title: The initial substrate-binding site of gamma-secretase is located on presenilin near the active site.
abstract: gamma-Secretase is a structurally enigmatic multiprotein complex that catalyzes intramembrane proteolysis of a variety of substrates, including the amyloid beta-protein precursor of Alzheimer's disease and the Notch receptor essential to cell differentiation. The active site of this transmembrane aspartyl protease apparently lies at the interface between two subunits of presenilin-1 (PS1); however, evidence suggests the existence of an initial substrate-binding site that is distinct from the active site. Here, we report that photoaffinity probes based on potent helical peptide inhibitors and designed to mimic the amyloid beta-protein precursor substrate bind specifically to the PS subunit interface, at a site close to the active site. The location of the helical peptide-binding site suggests that substrate passes between the two PS1 subunits to access the active site. An aggressive Alzheimer-causing mutation in PS1 strongly reduced photolabeling by a transition-state analogue but not by helical peptides, providing biochemical evidence that the pathological effect of this PS mutation is due to alteration of the active-site topography.
authors.affiliation: Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
authors.forename: Anna Y
authors.initials: AY
authors.lastname: Kornilova
authors.forename: Frédéric
authors.initials: F
authors.lastname: Bihel
authors.forename: Chittaranjan
authors.initials: C
authors.lastname: Das
authors.forename: Michael S
authors.initials: MS
authors.lastname: Wolfe
mesh_terms: D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; D001665:Binding Sites; D002460:Cell Line; D019281:Dimerization; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D019920:Photoaffinity Labels; D053764:Presenilin-1; D013379:Substrate Specificity
chemical_list: D008565:Membrane Proteins; C508853:PSEN1 protein, human; D019920:Photoaffinity Labels; D053764:Presenilin-1; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human
references.citation: Biochemistry. 1994 May 17;33(19):5661-73
references.pmid: 8180191
references.citation: Nature. 2000 Sep 7;407(6800):48-54
references.pmid: 10993067
references.citation: Proc Natl Acad Sci U S A. 1999 Jun 8;96(12):6959-63
references.pmid: 10359821
references.citation: Biochemistry. 1999 Apr 13;38(15):4720-7
references.pmid: 10200159
references.citation: Science. 2004 Aug 20;305(5687):1119-23
references.pmid: 15326347
references.citation: Nature. 1995 Aug 31;376(6543):775-8
references.pmid: 7651536
references.citation: Science. 2001 Aug 24;293(5534):1449-54
references.pmid: 11520976
references.citation: Science. 2002 Jun 21;296(5576):2215-8
references.pmid: 12077416
references.citation: Nature. 2000 Jun 8;405(6787):689-94
references.pmid: 10864326
references.citation: J Biol Chem. 2003 Jun 13;278(24):21823-30
references.pmid: 12657647
references.citation: Science. 1995 Aug 18;269(5226):973-7
references.pmid: 7638622
references.citation: Proc Natl Acad Sci U S A. 2002 Jun 11;99(12):8025-30
references.pmid: 12048239
references.citation: Biochemistry. 2000 Aug 1;39(30):8698-704
references.pmid: 10913280
references.citation: Nature. 1999 Apr 8;398(6727):513-7
references.pmid: 10206644
references.citation: Biochemistry. 2001 Aug 14;40(32):9522-31
references.pmid: 11583151
references.citation: Nat Cell Biol. 2003 May;5(5):486-8
references.pmid: 12679784
references.citation: Nat Cell Biol. 2000 Jul;2(7):428-34
references.pmid: 10878808
references.citation: Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):3053-8
references.pmid: 10077635
references.citation: Dev Cell. 2002 Jul;3(1):85-97
references.pmid: 12110170
references.citation: Proc Natl Acad Sci U S A. 2003 May 27;100(11):6382-7
references.pmid: 12740439
references.citation: Biochemistry. 2003 Jul 15;42(27):8133-42
references.pmid: 12846562
references.citation: Biochemistry. 1999 Aug 31;38(35):11223-30
references.pmid: 10471271
references.citation: J Biol Chem. 2003 May 9;278(19):16470-3
references.pmid: 12644463
references.citation: J Am Chem Soc. 2003 Oct 1;125(39):11794-5
references.pmid: 14505382
references.citation: Proc Natl Acad Sci U S A. 2002 Mar 5;99(5):2720-5
references.pmid: 11867728
references.citation: J Med Chem. 2004 Jul 29;47(16):3931-3
references.pmid: 15267231
references.citation: Nat Cell Biol. 2000 Nov;2(11):848-51
references.pmid: 11056541
references.citation: J Biol Chem. 2001 Feb 23;276(8):5413-6
references.pmid: 11134059
references.citation: Proc Natl Acad Sci U S A. 2002 Jan 22;99(2):775-9
references.pmid: 11792846
references.citation: Neuron. 2003 Apr 10;38(1):9-12
references.pmid: 12691659
references.citation: Nature. 2003 Mar 27;422(6930):438-41
references.pmid: 12660785