (SPR); Tryptophan.

FIGURE 1.

Insertion of GCase Trp side chains into the membrane. A, top, GCase crystal structure (Protein Data Bank code 1OGS) (46). TIM barrel motif and β-sheet domains are colored taupe and teal, respectively. Trp and active site Glu residues are colored purple and red, respectively. Eight of the 12 Trp residues have some solvent exposure. Bottom, steady-state Trp fluorescence of GCase (1 μm) in the presence of POPC/POPS (F₀) and POPC/POPS vesicles containing 30% brominated phosphatidylcholine lipids (F). The location of the three different bromine sites is denoted by dotted lines. Solid lines, fits to distribution analysis. Error bars, S.D. values (n = 2). B, CD spectroscopy was employed to measure the secondary structure of GCase alone (top panel), α-syn alone (middle panel) in solution (dashed line) and in the presence of POPC/POPS vesicles (solid line), and α-syn in the presence of GCase and POPC/POPS vesicles (bottom panel). Experiments were performed in pH 5.5 buffer (50 mm MES and 25 mm NaCl) with 2 μm GCase, 5 μm α-syn, and 700 μm POPC/POPS (1:1) vesicles. Mathematical summation of independently measured spectra of α-syn and GCase at the same concentrations in the presence of vesicles is also plotted (bottom panel). Averages of independently measured spectra (n = 2) are shown.

FIGURE 2.

Effect of POPS on GCase membrane association and activity. GCase binding to POPC (circles) and equimolar POPC/POPS (squares) stBLMs is quantified by SPR spectroscopy. One pixel change corresponds to a SPR minimum angle change of 0.0067°. Solid lines, fits to a Langmuir adsorption process (equilibrium dissociation constants (K_d) are 210 ± 6 nm and 6.8 ± 1.7 μm for POPC/POPS and POPC, respectively). Error bars, SPR signal variations at the plateau of each addition. Inset, normalized GCase activity measured in vesicles containing different amounts of POPS (0–50%). Error bars, S.D. values (n = 2).

FIGURE 3.

GCase membrane binding probed by neutron reflectometry. A, neutron reflectivity (R/R_F) for a POPC/POPS stBLM and changes resulting from the addition of GCase (300 nm) in D₂O. Error bars, 68% confidence intervals for the measured reflectivity based on Poisson statistics. The differences between R/R_F curves (ΔR/R_F) are shown in units normalized to the S.D. value (σ) of the experimental error. Spline model fits to the data are shown as solid lines. Best fit neutron scattering length density profiles for all four measurements calculated from the composition-space model are shown in the inset. B, simplified molecular distributions for each organic interface layer of the POPC/POPS stBLM and GCase obtained from the best fit of reflectivity data to the spline fit model. The median protein envelope is shown with its associated 68% confidence intervals (black). Volume occupancy is indicated on the right axis. Comparison of the median orientation fit using a structural model of glycosylated GCase is shown with 68% confidence intervals (green). See Table 1 for parameters.

FIGURE 4.

Structure of GCase at the membrane interface revealed by neutron reflectometry. A, probability plot for the orientation of GCase at the membrane obtained from rigid body modeling using a structural model of glycosylated GCase as a function of the Euler angles (β, γ). The contour represents a confidence limit of 68%. Protein orientation is sufficiently described by two of three Euler angles, β and γ. Any orientation is achieved first rotating by γ about the z axis (membrane normal) and second by β about the x axis (in the plane of the membrane). The third Euler angle, α, does not affect the NR profile along z because it constitutes a final rotation of the sample about the membrane normal. Here, we have defined (β, γ = 0°, 0°) as the orientation of Protein Data Bank file 1OGS (chain A) (46) with the z coordinate axis taken as the normal to the membrane surface. Euler angles for the structures shown in Fig. 4B are indicated in blue and that for Fig. 4C is shown in red. B, molecular structures of probable orientations of GCase at the membrane interface. The TIM barrel motif and β-sheet domains are colored taupe and teal, respectively. Catalytic Glu residues, 235 and 340, are red. The flexible loops near the active site are loop 1 (green; residues 311–319), loop 2 (orange; residues 341–357), and loop 3 (purple; residues 380–402) and are as indicated. Trp residues and glycosylation sites (Asn-19, Asn-59, Asn-146, and Asn-270) (48) are also shown. C, expanded view of the molecular structure with probable orientation (β = 20°, γ = 90°) of GCase at the membrane interface. The TIM barrel motif and β-sheet domains are colored taupe and teal, respectively. Catalytic Glu residues, 235 and 340, and flexible loops 1–3 near the active site are indicated, colored as in Fig. 4B, with Trp residues and glycosylation sites also shown. D, molecular surface for GCase. The (20°, 90°) orientation, the same as in Fig. 4C, is shown in the left panel (side view). In the right panel, the membrane-facing view of GCase (bottom view, rotated 90° from the left panel) is shown. The surface is colored blue for lysine and arginine residues, cyan for histidine, red for aspartate and glutamate, and green for side chains of hydrophobic residues (alanine, valine, leucine, isoleucine, methionine, phenylalanine, and tryptophan). The enzyme active site cavity is indicated as well as the tryptophan residues of loops 2 and 3.

FIGURE 5.

Interaction of GCase and deuterated α-syn on the membrane probed by neutron reflectometry. A, neutron reflectivity (R/R_F) for a POPC/POPS stBLM and changes resulting from the addition of GCase and d-α-syn (300 nm each) in D₂O. Error bars, 68% confidence intervals for the measured reflectivity based on Poisson statistics. The differences between R/R_F curves (ΔR/R_F) are shown in units normalized to the S.D. (σ) of the experimental error. Spline model fits to the data are shown as solid lines. Best fit neutron scattering length density profiles for all four measurements calculated from the composition-space model are shown in the inset. B, simplified molecular distributions for each organic interface layer of the POPC/POPS stBLM and the median protein envelope for protiated GCase obtained from the best fit of reflectivity data to the spline fit model. Dashed lines, 68% confidence intervals. Volume occupancy is indicated on the right axis. See Table 1 for parameters. C, median protein envelope attributable to d-α-syn (green) is shown. For comparison, the median protein envelope obtained from fits to NR of d-α-syn in the absence of GCase (300 nm; red) on POPC/POPS stBLM is also shown. Dashed lines represent 68% confidence intervals. Volume occupancy is indicated on the right axis. See Table 1 for parameters. D, the integrated profiles for d-α-syn alone (red) and d-α-syn extracted from α-syn·GCase complex (green) are shown. The integral is normalized to the total number of residues (140 residues). Dashed lines represent 68% confidence intervals.

FIGURE 6.

Model structures of the membrane-bound α-syn·GCase complex. GCase is shown in gray and α-syn in blue and red for the exposed (A) and embedded model (B), respectively. The α-syn N and C termini are labeled. The viewpoint corresponds to a rotation of 60° about the membrane normal of Fig. 4C. C, comparison between the predicted cross-sectional profiles for α-syn in the two α-syn·GCase models (A and B) and the spline fit extracted from the α-syn·GCase NR data (reproduced from Fig. 5C). Dashed lines represent 68% confidence intervals. Volume occupancy is indicated on the right axis.