Department of Cancer Biology, Dana Farber Cancer Institute, Boston, MA 02115, USA.
Structure. 2011 Apr 13;19(4):546-54. doi: 10.1016/j.str.2011.01.016.
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.
Notch 蛋白是跨膜受体，通常处于静止状态，准备在与配体结合后进行激活蛋白水解。受体的静止状态由三个 LIN12/Notch 重复序列 (LNRs) 维持，这些重复序列缠绕在一个由 furin 在成熟过程中在 S1 位点切割分开的异二聚化结构域 (HD) 周围。配体结合通过诱导 HD 在受调控的 S2 切割位点对蛋白水解的敏感性来启动信号转导。在这里，我们使用氢交换质谱法在 S1 切割前后、在蛋白水解敏感的“开启”状态下以及与抑制性抗体形成复合物时，检查 Notch1 负调节区在自动抑制状态下的溶液动力学。向“开启”状态的转变导致 S2 区域和 HD 内附近二级结构元件中的氘代加速。相比之下，与抑制性抗体的复合物会减缓 S2 位点周围的氘代。这些研究共同揭示了受体激活如何促进 S2 位点的暴露，以及抑制性抗体如何抑制 S2 位点的暴露。
