Structural analysis of the Notch transmembrane domain explains substrate preference of gamma-secretase and reveals conformational variants of bacterially expressed substrates

Restricted Intramembrane Proteolysis (RIP) is an important signaling paradigm conserved throughout evolution. How I-CliP proteases (I&barbelow;ntramembrane Cleavi&barbelow;ng P&barbelow;rotease) recognize their substrates and catalyze cleavage in the hydrophobic lipid bilayer remains a mystery. gamma-Secretase, the I-CliP responsible for generating the Alzheimer's disease peptide Abeta42, also cleaves Notch, which is critical for early embryonic development, adult immune system function, and stem cell maintenance. Notch is additionally involved in several diseases including cancer, stroke, and multiple sclerosis. Because Notch is the only known substrate harboring a mutation impacting proteolysis, this research aimed to use nuclear magnetic resonance spectroscopy (NMR) to determine and compare the structures of wild-type and mutant Notch transmembrane domains (TMD) to elucidate the structural basis for recognition and cleavage by gamma-secretase. This comparison was initially thought feasible by the publication of conditions compatible with in vitro cleavage of a bacterially expressed Notch protein by a partially purified form of gamma-secretase. Bacterially expressed Notch would have been suitable for NMR analysis if the in vitro cleavage assay could have been replicated. Our establishment of the in vitro assay resulted in the discovery of uncleaved conformational variants that are mistaken for the actual Notch cleavage product(s) during SDS-PAGE. The first part of this dissertation is a detailed analysis and characterization of this artifactual phenomenon. Alternatively, synthetic peptides based on the Notch TMD are reported to undergo cleavage in the in vitro assay (1). Peptides containing the wild-type Notch TMD sequences were readily cleaved by gamma-secretase whereas peptides containing the mutant sequence were not. In the second part of this thesis, structural analysis of similar peptides solubilized in micelles using solution state NMR spectroscopy identified a difference that may underlie substrate discrimination: the wild-type Notch TMD adopts a helical structure while the mutant is less ordered, suggesting that gamma-secretase initially recognizes an intact helix that resides within the lipid bilayer. The results presented here suggest that previous reports using the in vitro solubilized gamma-secretase cleavage assay are confounded by the presence of uncleaved variants, and the structural comparison of Notch TMD provides further insight into the basic mechanism of gamma-secretase mediated cleavage.