Permissive Conformations of a Transmembrane Helix Allow Intramembrane Proteolysis by γ-Secretase

Area of Science:

• Biochemistry
• Molecular Biology
• Proteolysis

Background:

• Gamma-secretase (γ-secretase) is an intramembrane protease crucial for activating signaling molecules like Notch receptors.
• The structural determinants governing substrate transmembrane domain (TMD) cleavability by γ-secretase remain poorly understood.

Purpose of the Study:

• To elucidate how primary sequence elements within substrate TMDs influence their cleavage by γ-secretase.
• To identify specific regions and motifs within Notch1 and Notch3 TMDs that are critical for γ-secretase-mediated processing.

Main Methods:

• Development and application of a novel yeast-based cleavage assay.
• Utilized mutational analysis and gain-of-function approaches to investigate TMD function.
• Employed deuterium/hydrogen exchange and Nuclear Magnetic Resonance (NMR) spectroscopy to assess TMD conformation.

Main Results:

• Identified three critical regions within the TMDs of Notch1 and Notch3.
• Discovered that N-terminal AAAA or AGAV motifs enhance TMD conformational flexibility.
• Found that C-terminal residues may facilitate substrate docking and stabilize enzyme-substrate complexes.

Conclusions:

• Substrate cleavability by γ-secretase is regulated by distinct functional regions within the TMD.
• TMD conformational flexibility and specific sequence elements cooperate to determine substrate recognition and cleavage efficiency.
• This work enhances mechanistic understanding of intramembrane proteolysis and substrate discrimination by γ-secretase.