MOLECULAR DYNAMICS SIMULATIONS TO UNDERSTAND THE EFFECTS OF ACTIVE-SITE MUTATIO

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 摘要：所有的II型限制性内切酶都含有一簇活性部位的谷氨酸和天冬氨酸残基，它们负责催化辅因子镁的配位。直到酶与其特定的DNA识别序列结合，金属离子结合的部位才能完全和准确地组装起来。这提供了一种通过将识别与催化相结合来提高特异性的机制。在特定的EcoRI-DNA界面上，这个活性位点簇包括D91、E111和位于EcoRI特定识别序列内的GPAATTC位置上的剪切型磷氧。D59和E144也位于酸性活性中心簇附近，但不起到配位作用。此外，两个碱性侧链残基位于该酸性簇附近，即R145和K113。活性中心簇中负电荷的对置产生了很强的静电斥力，这种排斥可以通过二价金属的加入或活性中心残基的质子化来缓解。因此，当从蛋白质(通过将活性部位的酸性残基突变为丙氨酸)、从DNA(通过去除剪刀状的磷酸)、添加二价金属离子(作为镁的非催化模拟物)或通过滴定到pH低于6时，结合亲和力显著提高(在KA中~500倍)。当前的兴趣：我们想要扩大我们对活性部位排斥在特定EcoRI-DNA络合物形成的结构、动态和能量方面的作用的理解。具体地说，我们问：1)在游离酶和存在和不存在二价金属的酶-DNA复合体中，带电的活性中心残基的浮子位置和原子涨落的程度是什么；2)这三种结构中活性中心的静电势是什么；3)带电残基突变为中性残基或相反电荷的残基是如何影响三种结构中活性中心残基的浮子构象、原子涨落和静电势的；以及4)在所有三种结构中，活性中心处或附近的水结构是如何受到静电扰动的影响的？分配理由：为了解决上述问题，我们将使用Amber程序套件在存在和不存在DNA和二价金属的情况下，对野生型和突变型EcoRI复合体进行MD模拟和静电势计算。由于这些系统的规模，以及需要运行长时间的模拟才能看到可能的构象波动，我们相信XT3平台将满足我们的需求。我们认识到XT3 Cray系统将于2010年3月31日退役，因此我们在申请中包含了在People SGI Altix 7400系统上的分配，以便将来过渡到该平台。从这些研究中获得的数据，以及我们实验室进行的严格的热力学分析的结果，将使我们能够更好地了解EcoRI-DNA活性位点上酸性残基簇的结构、能量和动力学作用，并使我们能够将该系统与其他具有相似但略有不同的活性位点几何形状的其他II型限制性内切酶的活性位点进行比较。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Abstract: All type II restriction endonucleases contain a cluster of active-site Glu and Asp residues, which are responsible for coordination of the catalytic cofactor Mg2+. The site for metal ion binding is not assembled completely and precisely until the enzyme binds to its specific DNA recognition sequence. This provides a mechanism for enhancing specificity by coupling recognition to catalysis. At the specific EcoRI-DNA interface, this active-site cluster includes D91, E111, and the scissile phosphate oxygen, located at the GPAATTC position, within the EcoRI specific recognition sequence. D59 and E144 are also located in the vicinity of the acidic active-site cluster but do not play a role in coordinating Mg2+. In addition, two basic side-chain residues are located near this acidic cluster namely, R145 and K113. Apposition of the negative charges in the active-site cluster creates strong electrostatic repulsion, which can be relieved by the addition of divalent metal, or by protonation of active-site residues. Thus, large enhancements in binding affinity (~500-fold in KA) are observed upon removal of negative charge from the protein (by mutation of active-site acidic residues to alanine), from the DNA (by removal of the scissile phosphate), by addition of the divalent metal ion Ca2+, which acts as a non-catalytic mimic of Mg2+, or by titration to pH below 6. Current Interests: We want to extend our understanding of the role of active-site repulsion in the structural, dynamic, and energetic aspects of specific EcoRI-DNA complex formation. Specifically, we ask 1) what are the rotameric positions and the degree of atomic fluctuation of the charged active-site residues in the free enzyme, and in the enzyme-DNA complex in the presence and absence of divalent metal, 2) what are the electrostatic potentials at the active-site in all three of these structures, 3) how does mutation of charged residues to a neutral residue, or to a residue of the opposite charge, affect the rotameric conformation, atomic fluctuation, and electrostatic potentials of active-site residues in all three structures, and 4) how is the water structure at or near the active-site affected by electrostatic perturbation in all three structures? Justification for Allocation: In order to address the questions above, we will use the Amber suite of programs to perform MD simulations and electrostatic potential calculations on wild-type and mutant EcoRI complexes, in the presence and absence of DNA and divalent metal. Due to the size of these systems and the need to run long simulations in order to see possible conformational fluctuations, we believe the XT3 platform will suit our needs. We recognize that the XT3 Cray system will be decommissioned on March 31st, 2010, so we include in our request an allocation on the People SGI Altix 7400 system in order to transition to this platform in the future. Data obtained from these studies, along with the results of rigorous thermodynamic analyses performed in our laboratory, will allow us to better understand the structural, energetic, and dynamic role of the acidic cluster of residues at the EcoRI-DNA active-site, and allow us to compare this system with the active-site of other type II restriction endonucleases having similar but slightly different, active-site geometries.