Molecular Mechanism of Protein Arginine Deiminase 2: A Study Involving Multiple Microsecond Long Molecular Dynamics Simulations

Peptidylarginine deiminase 2 (PAD2) is a Ca2+-dependent enzyme that catalyzes the conversion of protein arginine residues to citrulline. This kind of structural modification in histone molecules may affect gene regulation, leading to effects that may trigger several diseases, including breast cancer, which makes PAD2 an attractive target for anticancer drug development. To design new effective inhibitors to control activation of PAD2, improving our understanding of the molecular mechanisms of PAD2 using up-to-date computational techniques is essential. We have designed five different PAD2–substrate complex systems based on varying protonation states of the active site residues. To search the conformational space broadly, multiple independent molecular dynamics simulations of the complexes have been performed. In total, 50 replica simulations have been performed, each of 1 μs, yielding a total simulation time of 50 μs. Our findings identify that the protonation states of Cys647, Asp473, and His471 are critical for the binding and localization of the N-α-benzoyl-l-arginine ethyl ester substrate within the active site. A novel mechanism for enzyme activation is proposed according to near attack conformers. This represents an important step in understanding the mechanism of citrullination and developing PAD2-inhibiting drugs for the treatment of breast cancer.肽基精氨酸脱亚胺酶2（Peptidylarginine deiminase 2，PAD2）是一种钙离子依赖型酶，可催化蛋白质精氨酸残基转化为瓜氨酸。组蛋白分子中的这类结构修饰可影响基因调控，进而引发包括乳腺癌在内的多种疾病，这使得PAD2成为抗肿瘤药物开发的极具潜力的靶点。为设计可有效调控PAD2激活的新型抑制剂，借助前沿计算技术深入解析PAD2的分子机制至关重要。我们基于活性位点残基的不同质子化状态，构建了五种不同的PAD2-底物复合物体系。为全面探索构象空间，我们对该复合物开展了多组独立的分子动力学模拟。总计完成50组重复模拟，单组模拟时长为1微秒，总模拟时长达到50微秒。本研究结果显示，Cys647、Asp473与His471的质子化状态，对于N-α-苯甲酰基-L-精氨酸乙酯底物在活性位点内的结合与定位具有关键作用。基于近攻击构象（near attack conformers），我们提出了一种全新的酶激活机制。本研究为阐明瓜氨酸化机制以及开发用于乳腺癌治疗的PAD2抑制剂迈出了重要一步。