title: Evidence for increased exposure of the Notch1 metalloprotease cleavage site upon conversion to an activated conformation.
abstract: Notch 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.
authors.affiliation: Department of Cancer Biology, Dana Farber Cancer Institute, Boston, MA 02115, USA.
authors.forename: Kittichoat
authors.initials: K
authors.lastname: Tiyanont
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Wales
authors.forename: Miguel
authors.initials: M
authors.lastname: Aste-Amezaga
authors.forename: Jon C
authors.initials: JC
authors.lastname: Aster
authors.forename: John R
authors.initials: JR
authors.lastname: Engen
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Blacklow
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D045683:Furin / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D045726:Metalloproteases / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051881:Receptor, Notch1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics
chemical_list: D010455:Peptides; D051881:Receptor, Notch1; D045726:Metalloproteases; D045683:Furin