Understanding the structural dynamics of TNF receptors

摘要 肿瘤坏死因子受体(TNFR)是一个跨膜蛋白超家族，发挥着重要的作用 在细胞凋亡和炎症性疾病中发挥重要作用，被认为是重要的治疗靶点。即使目标是 TNFR是一个价值数十亿美元的行业，临床上可用的药物会产生毁灭性的副作用，因为它们 缺乏受体特异性。我的研究重点是了解TNFR的基本构象动力学 在膜上传递信号，最终目标是实现高效和特定的 瞄准目标。为了加速科学发现，我们关注了两个与临床最相关的成员 超级家族：TNFR1，参与各种自身免疫性疾病，包括类风湿性关节炎；以及死亡 受体5，最积极的抗癌靶点之一。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 预测和了解这些单程跨膜蛋白的构象变化。我们的工作已经完成 在影响较大的期刊上发表了重要的研究成果。例如，我们阐明了配体的作用机制。 在TNFR1和DR5中都有结合。我们发现，结合是由蛋氨酸和蛋氨酸之间的相互作用控制的 芳香氨基酸，导致配体结合口袋的构象重排。我们对此的研究 交互作用基序导致了一个根本性的发现，回答了一个长期存在的关于 蛋氨酸在蛋白质折叠中的作用，以及蛋氨酸氧化如何导致蛋白质错误折叠。我们建造了一个新的 导致我们发现配体结合导致大规模骨架的TNFR寡聚模型 受体胞外区的构象变化。这一发现修正了之前的假设 关于TNFR，激活发生在受体骨架没有任何构象变化的情况下。使用 计算以及生物物理和细胞实验，我们还首次展示了一个剪刀状的开口 它发生在跨膜结构域螺旋中，并解释了其基本热力学 进程。值得注意的是，使用基于FRET的小分子发现，我们构建了我们的新的TNFR模型 激活，并表明变构改变的TNFR的构象可以抑制激活，和 从而为治疗干预开辟了新的途径。我们计划通过以下方式扩展我们的发现 整合受体各区域的动态模式并回答基本问题：什么 是TNFR激活的结构和动态机制吗？我们将解决一些有影响力的问题，其中一些 这可能是高风险的，但有可能在该领域产生变革，并在药物领域推出新的方向 发现号。我们的工作效率通过长期的跨学科协作而得到提高，这些协作涉及更多 生物物理工具，包括EPR和核磁共振。米拉赠款将提供灵活性，以有条不紊和深入地， 解决有关TNFR信号的基本问题，这将深刻加强识别和 理性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.!