Structural features of membrane-bound glucocerebrosidase and {alpha}-synuclein probed by neutron reflectometry and fluorescence spectroscopy [Protein Structure and Folding]

November 26th, 2014 by Yap, T. L., Jiang, Z., Heinrich, F., Gruschus, J. M., Pfefferkorn, C. M., Barros, M., Curtis, J. E., Sidransky, E., Lee, J. C.

Mutations in glucocerebrosidase (GCase), the enzyme deficient in Gaucher disease, are a common genetic risk factor for the development of Parkinson disease (PD) and related disorders, implicating the role of this lysosomal hydrolase in the disease etiology. A specific physical interaction exists between the PD-related protein, α-synuclein (α-syn) and GCase both in solution and on the lipid membrane, resulting in efficient enzyme inhibition. Here, 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.
  • Posted in Journal of Biological Chemistry, Publications
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