Mutations in the GBA1 gene are the most common of the known risk factors for Parkinson disease (PD). While clinical studies argue a strong case towards a link between GBA1 mutations and the development of PD, mechanistic insights have been lacking. GBA1 encodes glucocerebrosidase (GCase), a lysosomal enzyme which hydrolyzes glucosylceramide (GluCer) into glucose and ceramide and is deficient in Gaucher disease (GD). Recent research suggests a relationship between GCase and the PD-related amyloid-forming protein, α-synuclein (α-syn); however, the specific molecular mechanisms responsible for association remain elusive. A growing number of studies show a correlation between GCase deficiency and increased alpha-syn levels, leading some to speculate that GluCer accumulation affects normal α-syn turnover. In our work, we had discovered a specific physical interaction exists between α-syn and GCase both in solution and on the lipid membrane, resulting in efficient enzyme inhibition. It is currently unresolved whether reduced GCase activity alone leads to increased α-syn levels. In this research period, we sought to gain structural information on the membrane-bound α-syn-GCase complex. While there are extensive structural studies on the nature of α-syn membrane interaction, little is known on how GCase associates with the membrane, though X-ray crystal structures of soluble GCase are available. Neutron reflectometry (NR) 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 NR 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. Since only a minority of GD patients and carriers develop PD, other factors are also expected to play a role in promoting pathogenesis. Obvious molecules of interest include those that modulate GCase activity and α-syn-GCase interaction. Since the activator, Saposin C (Sap C), is considered vital for maximal catalytic activity and disrupt α-syn-GCase interaction, we carried out a detailed characterization of the interaction of Sap C and GCase in solution by analytical ultracentrifugation and saturation cross-transfer nuclear magnetic resonance (NMR) technique. The stoichiometry and binding affinity were measured using isothermal titration calorimetry (ITC). At or above micromolar concentration, GCase exists in equilibrium between monomer and dimer forms. However, in the presence of Sap C, only monomeric GCase is seen. ITC confirms that Sap C associates with GCase in solution in a 1:1 complex with approximate micromolar dissociation constant. Saturation cross-transfer NMR determined that the region of Sap C contacting GCase includes residues 63-66 and 74-76, which is distinct from the region known to enhance GCase activity. Because α-syn competes with Sap C for GCase binding, its interaction with GCase was also measured by ultracentrifugation and saturation cross-transfer. Unlike Sap C, binding of α-syn to GCase does not affect multimerization. However, adding α-syn reduces saturation cross-transfer from Sap C to GCase, confirming displacement. Based on the most frequently predicted multimer interface, the GCase active sites are partially buried, suggesting that Sap C might disrupt the multimer by binding near the active site.
