Rathore Surabhi, Gahlot Deepanshi, Castin Jesu, Pandey Arastu, Arvindekar Shreyas, Viswanath Shruthi, Thukral Lipi
CSIR-Institute of Genomics and Integrative Biology, New Delhi, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India.
CSIR-Institute of Genomics and Integrative Biology, New Delhi, India.
Biophys J. 2025 Jan 21;124(2):393-407. doi: 10.1016/j.bpj.2024.12.014. Epub 2024 Dec 13.
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 1756 amino acids. We performed coarse-grained dynamics simulations of NOTCH in both glycosylated and nonglycosylated 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 semicompact 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 reported previously. 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.
NOTCH是一种单次跨膜蛋白，通过细胞间通讯在细胞命运决定中发挥关键作用。它与位于相邻细胞上的两种典型配体Delta样（DLL）和锯齿状（JAG）相互作用，以调节多种细胞过程。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知之甚少。在本研究中，我们采用基于片段的建模和多尺度模拟来研究包含1756个氨基酸的人NOTCH胞外域的全长结构。我们对糖基化和非糖基化形式的NOTCH进行了粗粒度动力学模拟，以研究糖基化在调节其构象动力学中的作用。在无配体形式下，粗粒度模拟表明，糖基化的NOTCH蛋白可以从距膜表面约86 nm的伸长结构转变为半紧凑状态（约23.81±9.98 nm），这与冷冻电镜数据一致。为了使NOTCH从无配体形式转变为配体结合形式，我们采用原子和整合建模方法来模拟NOTCH-DLL4和NOTCH-JAG1之间的相互作用。较小的结合片段EGF8-13区域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先前报道的更大。致病突变的模拟表明，E360K和R448Q破坏了NOTCH-配体相互作用表面，导致解离。相反，Abruptex结构域中的C1133Y通过破坏该结构域与NOTCH-ECD无配体形式下的配体结合结构域的相互作用而损害了蛋白质稳定性。这些发现提供了对NOTCH及其配体的详细分子理解，为开发选择性靶向致病性NOTCH信号传导的新型治疗方法提供了见解。
