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

通过分子动力学模拟了解活性位点突变的影响

• 批准号: 8171926
• 负责人: LINDA JEN-JACOBSON
• 金额: $ 0.11万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171926
• 关键词: Active Sites; Address; Affect; Affinity; Alanine; Amber; Binding; Catalysis; Charge; Complex; Computer Retrieval of Information on Scientific Projects Database; Coupling; DNA; Data; Electrostatics; Enzymes; Excision; Funding; Future; Grant; Institution; Ions; LGLA; Laboratories; Metal Ion Binding; Molecular Conformation; Mutation; Oxygen; Play; Positioning Attribute; Proteins; Research; Research Personnel; Resources; Role; Running; Side; Site; Source; Specificity; Structure; System; Thermodynamics; Titrations; Type II site-specific deoxyribonuclease; United States National Institutes of Health; Water; abstracting; cofactor; divalent metal; inorganic phosphate; interest; molecular dynamics; mutant; programs; protonation; simulation; Active Sites; Address; Affect; Affinity; Alanine; Amber; Binding; Catalysis; Charge; Complex; Computer Retrieval of Information on Scientific Projects Database; Coupling; DNA; Data; Electrostatics; Enzymes; Excision; Funding; Future; Grant; Institution; Ions; LGLA; Laboratories; Metal Ion Binding; Molecular Conformation; Mutation; Oxygen; Play; Positioning Attribute; Proteins; Research; Research Personnel; Resources; Role; Running; Side; Site; Source; Specificity; Structure; System; Thermodynamics; Titrations; Type II site-specific deoxyribonuclease; United States National Institutes of Health; Water; abstracting; cofactor; divalent metal; inorganic phosphate; interest; molecular dynamics; mutant; programs; protonation; simulation

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 摘要：所有II型限制性核酸内切酶都包含一个有源位点GLU和ASP残基的簇，这些残基负责协调催化辅因子MG2+。金属离子结合的位点直到酶与其特异性DNA识别序列结合之前，才完全和精确地组装。这提供了一种通过耦合识别与催化来增强特异性的机制。在特定的ECORI-DNA界面上，该活性位点簇包括D91，E111和位于ECORI特异性识别序列内GPAATTC位置的s骨磷酸盐氧。 D59和E144也位于酸性活性位点簇的附近，但在协调MG2+方面不起作用。此外，两个基本的侧链残基位于这个酸性簇附近，即R145和K113。负电荷在活性位点簇中的含量会产生强烈的静电排斥，这可以通过添加二价金属或通过质子化的活性位点残基来缓解。 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想要扩展我们对特定ECORI-DNA复合物形成的结构，动态和能量方面的作用的理解。 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在所有三个结构中，有源位点残基的旋转构象，原子波动和静电势，以及4）在所有三个结构中受静电扰动影响的活性位点或附近的水结构如何？分配的理由：为了解决上面的问题，我们将使用琥珀色的程序套件在存在和不存在DNA和二价金属的情况下，对野生型和突变的ECORI复合物进行MD模拟和静电潜在计算。由于这些系统的大小以及需要进行长时间模拟以查看可能的构象波动的需求，我们认为XT3平台适合我们的需求。我们认识到，XT3 Cray系统将于2010年3月31日退役，因此我们在请求中包括对People Sgi Altix 7400系统的分配，以便将来过渡到该平台。从这些研究获得的数据，以及在我们的实验室中进行的严格热力学分析的结果，将使我们能够更好地理解ECORI-DNA活性站点中酸性残基的结构，能量和动态作用，并使我们能够将其与具有类似不同的II II型限制性内核的活性站点进行比较，但具有相似的geom，具有相似的geom，该系统与其他类似的II型限制性接种。