Modeling Sweet Protein-Receptor Interactions

描述(申请人提供)：模拟甜味蛋白-受体的相互作用本研究提案的目的是了解甜味蛋白和甜味受体(STR)之间相互作用的本质。STR是T1R2和T1R3的异源二聚体，T1R3是C家族的G蛋白偶联受体(GPCRs)。越来越多的研究表明，STR与不同甜味剂有多个配体结合位点。虽然目前还没有STR的晶体结构，但可以基于适当的模板开发STR的同源模型。金星捕蝇器模块(Vftm)和富含半胱氨酸结构域(CRD)可以根据代谢性谷氨酸受体(MGluRs)的晶体结构进行模拟。T1R2和T1R3的跨膜区(TMD)可以根据牛视紫红质的晶体结构进行模拟。我们已经成功地利用甜味受体的同源模型、甜味配体与受体的分子对接和受体的定点突变来确定甜味受体的潜在配体结合部位。我们的研究表明，hT1R2的vftm是甜味剂阿斯巴甜和纽甜的结合部位，而hT1R3的tmd是甜味剂甜蜜素和甜味剂乳糖醇的结合部位。最近的嵌合体和突变研究表明，STR的vftm和CRD都参与了甜蛋白-受体的相互作用。为了研究甜蛋白与STR的相互作用，我们建议构建甜蛋白与vftm-CRD复合体的模型。最近解析的mGluR-I/II的vftm-CRD的晶体结构为建立STR的vftm-CRD的同源模型提供了理想的模板。基于mGluR模板的晶体结构，我们提出了几个代表不同构象状态的STR同源模型的VFTM-CRD。改进的STR的vftm-CRD模型将被用来通过布朗动力学(BD)模拟对接甜蛋白，以了解甜蛋白与受体的相互作用。结合定点突变实验和BD对接模拟，我们将确定STR的vftm-CRD与甜蛋白的潜在复合体。预测的复合体模型将通过诱变研究进行测试，以评估预测的成功。这些研究将有助于更好地理解STR的作用机制。与公共卫生相关：在发达国家，肥胖和糖尿病已经达到了流行的程度。用低热量或无热量的甜味剂取代糖可能是有益的。为了设计更有效的甜味剂，重要的是要在分子水平上了解甜味受体是如何发挥作用的。

DESCRIPTION (provided by applicant): Modeling Sweet Protein-Receptor Interactions The goal of this research proposal is to understand the nature of the interactions between sweet proteins and sweet taste receptor (STR). STR is a heterodimer of T1R2 and T1R3, which is a G protein-coupled receptor (GPCRs) from Family C. Growing evidence shows that STR has multiple ligand binding sites for different sweeteners. Although no crystal structure of STR is available at this time, homology models of STR can be developed based on the appropriate templates. The Venus Flytrap Module (VFTM) and Cysteine Rich Domain (CRD) can be modeled based on the crystal structures of metabotropic glutamate receptors (mGluRs). Transmembrane Domain (TMD) of T1R2 and T1R3 can be modeled based on the crystal structure of bovine rhodopsin. We have successfully used homology models of the sweet taste receptors, molecular docking of sweet ligands to the receptors, and site-directed mutagenesis of the receptors to identify potential ligand binding sites of the sweet taste receptor. Our studies show that the VFTM of hT1R2 is the binding site for sweeteners aspartame and neotame, while the TMD of hT1R3 is the binding site for the sweetener cyclamate and the sweet inhibitor lactisole. Recent chimera and mutagenesis studies show that both the VFTM and CRD of STR are involved in sweet protein-receptor interactions. To investigate the interactions of sweet proteins with STR we propose to construct models of the VFTM-CRD in complex with sweet proteins. The crystal structures of the VFTM-CRD of mGluR-I/II, which were resolved recently, provide us an ideal template to develop homology models for the VFTM-CRD of STR. Here we propose to develop several VFTM-CRD of STR homology models, which represent different conformational states, based on the crystal structures of mGluR templates. The refined VFTM-CRD models of STR will be used for docking the sweet proteins by Brownian Dynamics (BD) simulations to understand sweet protein-receptor interactions. In combination with site-directed point mutational experiments and BD docking simulations, we will identify potential complexes of the VFTM-CRD of STR with sweet proteins. The predicted complexes models will be tested via mutagenesis studies to assess the success of the predictions. These studies should lead to a better understanding the function mechanism of STR. PUBLIC HEALTH RELEVANCE: Obesity and diabetes have reached epidemic proportions in developedsocieties. Replacing sugar with low-or non-caloric sweeteners may be of benefit. To design moreeffective sweeteners it is important to understand at the molecularlevel how the sweet taste receptor functions.