Deducing the transmembrane domain organization of presenilin-1 in gamma-secretase by cysteine disulfide cross-linking.
Gamma-secretase is a founding member of membrane-embedded aspartyl proteases that cleave substrates within transmembrane domains, and this enzyme is an important target for the development of therapeutics for Alzheimer's disease. The structure of gamma-secretase and its precise catalytic mechanism still remain largely unknown. Gamma-secretase is a complex of four integral membrane proteins, with presenilin (PS) as the catalytic component. To gain structural and functional information about the nine-transmembrane domain (TMD) presenilin, we employed a cysteine mutagenesis/disulfide cross-linking approach. Here we report that native Cys92 is close to both Cys410 and Cys419, strongly implying that TMD1 and TMD8 are adjacent to each other. This structural arrangement also suggests that TMD8 is distorted from an ideal helix. Importantly, binding of an active site directed inhibitor, but not a docking site directed inhibitor, reduces the ability of the native cysteine pairs of PS1 to cross-link upon oxidation. These findings suggest that the conserved cysteines of TMD1 and TMD8 contribute to or allosterically interact with the active site of gamma-secretase.
Kornilova
Anna Y
AY
Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA. anna_kornilova@merck.com
Kim
Jennifer
J
Laudon
Hanna
H
Wolfe
Michael S
MS
D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D016282:Aspartic Acid Endopeptidases; D001665:Binding Sites; D001755:Blastocyst / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D002478:Cells, Cultured; D017124:Conserved Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004220:Disulfides / Q000737:chemistry*; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005260:Female; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry*; D051379:Mice; D018345:Mice, Knockout; D008861:Microsomes / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D011247:Pregnancy; D053764:Presenilin-1; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013234:Stem Cells / Q000166:cytology / Q000201:enzymology / Q000378:metabolism
D003432:Cross-Linking Reagents; D004220:Disulfides; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; C508853:PSEN1 protein, human; D053764:Presenilin-1; D001224:Aspartic Acid; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; C509282:Bace1 protein, mouse; D003545:Cysteine