Theory and simulation of protein dynamics, folding, and function

我们在与蛋白质动力学、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)骨架的切割(1)。这一反应是由核糖核酸酶H(RNaseH)催化的，核糖核酸酶H是使用双金属催化处理核酸的酶大家族中的典型成员。这个家族包括HIV逆转录酶，它是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RNaseH结构域。通过计算参与反应的原子对之间的距离的多维自由能表面，我们可以表征RNA裂解反应的机理。在第一个反应步骤中，水分子在镁离子A的辅助下攻击剪切的磷酸盐。这种攻击导致磷酸盐和核糖氧之间的键断裂。在第二步中，反应通过核糖的质子化来完成。我们发现，中性的Asp132提供了一个可能的质子供体。反应的关键步骤及其能量学与广泛的实验是一致的。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的RNaseH功能抑制剂，如HIV。 大型多蛋白组件的结构和动力学。我们将粗粒度模拟模型和实验相结合，研究了大分子多蛋白复合体和含有部分无序片段的模块蛋白的结构和运动。为此，我们开发了一种集合精化方法，允许我们将模拟集合和低分辨率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 CHMP3的详细结构，该蛋白是ESCRT膜蛋白运输途径中的关键蛋白(2)。在赫尔利博士和伊顿博士(都是NIDDK，NIH)的实验小组的合作下，我们还获得了ESCRT-I的结构，ESCRT-I是ESCRT途径的另一种必需蛋白质(3)。此外，在与赫尔利博士的合作下，我们还可以表征蛋白激酶C(PKC)在自动抑制状态下的溶液结构(4)。PKcs调节多种生理功能，从T细胞识别到细胞增殖和分化，以及神经元信号转导。结合中间态PKC的晶体结构，这项工作为这一重要酶的变构调节提供了新的见解。 蛋白质-蛋白质相互作用网络。有了蛋白质-蛋白质相互作用网络的理论模型，我们可以为观察到从线虫到人类的多细胞生物具有大致相同数量的蛋白质编码基因提供可能的物理解释(5)。我们的计算表明，为了防止蛋白质之间致病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，针对错误结合优化结合界面的需要可能会影响蛋白质-蛋白质相互作用网络的拓扑。 1.E.Rosta，M.Nowotny，W.Yang，G.Hummer，核糖核酸酶H催化核糖核酸酶断裂的机理化学。SoC。133 8934-8941(2011)。 2.B.Rozycki，Y.C.Kim，G.Hummer，《ESCRT-III CHMP3构象转变的SAXS系综精炼》，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，ESCRT-I复合体的溶液结构小角X射线散射，EPR和FRET光谱分析，Proc.娜塔莉。阿卡德。SCI。美国108,9437-9442(2011)。 4.T.A.Leonard，B.Rozycki，L.F.Saidi，G.Hummer，J.H.Hurley，蛋白激酶C的晶体结构和变构激活II，细胞144，55-66(2011)。 5.M.E.Johnson，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。娜塔莉。阿卡德。SCI。美国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).