title: C-terminal PAL motif of presenilin and presenilin homologues required for normal active site conformation.
abstract: The Alzheimer's disease-associated beta-amyloid peptide is produced through cleavage of amyloid precursor protein by beta-secretase and gamma-secretase. gamma-Secretase is a complex containing presenilin (PS) as the catalytic component and three essential cofactors: Nicastrin, anterior pharynx defective (APH-1) and presenilin enhancer-2 (PEN-2). PS and signal peptide peptidase (SPP) define a novel family of aspartyl proteases that cleave substrates within the transmembrane domain presumptively using two membrane-embedded aspartic acid residues for catalysis. Apart from the two aspartate-containing active site motifs, the only other region that is conserved between PS and SPP is a PAL sequence at the C-terminus. Although it has been well documented that this motif is essential for gamma-secretase activity, the mechanism underlying such a critical role is not understood. Here we show that mutations in this motif affect the conformation of the active site of gamma-secretase resulting in a complete loss of PS binding to a gamma-secretase transition state analog inhibitor, Merck C. Analogous mutations in SPP significantly inhibit its enzymatic activity. Furthermore, these mutations also abolish SPP binding to Merck C, indicating that SPP and gamma-secretase share a similar active site conformation, which is dependent on the PAL motif. Exploring the amino acid requirements within this motif reveals a very small side chain requirement, which is conserved during evolution. Together, these observations strongly support the hypothesis that the PAL motif contributes to the active site conformation of gamma-secretase and of SPP.
authors.affiliation: Department of Psychiatry, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.forename: Dirk
authors.initials: D
authors.lastname: Beher
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Nyborg
authors.forename: Mark S
authors.initials: MS
authors.lastname: Shearman
authors.forename: Todd E
authors.initials: TE
authors.lastname: Golde
authors.forename: Alison
authors.initials: A
authors.lastname: Goate
mesh_terms: D020816:Amino Acid Motifs; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006868:Hydrolysis; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D009154:Mutation / Q000502:physiology; D010447:Peptide Hydrolases / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D053764:Presenilin-1; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D004791:Enzyme Inhibitors; D008565:Membrane Proteins; C508853:PSEN1 protein, human; D053764:Presenilin-1; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D010447:Peptide Hydrolases; D016282:Aspartic Acid Endopeptidases; C460085:signal peptide peptidase; C509280:BACE1 protein, human