Understanding the structural dynamics of TNF receptors

摘要 肿瘤坏死因子受体（TNFRs）是一个跨膜蛋白超家族，在肿瘤的发生、发展和治疗中起着重要作用 在细胞凋亡和炎性疾病中，被认为是重要的治疗靶点。尽管针对 TNFRs是一个价值数十亿美元的产业，临床上可用的药物会引起毁灭性的副作用，因为它们 缺乏受体特异性。我的研究重点是了解TNFRs的基本构象动力学 它将信号传递到细胞膜上，最终目标是实现高效和特异性的 面向.为了加速科学发现，我们专注于两个最临床相关的成员， TNFR 1超家族，参与各种自身免疫性疾病，包括类风湿性关节炎; 受体5，最积极追求的抗癌靶点之一。我们采用的调查策略包括 计算分子建模，热力学计算和体外实验工具，使我们能够 预测和理解这些单程跨膜蛋白的构象变化。我们的工作 在高影响力期刊上发表了重要发现。例如，我们阐明了配体 结合TNFR 1和DR 5。我们发现，结合是由甲硫氨酸和 芳香族氨基酸，引起配体结合口袋的构象重排。我们对此的研究 互动主题导致了一个根本性的发现，回答了一个长期存在的问题， 甲硫氨酸在蛋白质折叠中的作用，以及甲硫氨酸氧化如何导致蛋白质错误折叠。我们建立了一个新的 TNFR寡聚化模型，使我们发现配体结合导致大规模骨架 受体胞外结构域的构象变化。这一发现修订了先前的假设 关于TNFR，活化发生时受体骨架没有任何构象变化。与 在计算、生物物理和细胞实验中，我们还首次展示了一个剪刀状的开口， 发生在跨膜结构域螺旋中，并解释了这一基本热力学 过程值得注意的是，使用基于FRET的小分子发现，我们建立了TNFR的新模型 激活，并表明TNFR构象状态的变构改变可以抑制激活， 从而为治疗干预开辟了新的途径。我们打算通过以下方式来扩展我们的发现 整合跨受体域的动态模式，并回答基本问题： 是TNFR激活的结构和动力学机制？我们将讨论一些有影响力的问题， 这可能是高风险的，但有可能在该领域发生变革，并在药物治疗领域开辟新的方向。 的发现我们的生产力通过长期的跨学科合作得到提高， 生物物理工具，包括EPR和NMR。MIRA赠款将提供灵活性，有条不紊，深入， 解决有关TNFR信号的基本问题，这将大大加强识别和 合理地瞄准这些重要蛋白质中最脆弱的结构基序。

ABSTRACT The tumor necrosis factor receptors (TNFRs) are a superfamily of transmembrane proteins that play critical roles in apoptosis and inflammatory diseases and are considered important therapeutic targets. Even though targeting of TNFRs is a billion-dollar industry, the clinically available drugs cause devastating side effects because they lack receptor specificity. My research focuses on understanding the essential conformational dynamics of TNFRs that transduce signals across the membrane, with the ultimate goal of enabling highly effective and specific targeting. To accelerate scientific discovery, we have focused on two of the most clinically relevant members of the superfamily: TNFR1, involved in various autoimmune diseases, including rheumatoid arthritis; and Death Receptor 5, one of the most actively pursued anti-cancer targets. We apply an investigative strategy that includes computational molecular modeling, thermodynamic calculations, and in vitro experimental tools, enabling us to predict and understand conformational changes in these single-pass transmembrane proteins. Our work has yielded important findings published in high-impact journals. For instance, we elucidated mechanisms of ligand binding in both TNFR1 and DR5. We found that binding is controlled by an interaction between methionine and aromatic amino acids, causing a conformational rearrangement of the ligand-binding pocket. Our studies of this interaction motif led to a fundamental discovery that answered a long-standing question regarding the role of methionine in protein folding, and further, how methionine oxidation causes protein misfolding. We built a new model of TNFR oligomerization that led us to discover that ligand binding causes a large-scale backbone conformational change in the extracellular domain of the receptor. This finding revised previous assumptions regarding TNFRs that activation occurs without any conformational changes in the receptor backbone. With computation and biophysical and cellular experiments, we also showed for the first time a scissors-like opening that occurs in the transmembrane domain helices and explained the fundamental thermodynamics of this process. Significantly, using FRET-based small molecule discovery, we built on our new model of TNFR activation and showed that allosteric alteration of the conformational states of TNFRs can inhibit activation, and have thereby opened new avenues to therapeutic intervention. We propose to extend our discoveries by integrating the dynamic modes across domains of the receptor and answering the fundamental question: what is the structural and dynamic mechanism of TNFR activation? We will address impactful questions, some of which may be high-risk, but with potential to be transformative in the field and to launch new directions in drug discovery. Our productivity is enhanced by longstanding interdisciplinary collaborations that engage additional biophysical tools, including EPR and NMR. The MIRA grant will provide flexibility to methodically, and deeply, address fundamental questions regarding TNFR signaling, which will profoundly enhance efforts to identify and rationally target the most vulnerable structural motifs in these important proteins.!