Computationally Guided Design of Thermostable mutants of Neurotensin receptor1

说明(申请人提供)：神经降压素受体属于多肽激活的G蛋白偶联受体(GPCRs)超家族，是治疗精神分裂症甚至癌症等疾病的重要药物靶点。NTS1和许多多肽激活的GPCRs药物设计的复杂性部分源于缺乏结构信息，这些信息可以为合理的药物设计提供重要信息。膜结合神经降压素受体的结构和生物物理研究的障碍主要是由于三个挑战：1.获得足够水平的哺乳动物受体表达，2.将其功能重组为膜模拟物，3.由于GPCRs的构象灵活性，最重要的是稳定特定的非活性或活性状态构象。虽然步骤(1)和(2)是可以克服的，但步骤(3)GPCRs的构象灵活性严重阻碍了进一步结晶或GPCRs的任何其他生物物理研究的蛋白质纯化过程。因此，迎接挑战(3)对于解决大分子结构和配体与GPCRs的结合至关重要，需要结合预测计算模型和实验技术。我们建议开发一种计算方法“LiticonDesign”，它将能够设计出大鼠神经降压素受体1(NTS1)(一般为A类GPCRs)的热稳定突变体。LiticonDesign将基于“Liticon”，这是一种预测方法，允许对GPCRs的各种功能配体稳定构象状态进行准确建模。预测的NTS1突变体将被表达并测试热稳定性，并获得足够数量的纯功能受体用于结构研究。本工作为获得稳定、功能纯净的NTS1蛋白提供了一种突破性的方法，可推广到其他A类GPCRs。拟议的跨学科合作是希望之城的Nagarajan Vaidehi博士(PI)、NIH的Reinhard Grisshammer NINDS博士和Christopher Tate博士(Univ.英国剑桥大学)。Vaidehi博士在开发GPCRs的建模技术方面拥有丰富的经验，而Tate博士是获得GPCRs的耐热突变体的专家。格里沙默博士是神经降压素受体及其药理学方面的专家。预算只分配给Vaidehi博士，因为Grisshammer博士是由NINDS的内部资金资助的，而Tate博士有足够的资金来开展这项工作。拟议中的工作正在他的实验室进行。

DESCRIPTION (provided by applicant): Neurotensin receptors belong to the superfamily of peptide activated G protein-coupled receptors (GPCRs) and they are important drug targets to diseases such as schizophrenia and even cancer. The complexity in drug design for NTS1 and for many peptide activated GPCRs partly stems from the paucity of structural information, which could provide vital information for rational drug design. The barriers to pursuing structural and biophysical studies of membrane bound neurotensin receptors are due primarily to three challenges that are: 1.obtaining sufficient levels of expressed mammalian receptors, 2. functionally reconstituting them into membrane mimetics and 3. most importantly, stabilizing a particular inactive or active state conformation due to the conformational flexibility of GPCRs. While steps (1) and (2) are surmountable, step (3) the conformational flexibility in GPCRs, severely hampers the protein purification process for further crystallization or any other biophysical studies of GPCRs. Therefore meeting challenge (3) is critical in solving the macromolecular structure and ligand binding to GPCRs and requires a confluence of predictive computational models accompanied by experimental techniques. We propose to develop a computational method "LiticonDesign" that would enable design of thermally stable mutants of rat neurotensin receptor 1 (NTS1) (class A GPCRs in general). LiticonDesign will be based on the "Liticon", a predictive method that allows accurate modeling of various functional ligand stabilized conformational states of GPCRs. The predicted mutants of NTS1 will be expressed and tested for thermal stability and derive sufficient quantities of the pure functional receptor for structural studies. The results of this proposed work will provide a breakthrough method to obtain stable and functional pure protein of NTS1 which could be extended to other class A GPCRs. The proposed interdisciplinary collaboration is between Dr. Nagarajan Vaidehi (PI) at City of Hope, Dr. Reinhard Grisshammer NINDS/NIH and Dr. Christopher Tate (Univ. of Cambridge UK). Dr. Vaidehi has extensive experience in developing modeling techniques for GPCRs, while Dr. Tate is an expert in deriving thermostable mutants of GPCRs. Dr. Grisshammer is an expert on neurotensin receptors and their pharmacology. The budget is allocated only for Dr. Vaidehi, since Dr. Grisshammer is funded by the intramural funding in NINDS and Dr. Tate has sufficient funding for this work. The proposed work is ongoing in his laboratory.