Evidence for Increased Exposure of the Notch1 Metalloprotease Cleavage Site upon Conversion to an Activated Conformation

SummaryNotch proteins are transmembrane receptors that normally adopt a resting state poised to undergo activating proteolysis upon ligand engagement. Receptor quiescence is maintained by three LIN12/Notch repeats (LNRs), which wrap around a heterodimerization domain (HD) divided by furin cleavage at site S1 during maturation. Ligand binding initiates signaling by inducing sensitivity of the HD to proteolysis at the regulated S2 cleavage site. Here, we used hydrogen exchange mass spectrometry to examine the solution dynamics of the Notch1 negative regulatory region in autoinhibited states before and after S1 cleavage, in a proteolytically sensitive “on” state, and in a complex with an inhibitory antibody. Conversion to the “on” state leads to accelerated deuteration in the S2 region and in nearby secondary structural elements within the HD. In contrast, complexation with the inhibitory antibody retards deuteration around the S2 site. Together, these studies reveal how S2 site exposure is promoted by receptor activation and suppressed by inhibitory antibodies.

Graphical AbstractFigure optionsDownload high-quality image (260 K)Download as PowerPoint slideHighlights
► First use of HX-MS to investigate the dynamics of the Notch activation switch
► In the autoinhibited conformation, the S2 site is protected against exchange
► The dynamics of the protein are unaffected upon maturation cleavage by furin at S1
► Conversion to the “on” state leads to accelerated deuteration of the S2 site