DEVELOP AND TEST MODELS OF THE STRUCTURE AND THE ALLOSTERIC SWITCHING MECHANISM

结构和变构切换机制的开发和测试模型

• 批准号: 8364341
• 负责人: Myunggi Yi
• 金额: $ 0.1万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-15 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8364341
• 关键词: Active Sites; Amides; Amino Acids; Antibiotics; Binding; Biochemistry; Biomedical Research; Coupling; Data; Engineering; Enzymes; Funding; Generations; Goals; Grant; Gray unit of radiation dose; High Performance Computing; Hydrolysis; Individual; Libraries; Maltose; Measurement; Modeling; Molecular; Monobactams; Motion; National Center for Research Resources; Parents; Penicillins; Principal Investigator; Process; Property; Protein Binding; Proteins; Relaxation; Research; Research Infrastructure; Residual state; Resources; Running; Science; Source; Structural Models; Structure; Tertiary Protein Structure; Testing; Time; United States National Institutes of Health; beta-Lactamase; beta-Lactams; cost; design; flexibility; maltose-binding protein; molecular dynamics; novel; nuclear Overhauser enhancement; quantum chemistry; research study; simulation

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. RG13 is a 637-aminoacid engineered allosteric molecular switch between beta-lactamase (BLA) and maltose binding protein (MBP) [Gutas G, Mitchell SF, Ostermeier M, (2004) Chem Biol 11:1483-1487; Guntas G, Mansell TJ, Kim JR, Ostermeier M, (2005) Proc Natl Acad Sci USA 102:11224-11229]. In the absence of maltose, MBP exists in an open form. Maltose binding is concomitant with a 35 degree bending motion about the hinge, resulting in the closed form of the protein [Sharff AJ, Rodseth LE, Spurlino JC, Quiocho FA, (1992) Biochemistry 31:10657-10663]. BLA is a monomeric enzyme that hydrolyzes the amide bond of the beta-lactam ring of beta-lactam antibiotic such as penicillin. The circularly permuted BLA was inserted into the MBP, and a switch (RG13) was identified in which its beta-lactam hydrolysis activity was compromised in the absence of maltose but increased 25-fold in the presence of maltose. We reasoned that in the switch the conformational change in the MBP domain upon maltose binding would propagates to the active site of the BLA domain and alter its catalytic properties, a mechanism analogous to natural allosteric effects. RG13 was identified from a combinational library rather than rationally designed, and thus, the molecular mechanism by which switching occurs is not known. Our goal is to develop a structural understanding of the switching mechanism in order to guide generation of further hypotheses such as optimizing and creating novel allosteric switch molecules for various applications. In order to achieve the goal, Dr. Ostermeier and co-workers are conducting NMR experiments including residual dipolar coupling (RDC), nuclear Overhauser enhancement (NOE), and paramagnetic relaxation enhancement (PRE) measurements. These measurements provide orientation and distance constraints for structural modeling RG13. We would first predict the structures of the switch using Rosetta NMR [Raman S, Lange OF, Rossi P, Tyka M, Wang X, et al. (2010) Science 327:1014-1018; Raman S, Huang YJ, Mao B, Rossi P, Aramini JM, et al. (2010) J Am Chem Soc 132:202-207; Shen Y, Vernon R, Baker D, Bax A, (2009) J Biomol NMR 43:63-78] and domain insertion [Berrando M, Ostermeier M, Gray JJ, (2008) Structure 16:513-527] approaches. Our previous NMR studies (15N, 1H-TROSY-HSQC) indicate that the individual domain structures of RG13 are substantially conserved from MBP and BLA [Wright CM, Majumdar A, Tolman JR, Ostermeier M, (2010) Proteins 78:1423-1430]. Therefore we can get good starting structures from Monte Carlo (MC) simulations with NMR constraints. Proteins, however, are flexible and dynamic. In order to achieve our goal to understand the allosteric switching mechanism of RG13, we have to understand conformational changes upon fusion of two parent proteins (BLA and MBP) and binding of maltose and their propagation from MBP to BLA through the flexible linkers. This dynamic process will be investigated by running molecular dynamics (MD) simulations. Structural model and its dynamics will be compared and tested with NMR relaxation data by running long time MD simulations (>60 ns) [Bhattacharya N, Yi M, Zhou HX, Logan TM, (2007) J Mol Biol 374:977-992]. We would model the structure in the presence of antibiotic molecules such as penicillin, which has not been parameterized for MD simulations. In order to prepare the parameters compatible to CHARMM force field format, we would perform quantum chemistry calculations with Gaussian03.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 RG13是β-内酰胺酶（BLA）和麦芽糖结合蛋白（MBP）之间的637-氨基酸工程性变构分子转换[Gutas G，Mitchell SF，Ostermeier M，（2004）Chem Biol，Chem Biol 11：1483-1487; Guntas G，Mansell TJ，Kim JR，Ostermeier M，（2005）Proc Natl Acad Sci USA 102：11224-11229]。在没有麦芽糖的情况下，MBP以开放形式存在。麦芽糖的结合与铰链的35度弯曲相关，导致蛋白质的封闭形式[Sharff AJ，Rodseth Le，Spurlino JC，Quiocho FA，（1992）生物化学31：10657-10663]。 BLA是一种单体酶，可以水解β-内酰胺抗生素（例如青霉素）的β-内酰胺环的酰胺键。将圆形置换的BLA插入MBP，并鉴定出一个开关（RG13），其中在没有麦芽糖的情况下会损害其β-内酰胺水解活性，但在麦芽糖存在下增加了25倍。我们认为，在开关中，麦芽糖结合时MBP结构域中的构象变化将传播到BLA结构域的活性位点并改变其催化特性，这是一种类似于天然变构效应的机制。从组合文库而不是合理设计的RG13鉴定出RG13，因此，开关发生的分子机制尚不清楚。我们的目标是对开关机制产生结构性理解，以指导生成进一步的假设，例如优化和为各种应用创建新型的变构开关分子。为了实现目标，Ostermeier博士和同事正在进行NMR实验，包括残留偶极耦合（RDC），核过度大冲突器增强（NOE）和顺磁性松弛增强（PER）测量。这些测量值为结构建模RG13提供了方向和距离约束。我们首先使用Rosetta NMR [Raman S，Lange of，Rossi P，Tyka M，Wang X等人。 （2010）科学327：1014-1018; Raman S，Huang YJ，Mao B，Rossi P，Aramini JM等。 （2010）J AM Chem Soc 132：202-207; Shen Y，Vernon R，Baker D，Bax A，（2009）J Biomol NMR 43：63-78]和域插入[Berrando M，Ostermeier M，M，Gray JJ，（2008）结构16：513-527]方法。我们以前的NMR研究（15N，1H-TROSY-HSQC）表明，RG13的各个域结构基本上来自MBP和BLA [Wright CM，Majumdar A，Tolman A，Ostermeier JR，Ostermeier M，（2010年）蛋白78：1423-1430蛋白]。因此，我们可以从具有NMR约束的蒙特卡洛（MC）模拟中获得良好的起始结构。但是，蛋白质是柔性且动态的。为了实现我们的目标，以了解RG13的变构开关机制，我们必须了解两种母蛋白（BLA和MBP）融合后的构象变化，以及麦芽糖的结合及其通过灵活的接头从MBP到BLA的传播。该动态过程将通过运行分子动力学（MD）模拟来研究。结构模型及其动力学将通过运行长期MD模拟（> 60 ns）[Bhattacharya N，Yi M，Yi M，Yi M，Zhou HX，Logan TM，（2007）J Mol Biol 374：977-992]进行比较和测试。 。我们将在存在抗生素分子（例如青霉素）的情况下建模，该结构尚未用于MD模拟的参数化。为了准备与Charmm力场格式兼容的参数，我们将使用Gaussian03进行量子化学计算。