γsecretase structure · Structure and function of γ-secretase studied by single particle cryo-electron microscopy

Structure and function of γ-secretase studied by single particle cryo-electron microscopy

The γ-secretase intra-membrane protease complex plays a critical role in Alzheimer's disease (AD), since it secretes amyloid β peptides. The enzymatic activity of γ-secretase is, therefore, a prime target for intervention in AD and is of major interest. The complex has important proteolitic functions also in several cellular processes including Notch signaling. However, despite detailed knowledge of γ-secretase's role in cellular processes and disease, its mechanism of action remains poorly understood, primarily due to the lack of structural information. Indeed, the hydrophobic nature of γ-secretase and its complex maturation in the cell, render traditional structural approaches such as x-ray crystallography and NMR spectroscopy considerably challenging to apply. In order to shed light on the unique mechanism underlying γ-secretase activity and its involvement in AD, we have proposed to perform a structural analysis of the complex using single particle electron microscopy (EM). Single particle EM has proven to be a method of choice for the study of multi-subunit membrane complexes, and the feasibility of 3D visualization of γ-secretase specifically, has been recently demonstrated. Additionally, the technique has a unique ability to deal with unstable and heterogeneous protein complexes, and requires small amount of sample. To this end we have developed a novel procedure for purifying γ-secretase for structural studies. The complex is overexpressed in insect cells and purification is performed using newly-generated nanobodies (single-chain camelidae antibodies) against native epitopes within γ-secretase. The procedure yields highly pure, stable and active complex, enabling single particle EM analysis and reconstruction of 3D maps. Using single particle EM we analyzed the conformational landscape of wild type γ-secretase complex and effect of a Familial Alzheimer's Disease (FAD)-linked mutant. Furthermore, using antibody labeling and substrate-bound complex, we mapped the subunits in the structure. By classifying a large data-set, we find that γ-secretase complex comprises a dynamic population of conformations, with three well-defined classes of varying compactness. The conformational changes involve several domains of the complex, suggesting a high level of structural interdependence between its subunits. Remarkably, the most compact subpopulation of conformers is significantly enriched after binding of a transition-state analogue inhibitor, whereas the FAD mutation stabilizes a subpopulation of intermediate compactness. These observations indicate that the function of γ-secretase involves large-scale conformational dynamics. Loss of function of the protease and increase release of longer AB peptides correlates with alterations in γ-secretase conformational landscape.

Data: CORDIS, © European Union