National Institutes of Health/National Cancer Institute (NIH/NCI)
R35 CA220340
United States
Other private
Gift from Edward B. Goodnow
Citation
Journal: Cell / Year: 2023 Title: Structural basis for membrane-proximal proteolysis of substrates by ADAM10. Authors: Colin H Lipper / Emily D Egan / Khal-Hentz Gabriel / Stephen C Blacklow / Abstract: The endopeptidase ADAM10 is a critical catalyst for the regulated proteolysis of key drivers of mammalian development, physiology, and non-amyloidogenic cleavage of APP as the primary α-secretase. ...The endopeptidase ADAM10 is a critical catalyst for the regulated proteolysis of key drivers of mammalian development, physiology, and non-amyloidogenic cleavage of APP as the primary α-secretase. ADAM10 function requires the formation of a complex with a C8-tetraspanin protein, but how tetraspanin binding enables positioning of the enzyme active site for membrane-proximal cleavage remains unknown. We present here a cryo-EM structure of a vFab-ADAM10-Tspan15 complex, which shows that Tspan15 binding relieves ADAM10 autoinhibition and acts as a molecular measuring stick to position the enzyme active site about 20 Å from the plasma membrane for membrane-proximal substrate cleavage. Cell-based assays of N-cadherin shedding establish that the positioning of the active site by the interface between the ADAM10 catalytic domain and the bound tetraspanin influences selection of the preferred cleavage site. Together, these studies reveal the molecular mechanism underlying ADAM10 proteolysis at membrane-proximal sites and offer a roadmap for its modulation in disease.
Name: 4-(N-HYDROXYAMINO)-2R-ISOBUTYL-2S-(2-THIENYLTHIOMETHYL)SUCCINYL-L-PHENYLALANINE-N-METHYLAMIDE type: ligand / ID: 10 / Number of copies: 1 / Formula: BAT
Model: Quantifoil / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV / Details: Blot for 7 seconds with a blot force of 15.
Details
This sample was monodisperse
-
Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 10037 / Average exposure time: 1.3 sec. / Average electron dose: 51.99 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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