title: Substrate recruitment of γ-secretase and mechanism of clinical presenilin mutations revealed by photoaffinity mapping.
abstract: Intramembrane proteases execute fundamental biological processes ranging from crucial signaling events to general membrane proteostasis. Despite the availability of structural information on these proteases, it remains unclear how these enzymes bind and recruit substrates, particularly for the Alzheimer's disease-associated γ-secretase. Systematically scanning amyloid precursor protein substrates containing a genetically inserted photocrosslinkable amino acid for binding to γ-secretase allowed us to identify residues contacting the protease. These were primarily found in the transmembrane cleavage domain of the substrate and were also present in the extramembranous domains. The N-terminal fragment of the catalytic subunit presenilin was determined as principal substrate-binding site. Clinical presenilin mutations altered substrate binding in the active site region, implying a pathogenic mechanism for familial Alzheimer's disease. Remarkably, PEN-2 was identified besides nicastrin as additional substrate-binding subunit. Probing proteolysis of crosslinked substrates revealed a mechanistic model of how these subunits interact to mediate a stepwise transfer of bound substrate to the catalytic site. We propose that sequential binding steps might be common for intramembrane proteases to sample and select cognate substrates for catalysis.
authors.affiliation: Biomedical Center (BMC), Metabolic Biochemistry, Ludwig-Maximilians-University Munich, Munich, Germany German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.
authors.forename: Akio
authors.initials: A
authors.lastname: Fukumori
authors.affiliation: Biomedical Center (BMC), Metabolic Biochemistry, Ludwig-Maximilians-University Munich, Munich, Germany German Center for Neurodegenerative Diseases (DZNE), Munich, Germany harald.steiner@med.uni-muenchen.de.
authors.forename: Harald
authors.identifier: 0000-0003-3935-0318
authors.initials: H
authors.lastname: Steiner
mesh_terms: D000544:Alzheimer Disease / Q000473:pathology; D053829:Amyloid Precursor Protein Secretases / Q000378:metabolism*; D020134:Catalytic Domain; D002460:Cell Line; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D053763:Presenilins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D059748:Proteolysis
chemical_list: D050505:Mutant Proteins; D053763:Presenilins; D053829:Amyloid Precursor Protein Secretases
keywords: exosite; intramembrane proteolysis; photocrosslinking; substrate recognition; γ‐secretase