Mutational analysis of the Notch2 negative regulatory region identifies key structural elements for mechanical...

Area of Science:

• Cell biology
• Biophysics
• Molecular signaling

Background:

• The Notch signaling pathway regulates cell differentiation and is activated by ligand binding, inducing conformational changes in the Notch negative regulatory region (NRR).
• Force transmission to the NRR, potentially via ligand endocytosis, is thought to be essential for exposing the S2 proteolytic site and initiating downstream signaling.
• Previous work demonstrated that mechanical force applied to the Notch2 NRR N-terminus promotes metalloprotease cleavage.

Purpose of the Study:

• To investigate the role of specific structural elements within the Notch2 NRR in mechanical response and activation.
• To compare the mechanical stability of wild-type and mutated Notch2 NRR proteins.
• To identify key regions involved in force-dependent activation and understand differences between Notch1 and Notch2 NRRs.

Main Methods:

• Site-directed mutagenesis within the heterodimerization (HD) domain of the Notch2 NRR.
• Atomic force microscopy and molecular dynamics simulations to assess mechanical stability and forced unfolding.
• Analysis of metalloprotease cleavage site (S2) exposure and protein stability.

Main Results:

• Mutations in the Notch2 NRR HD domain, known to cause constitutive Notch1 activation, reduced the mechanical stability of Notch2 NRR.
• The linker region between Lin12-Notch repeats LNRA and LNRB, and the α3 helix within the HD domain, were identified as critical for masking the S2 cleavage site under force.
• Two mutated proteins showed reduced stability in the LNRC:HD domain interaction, highlighting regions important for mechanical, but not chemical, stability.

Conclusions:

• The study identifies specific structural elements in the Notch2 NRR that are critical for force-dependent activation, distinct from chemical stability.
• These findings provide insights into the mechanical underpinnings of Notch signaling and potential differences between Notch receptor subtypes.
• The research contributes to understanding how mechanical forces regulate biological processes at the molecular level.