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

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 RG 13是β-内酰胺酶（BLA）和麦芽糖结合蛋白（MBP）之间的637个氨基酸工程化变构分子开关[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]。在不存在麦芽糖的情况下，MBP以开放形式存在。麦芽糖结合伴随着围绕铰链的35度弯曲运动，导致蛋白质的闭合形式[Sharff AJ，Rodseth LE，Spurlino JC，Quiocho FA，（1992）Biochemistry 31：10657-10663]。BLA是水解β-内酰胺抗生素如青霉素的β-内酰胺环的酰胺键的单体酶。将环状排列的BLA插入到MBP中，并鉴定出开关（RG 13），其中在不存在麦芽糖的情况下其β-内酰胺水解活性受损，但在麦芽糖存在下增加25倍。我们推断，在开关的MBP结构域的构象变化麦芽糖结合后，将传播到BLA结构域的活性位点，并改变其催化性能，类似于自然变构效应的机制。RG 13是从组合文库中鉴定的，而不是合理设计的，因此，转换发生的分子机制尚不清楚。我们的目标是发展一个结构的开关机制的理解，以指导进一步的假设，如优化和创造新的变构开关分子的各种应用的生成。为了实现这一目标，Ostermeier博士及其同事正在进行NMR实验，包括残余偶极耦合（RDC），核Overhauser增强（NOE）和顺磁弛豫增强（PRE）测量。这些测量为结构建模RG 13提供方向和距离约束。我们将首先使用Rosetta NMR预测开关的结构[拉曼S，Lange OF，Rossi P，Tyka M，Wang X，等人（2010）Science 327：1014-1018;拉曼S，Huang YJ，Mao B，Rossi P，Aramini JM，等人（2010）J Am Chem Soc 132：202-207; Shen Y，弗农R，Baker D，Bax A，（2009）J Biomol NMR 43：63-78]和结构域插入[Berrando M，Ostermeier M，Gray JJ，（2008）Structure 16：513-527]方法。我们先前的NMR研究（15 N，1H-TROSY-HSQC）表明，RG 13的各个结构域结构与MBP和BLA基本上是保守的[Wright CM，Majumdar A，Tolman JR，Ostermeier M，（2010）Proteins 78：1423-1430]。因此，我们可以得到良好的起始结构从Monte Carlo（MC）模拟与NMR约束。然而，蛋白质是灵活和动态的。为了实现我们理解RG 13的变构转换机制的目标，我们必须理解两个亲本蛋白（BLA和MBP）融合和麦芽糖结合后的构象变化以及它们通过柔性接头从MBP到BLA的传播。这一动态过程将通过运行分子动力学（MD）模拟进行研究。将通过运行长时间MD模拟（>60 ns）用NMR弛豫数据比较和测试结构模型及其动力学[Bhattacharya N，Yi M，Zhou HX，Logan TM，（2007）J Mol Biol 374：977-992]。我们将在抗生素分子（如青霉素）的存在下对结构进行建模，这还没有被参数化用于MD模拟。为了准备与CHARMM力场格式兼容的参数，我们将使用Gaussian 03进行量子化学计算。

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.