Mechanisms of Diacylglycerol Signaling Through C1 Domain Proteins

通过 C1 结构域蛋白的二酰甘油信号传导机制

基本信息

批准号：
8349736
负责人：
James Hurley
金额：
$ 31.57万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

Mechanisms of Diacylglycerol Signaling Through C1 Domain Proteins Diacylglycerol (DAG) is a paradigmatic lipid second messenger in metazoan cell signaling, the first to be discovered. The protein kinase C family is the best known target for DAG signals, but other proteins, such as the chimaerins, have also come to the fore as important receptors. PKCs and other DAG receptors are therapeutic targets for cancer, diabetes, and immunosuppression, among others. PKCs and other DAG receptors bind DAG through their C1 domains. This binding event triggers both membrane translocation and allosteric activation through conformational changes. This project aims to characterize these activation mechanisms at atomic-level detail. In addition to the structure of the phorbol ester:PKC delta C1B domain complex , past work in this project has yielded the first and only structure of a full-length C1 domain protein, that of beta2 chimaerin. The approaches used are 1) the crystallization of full-length C1-domain containing proteins; 2) analysis of activation dynamics using molecular simulations; and 3) spectroscopic analysis of activation kinetics and the conformation of active, membrane-associated states inaccessible to crystallization. A major milestone was reached in the last FY. The crystal structure of full-length protein kinase C bII was determined at 4.0 in a partially open conformation. The C1B, C2, and tris-phosphorylated catalytic domains were visualized, while the pseudosubstrate and C1A domains were disordered. The C1B domain clamps the novel helix 624-634 in a position that sequesters the ATP-binding Phe629 of the conserved NFD motif in a catalytically unproductive conformation. A low resolution solution structure of the closed conformation of PKCbII was derived from small angle x-ray scattering. Together, these results show how PKCbII is allosterically regulated by clamping of the NFD helix by the C1B domain. The clamp is reversed upon membrane binding, defining a new structural class of protein kinase regulation.

二酰基甘油通过C1域蛋白的机制二酰基甘油（DAG）是内唑细胞信号传导中的范式脂质第二信使，是第一个被发现的。蛋白激酶C家族是最著名的DAG信号靶标，但是其他蛋白质（例如嵌合蛋白）也已成为重要受体。 PKC和其他DAG受体是癌症，糖尿病和免疫抑制等的治疗靶标。 PKC和其他DAG受体通过其C1结构域结合DAG。这种结合事件通过构象变化触发膜易位和变构激活。该项目旨在将这些激活机制表征为原子级细节。除了Phorbol酯的结构：PKC Delta C1B结构域复合物外，该项目的过去工作还产生了Beta2 Chimaerin的全长C1域蛋白的第一个也是唯一的结构。所使用的方法是1）全长C1结构域含有蛋白质的结晶； 2）使用分子模拟分析激活动力学； 3）激活动力学的光谱分析以及与结晶无法接近的活性，膜相关状态的构象。在最后一个FY中达到了一个重大的里程碑。全长蛋白激酶C BII的晶体结构在部分开放的构象中以4.0确定。可视化C1b，C2和Tris磷酸化催化结构域，而假基底和C1A结构域则无序。 C1b结构域将新型螺旋624-634夹在一个位置，该位置隔离了保守的NFD基序的ATP结合PHE629，以催化无产生的构象。 PKCBII闭合构象的低分辨率解结构源自小角度X射线散射。总之，这些结果表明了如何通过C1B结构域对NFD螺旋夹住的pKCBII如何变构调节。夹具在膜结合上逆转，定义了新的蛋白激酶调节的结构类别。