NOTCH, a single-pass transmembrane protein, plays a crucial role in cell fate determination through cell-to-cell communication. It interacts with two canonical ligands, Delta-like (DLL) and Jagged (JAG), located on neighboring cells to regulate diverse cellular processes. Despite extensive studies on the functional roles of NOTCH and its ligands in cellular growth, the structural details of full-length NOTCH and its ligands remain poorly understood. In this study, we employed fragment-based modeling and multiscale simulations to study the full-length structure of the human NOTCH ectodomain, comprising 1,756 amino acids. We performed coarse-grain dynamics simulations of NOTCH in both glycosylated and non-glycosylated forms to investigate the role of glycosylation in modulating its conformational dynamics. In apo form, coarse-grained simulations revealed that glycosylated NOTCH protein can transition from an elongated structure of ∼ 86 nm from the membrane surface to a semi-compact state (∼23.81 ± 9.98 nm), which aligns with cryo-EM data. To transition from the apo form to ligand-bound forms of NOTCH, we followed an atomistic and integrative modeling approach to model the interactions between NOTCH-DLL4 and NOTCH-JAG1. Atomistic simulations of the smaller bound fragment EGF8-13 patch revealed conformational plasticity critical for NOTCH binding, while integrative modeling of full-length complexes suggested a larger binding surface than previously reported. Simulations of pathogenic mutations revealed that E360K and R448Q disrupted the NOTCH-ligand interaction surfaces, causing dissociation. In contrast, C1133Y in the Abruptex domain compromised protein stability by disrupting the domain's interaction with the ligand-binding domain in the apo form of NOTCH-ECD. These findings provide a detailed molecular understanding of NOTCH and its ligands, offering insights that could enable the development of novel therapeutic approaches to selectively target pathogenic NOTCH signaling.

中文翻译：

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多尺度模拟揭示了 NOTCH 蛋白的结构和配体特异性特征。


NOTCH 是一种单通道跨膜蛋白，通过细胞间通讯在细胞命运决定中起着至关重要的作用。它与位于相邻细胞上的两个经典配体 Delta 样 （DLL） 和锯齿状 （JAG） 相互作用，以调节不同的细胞过程。尽管对 NOTCH 及其配体在细胞生长中的功能作用进行了广泛研究，但全长 NOTCH 及其配体的结构细节仍然知之甚少。在这项研究中，我们采用基于片段的建模和多尺度模拟来研究人类 NOTCH 胞外结构域的全长结构，包括 1,756 个氨基酸。我们对糖基化和非糖基化形式的 NOTCH 进行了粗晶动力学模拟，以研究糖基化在调节其构象动力学中的作用。在 apo 形式中，粗粒度模拟显示糖基化 NOTCH 蛋白可以从膜表面 ∼ 86 nm 的细长结构转变为半紧凑状态 （∼23.81 ± 9.98 nm），这与冷冻电镜数据一致。为了从 NOTCH 的 apo 形式过渡到配体结合形式，我们遵循原子和整合建模方法来模拟 NOTCH-DLL4 和 NOTCH-JAG1 之间的相互作用。较小结合片段 EGF8-13 贴片的原子模拟揭示了对 NOTCH 结合至关重要的构象可塑性，而全长复合物的整合建模表明结合表面比以前报道的更大。致病性突变的模拟显示，E360K 和 R448Q 破坏了 NOTCH-配体相互作用表面，导致解离。相比之下，Abruptex 结构域中的 C1133Y 通过破坏结构域与 NOTCH-ECD 的载脂蛋白形式的配体结合结构域的相互作用来损害蛋白质稳定性。 这些发现提供了对 NOTCH 及其配体的详细分子理解，提供了可能使开发选择性靶向致病性 NOTCH 信号转导的新型治疗方法的见解。