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。 多蛋白组装体：我们继续发展粗粒度模型和有效能量函数，以研究具有相对低结合亲和力（Kd > 1微摩尔）的多蛋白质复合物的热力学和结构性质。 折叠的蛋白质结构域表示为刚体。结构域之间的相互作用被处理在残基水平与氨基酸依赖的对电位和Debye-Huckel型静电相互作用。连接刚性蛋白质结构域的柔性接头肽表示为具有适当拉伸、弯曲和扭转角电位的聚合物上的氨基酸珠。为了增强具有长连接器或系绳的多蛋白质组装体的采样，从而导致网络拓扑结构，我们添加了构建高斯链类型连接的选项。 利用经验证的模型，与Hurley博士（NIDDK）的小组合作，我们模拟了由Hrs和STAM蛋白组成的人ESCRT-0复合物（Ren等人，结构，2009年）。 ESCRT-0有助于将泛素化的细胞表面受体分选到溶酶体进行降解。通过将先前解决的域结构和流体动力学测量的信息与我们的模拟模型相结合，我们能够构建完整的ESCRT-0复合物的结构。 我们对ESCRT-0的模拟揭示了一个非常适合不同功能的动态构象集合。 模拟方法：分子动力学（MD）模拟被广泛用于研究生物大分子的结构和动力学。 但是，尽管它们被广泛使用，两个主要问题是困扰MD模拟：描述分子相互作用的基本力场的准确性，以及计算采样的效率。 我们在两个方向都取得了进展。 基于与Best博士（剑桥）合作完成的针对肽的实验数据的电流力场的仔细验证，我们可以表明能量函数中的相对较小的校正导致在MD模拟中产生正确的二级结构偏好的能力的显著提高（Best等人，物理化学杂志B 2009）。 我们还研究了复制交换分子动力学，这是一种广泛使用的增强生物分子构象空间采样的方法（罗斯塔等人，J. Chem. Theory Comput. 2009），并且可以表明使用某些恒温器可以导致蛋白质折叠的偏置解链曲线。 酶功能 我们与Gutkind博士（NIDCR）和Turjanski教授（布宜诺斯艾利斯大学）合作，研究了丝裂原活化蛋白激酶（MAPK）的酶功能。 MAPK信号通路在将环境刺激物转导至细胞核中起重要作用，从而调节多种细胞过程，包括细胞增殖、分化和程序性细胞死亡。我们已经模拟了激酶ERK与靶肽的相互作用，并分析了对Ser/Thr-Pro基序的特异性。通过使用量子力学/分子力学（QM/MM）方法，我们确定了磷酰基转移反应的机制（Turjanski等人，J. Am. 2009）。我们的研究结果表明：（1）脯氨酸残基在特异性和磷转移效率方面都有作用，（2）反应在一个步骤中发生，ERK 2 Asp 147作为催化碱基，（3）激酶超家族中保守的Lys强烈稳定过渡态，（4）反应机制与活性位点中的一个或两个Mg 2+离子相似。两者合计，我们的研究结果提供了一个详细的描述，参与MAPK催化的磷酸化反应的分子事件，并有助于激酶活性的一般理解。

We have made significant progress in several areas related to protein dynamics, folding, binding, and function. Multi-protein assemblies: We continued our development of coarse-grained models and effective energy functions to study the thermodynamic and structural properties of multiprotein complexes with relatively low binding affinity (Kd > 1 micromolar). Folded protein domains are represented as rigid bodies. The interactions between the domains are treated at the residue level with amino-acid-dependent pair potentials and Debye-Huckel-type electrostatic interactions. Flexible linker peptides connecting rigid protein domains are represented as amino acid beads on a polymer with appropriate stretching, bending, and torsion-angle potentials. To enhance the sampling of multi-protein assemblies with long linkers or tethers that result in a network topology, we added the option of constructing Gaussian-chain type connections. With the validated model, in collaboration with the group of Dr. Hurley (NIDDK), we simulated the human ESCRT-0 complex comprised of Hrs and STAM proteins (Ren et al., Structure, 2009). ESCRT-0 helps sort ubiquitinated cell surface receptors to lysosomes for degradation. By combining information from previously solved domain structures and hydrodynamic measurements with our simulation model, we were able to build a structure for the complete ESCRT-0 complex. Our simulations of ESCRT-0 revealed a dynamic ensemble of conformations well suited for diverse functions. Simulation methodology: Molecular-dynamics (MD) simulations are widely used to study the structure and dynamics of bio-macromolecules. But despite their widespread use, two major issues are plaguing MD simulations: the accuracy of the underlying force fields describing the molecular interactions, and the efficiency of the computational sampling. We have made progress in both directions. Based on a careful validation of current force fields against experimental data on peptides, done in collaboration with Dr. Best (University of Cambridge), we could show that relatively minor corrections in the energy functions resulted in a dramatic improvement in the ability to produce correct secondary structure preferences in MD simulations (Best et al., J. Phys. Chem. B 2009). We also examined replica-exchange molecular dynamics, a widely used method to enhance the sampling of the conformation space of biomolecules (Rosta et al, J. Chem. Theory Comput. 2009) and could show that the use of certain thermostats can result in biased melting profiles for protein folding. Enzyme function. In collaboration with Dr. Gutkind (NIDCR) and Prof. Turjanski (Univ. Buenos Aires), we have studied the enzymatic function of mitogen-activated protein kinases (MAPK). MAPK signaling pathways play an essential role in the transduction of environmental stimuli to the nucleus, thereby regulating a variety of cellular processes, including cell proliferation, differentiation, and programmed cell death. We have modeled the interaction of the kinase ERK with a target peptide and analyzed the specificity toward Ser/Thr-Pro motifs. By using a quantum mechanics/molecular mechanics (QM/MM) approach, we identified a mechanism for the phosphoryl transfer reaction (Turjanski et al., J. Am. Chem. Soc. 2009). Our results suggest that (1) the proline residue has a role in both specificity and phosphor transfer efficiency, (2) the reaction occurs in one step, with ERK2 Asp147 acting as the catalytic base, (3) a conserved Lys in the kinase superfamily strongly stabilizes the transition state, and (4) the reaction mechanism is similar with either one or two Mg2+ ions in the active site. Taken together, our results provide a detailed description of the molecular events involved in the phosphorylation reaction catalyzed by MAPK and contribute to the general understanding of kinase activity.