Studying the collective motions of the adenosine A2A receptor as a result of ligand binding using principal component analysis

Adenosine receptors (ARs) belong to family A of GPCRs that are involved in many diseases, including cerebral and cardiac ischemic diseases, immune and inflammatory disorders, etc. Thus, they represent important therapeutic targets to treat these conditions. Computational techniques such as molecular dynamics (MD) simulations permit researchers to obtain structural information about these proteins, and principal component analysis (PCA) allows for the identification of collective motions. There are available structures for the active form (3QAK) and the inactive form (3EML) of A2AR which permit us to gain insight about their activation/inactivation mechanism. In this work, we have proposed an inverse strategy using MD simulations where the active form was coupled to the antagonist caffeine and the inactive form was coupled to adenosine agonist. Moreover, we have included four reported thermostabilizing mutations in the inactive form to study A2AR structural differences under different conditions. Some observations stand out from the PCA studies. For instance, the apo structures showed remarkable similarities, and the principal components (PCs) were rearranged in a ligand-dependent manner. Additionally, the active conformation was less stable compared to the inactive one. Some PCs inverted their direction in the presence of a ligand, and comparison of the PCs between 3EML and 3EML_ADN showed that adenosine induced major changes in the structure of A2AR. Rearrangement of PCs precedes and drives conformational changes that occur after ligand binding. Knowledge about these conformational changes provides important insights about the activity of A2AR.

腺苷受体（Adenosine receptors, ARs）属于G蛋白偶联受体（G protein-coupled receptors, GPCRs）的A家族，参与包括心脑血管缺血性疾病、免疫与炎症紊乱在内的多种疾病进程，因此是治疗此类病症的重要治疗靶点。诸如分子动力学（molecular dynamics, MD）模拟这类计算技术可帮助研究人员获取此类蛋白的结构信息，而主成分分析（principal component analysis, PCA）则可用于识别其集体运动模式。目前已解析出腺苷A2A受体（A2AR）的活性构象（3QAK）与非活性构象（3EML）的晶体结构，可帮助我们深入探究其激活/失活机制。本研究提出了一种基于MD模拟的反向研究策略：将活性构象与拮抗剂咖啡因结合，将非活性构象与腺苷类激动剂结合。此外，我们在非活性构象中引入了4种已报道的热稳定突变，以探究不同条件下A2AR的结构差异。主成分分析实验得到了若干突出的观测结果：例如，无配体（apo）结构展现出显著的结构相似性，且主成分（Principal Components, PCs）的重排呈现配体依赖性。此外，相较于非活性构象，活性构象的稳定性更差。部分主成分在配体存在的情况下会反转运动方向；对比3EML与3EML_ADN的主成分可知，腺苷可诱导A2AR的结构发生显著变化。主成分的重排在配体结合后引发的构象变化之前发生，并驱动这些构象变化。对这些构象变化的认知，可为阐明A2AR的激活机制提供重要见解。