title: Structure of the catalytic pore of gamma-secretase probed by the accessibility of substituted cysteines.
abstract: Several single-span membrane proteins are cleaved within their transmembrane domains (TMDs) by intramembrane-cleaving proteases, although the structure of the active site executing intramembrane cleavage remains unknown. Here we use the substituted cysteine accessibility method to examine the structure of presenilin-1, a catalytic subunit of gamma-secretase, involved in amyloid beta protein generation in Alzheimer's disease and Notch signaling. We show that TMD6 and TMD7 of presenilin-1 contribute to the formation of a hydrophilic pore within the membrane. Residues at the luminal portion of TMD6 are predicted to form a subsite for substrate or inhibitor binding on the alpha-helix facing a hydrophilic milieu, whereas those around the GxGD catalytic motif within TMD7 are highly water accessible, suggesting formation of a hydrophilic structure within the pore. Collectively, our data suggest that the active site of gamma-secretase resides in a catalytic pore filled with water within the lipid bilayer and is tapered around the catalytic aspartates.
authors.affiliation: Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Chihiro
authors.initials: C
authors.lastname: Sato
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Morohashi
authors.forename: Taisuke
authors.initials: T
authors.lastname: Tomita
authors.forename: Takeshi
authors.initials: T
authors.lastname: Iwatsubo
mesh_terms: D020816:Amino Acid Motifs / Q000502:physiology; D053829:Amyloid Precursor Protein Secretases / Q000737:chemistry* / Q000378:metabolism; D016229:Amyloid beta-Peptides / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry*; D001921:Brain / Q000378:metabolism / Q000503:physiopathology; D001923:Brain Chemistry / Q000502:physiology; D020134:Catalytic Domain / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry*; D006801:Humans; D008563:Membrane Lipids / Q000737:chemistry*; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009448:Neurochemistry / Q000379:methods; D053764:Presenilin-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D040901:Proteomics / Q000379:methods
chemical_list: D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D008563:Membrane Lipids; D053764:Presenilin-1; D001224:Aspartic Acid; D053829:Amyloid Precursor Protein Secretases; D003545:Cysteine