Theory and simulation of protein dynamics, folding, and function

我们已经在与蛋白质动力学、折叠、结合和功能相关的几个领域取得了重大进展。 核酸加工催化：利用量子力学/分子力学（QM/MM）模拟，我们研究了RNA/DNA二聚体中核糖核酸（RNA）主链的切割（1）。 该反应由核糖核酸酶H（RNase H）催化，核糖核酸酶H是使用双金属催化来处理核酸的酶大家族的原型成员。该家族包括HIV逆转录酶，抗逆转录病毒药物的主要靶点，具有对HIV病毒复制至关重要的RNA酶H结构域。通过计算反应中原子间距离的多维自由能面，我们可以表征RNA切割反应的机理。在第一反应步骤中，水分子在镁离子A的辅助下攻击易分裂的磷酸盐。这种攻击导致磷酸盐和核糖氧之间的键断裂。在第二步中，通过核糖的质子化完成反应。 我们发现，中性Asp 132提供了一个可能的质子供体。 反应中的关键步骤及其能量学与广泛的实验一致。 我们的计算揭示了生物学中的一个重要反应，并可能有助于设计一类新的逆转录病毒逆转录酶（如HIV）的RNase H功能抑制剂。 大型多蛋白质组装体的结构与动力学。我们结合了一个粗粒度的模拟模型和实验来研究大的多蛋白质复合物和模块化蛋白质的结构和运动与部分无序的片段。 为此，我们开发了一种集成细化方法，使我们能够将联合收割机模拟集成和低分辨率X射线散射数据结合起来（2）。 通过这种方法，我们能够获得ESCRT-III CHMP 3的详细结构，CHMP 3是ESCRT膜蛋白运输途径中的关键蛋白（2）。 与Hurley博士和Eaton博士（均为NIDDK，NIH）的实验组合作，我们还获得了ESCRT途径的另一种必需蛋白ESCRT-I的结构（3）。 同样与Hurley博士合作，我们可以表征蛋白激酶C（PKC）在自抑制状态下的溶液结构（4）。 PKC调节从T细胞识别到细胞增殖和分化以及神经元信号传导的广泛生理功能。 连同中间状态的PKC的晶体结构，这项工作提供了对这种重要酶的变构调节的新见解。 蛋白质相互作用网络。利用蛋白质相互作用网络的理论模型，我们可以为多细胞生物中C。从线虫到人类，具有大致相同数量的蛋白质编码基因（5）。 我们的计算表明，为了防止引起疾病的蛋白质之间的非特异性相互作用，蛋白质组的大小是有限的。通过蛋白质结合界面的氨基酸序列的集体进化，我们估计了错误结合的程度作为不同蛋白质数量的函数。我们还表明，需要优化结合界面对错误的结合可能会影响蛋白质-蛋白质相互作用网络的拓扑结构。 1. E.罗斯塔，M. Nowotny，W. Yang，G. Hummer，量子力学/分子力学模拟中核糖核酸酶H切割RNA骨架的催化机制，J. Am. 133 8934-8941（2011）。 2. B。Rozycki，Y. C. Kim，G. Hummer，SAXS ensemble refinement of ESCRT-III CHMP3 conformational transitions，Structure 19，109-116（2011）. 3. E.布拉，B。Rozycki，D. Z. Herrick，H. S.阿忠J. Vecer，W. A. Eaton，D. Cafiso，G. Hummer，J. H. Hurley，通过小角X射线散射、EPR和FRET光谱法测定的ESCRT-I络合物的溶液结构，Proc. Natl. Acad. Sci. USA 108，9437-9442（2011）。 4. T. A.伦纳德，B。罗济茨基湖F. Saidi，G. Hummer，J. H. Hurley，Crystal structure and allosteric activation of protein kinase C II，Cell 144，55-66（2011）. 5. M. E.约翰逊，G. Hummer，非特异性结合限制了细胞中蛋白质的数量并形成了它们的相互作用网络，Proc. Natl. Acad. Sci. USA 108，603-608（2011）。

We have made significant progress in several areas related to protein dynamics, folding, binding, and function. Nucleic acid processing catalysis: With quantum mechanics / molecular mechanics (QM/MM) simulations we studied the cleavage of the ribonucleic acid (RNA) backbone in an RNA/DNA dimer (1). This reaction is catalyzed by ribonuclease H (RNase H), a prototypical member of a large family of enzymes that use two-metal catalysis to process nucleic acids. This family includes HIV reverse transcriptase, a primary target for antiretroviral drugs, with an RNase H domain that is essential for HIV viral replication. By calculating the multidimensional free energy surface for distances between pairs of atoms involved the reaction we could characterize the mechanism of the RNA cleavage reaction. In a first reaction step, a water molecule attacks the scissile phosphate, aided by magnesium ion A. This attack results in breaking of the bond between the phosphate and the ribose oxygen. In a second step, the reaction is completed by protonation of the ribose. We found that a neutral Asp132 provides a likely proton donor. Key steps in the reaction and its energetics are consistent with a broad range of experiments. Our calculations shed light on an important reaction in biology and may aid in the design of a new class of inhibitors of the RNase H functionality of retroviral reverse transcriptases such as HIV. Structure and dynamics of large multiprotein assemblies. We combined a coarse-grained simulation model and experiments to study the structures and motions of large multiprotein complexes and modular proteins with partially disordered segments. For this we developed an ensemble refinement approach that allows us to combine simulation ensembles and low-resolution x-ray scattering data (2). With this approach we were able to obtain detailed structures of ESCRT-III CHMP3, a key protein in the ESCRT membrane protein trafficking pathway (2). In collaboration with the experimental groups of Dr. Hurley and Dr. Eaton (both NIDDK, NIH), we also obtained the structure of ESCRT-I, another essential protein of the ESCRT pathway (3). Also in collaboration with Dr. Hurley, we could characterize the solution structure of protein kinase C (PKC) in the autoinhibited state (4). PKCs regulate a wide range of physiological functions that range from T cell recognition to cell proliferation and differentiation, and neuronal signaling. Together with the crystal structure of PKC in an intermediate state, this work provided new insights into the allosteric regulation of this important enzyme. Protein-protein interaction networks. With a theoretical model of protein-protein interaction networks, we could provide a possible physical explanation for the observation that multicellular organisms, from C. elegans to humans, have roughly the same number of protein encoding genes (5). Our calculations suggest that to prevent disease-causing nonspecific interactions between proteins, the proteome size is limited. By collective evolution of the amino-acid sequences of protein binding interfaces we estimated the degree of mis-binding as a function of the number of distinct proteins. We also showed that the need to optimize binding interfaces against misbinding may influence the topology of the protein-protein interaction network. 1. E. Rosta, M. Nowotny, W. Yang, G. Hummer, Catalytic mechanism of RNA backbone cleavage by ribonuclease H from quantum mechanics/molecular mechanics simulations, J. Am. Chem. Soc. 133 8934-8941 (2011). 2. B. Rozycki, Y. C. Kim, G. Hummer, SAXS ensemble refinement of ESCRT-III CHMP3 conformational transitions, Structure 19, 109-116 (2011). 3. E. Boura, B. Rozycki, D. Z. Herrick, H. S. Chung. J. Vecer, W. A. Eaton, D. Cafiso, G. Hummer, J. H. Hurley, Solution structure of the ESCRT-I complex by small angle x-ray scattering, EPR, and FRET spectroscopy, Proc. Natl. Acad. Sci. USA 108, 9437-9442 (2011). 4. T. A. Leonard, B. Rozycki, L. F. Saidi, G. Hummer, J. H. Hurley, Crystal structure and allosteric activation of protein kinase C βII, Cell 144, 55-66 (2011). 5. M. E. Johnson, G. Hummer, Nonspecific binding limits the number of proteins in a cell and shapes their interaction networks, Proc. Natl. Acad. Sci. USA 108, 603-608 (2011).