title: Sampling the conformational space of the catalytic subunit of human γ-secretase.
abstract: Human γ-secretase is an intra-membrane protease that cleaves many different substrates. Aberrant cleavage of Notch is implicated in cancer, while abnormalities in cutting amyloid precursor protein lead to Alzheimer's disease. Our previous cryo-EM structure of γ-secretase revealed considerable disorder in its catalytic subunit presenilin. Here, we describe an image classification procedure that characterizes molecular plasticity at the secondary structure level, and apply this method to identify three distinct conformations in our previous sample. In one of these conformations, an additional transmembrane helix is visible that cannot be attributed to the known components of γ-secretase. In addition, we present a γ-secretase structure in complex with the dipeptidic inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). Our results reveal how conformational mobility in the second and sixth transmembrane helices of presenilin is greatly reduced upon binding of DAPT or the additional helix, and form the basis for a new model of how substrate enters the transmembrane domain.
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.
authors.forename: Xiao-chen
authors.initials: XC
authors.lastname: Bai
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.
authors.forename: Eeson
authors.initials: E
authors.lastname: Rajendra
authors.affiliation: Ministry of Education Key Laboratory of Protein Science, Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China.
authors.forename: Guanghui
authors.initials: G
authors.lastname: Yang
authors.affiliation: Ministry of Education Key Laboratory of Protein Science, Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China.
authors.forename: Yigong
authors.initials: Y
authors.lastname: Shi
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.
authors.forename: Sjors H W
authors.identifier: http://orcid.org/0000-0002-0462-6540
authors.initials: SH
authors.lastname: Scheres
mesh_terms: D004151:Dipeptides / Q000378:metabolism*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008958:Models, Molecular; D053763:Presenilins / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary
chemical_list: D004151:Dipeptides; C419410:N-(N-(3,5-difluorophenacetyl)alanyl)phenylglycine tert-butyl ester; D053763:Presenilins
keywords: biophysics; electron microscopy; gamma-secretase; human; image analysis; structural biology