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.

通过 C1 结构域蛋白的二酰甘油信号传导机制二酰基甘油 (DAG) 是后生动物细胞信号传导中的典型脂质第二信使，也是第一个被发现的。蛋白激酶 C 家族是 DAG 信号最知名的靶标，但嵌合蛋白等其他蛋白也作为重要受体脱颖而出。 PKC 和其他 DAG 受体是癌症、糖尿病和免疫抑制等的治疗靶点。 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。该项目旨在在原子级细节上表征这些激活机制。除了佛波酯：PKC delta C1B 结构域复合物的结构外，该项目过去的工作还产生了第一个也是唯一一个全长 C1 结构域蛋白的结构，即 beta2 嵌合蛋白。使用的方法是 1) 含有全长 C1 结构域的蛋白质的结晶； 2）利用分子模拟分析活化动力学； 3) 活化动力学和无法结晶的活性、膜相关状态的构象的光谱分析。上个财年实现了一个重要的里程碑。全长蛋白激酶 C bII 的晶体结构在 4.0 处以部分开放构象确定。 C1B、C2 和三磷酸化催化结构域可见，而假底物和 C1A 结构域是无序的。 C1B 结构域将新型螺旋 624-634 夹在一个位置，该位置将保守 NFD 基序的 ATP 结合 Phe629 隔离在催化无效构象中。 PKCbII 闭合构象的低分辨率溶液结构是从小角度 X 射线散射得出的。总之，这些结果显示了 PKCbII 如何通过 C1B 结构域夹紧 NFD 螺旋进行变构调节。膜结合后钳位被逆转，定义了蛋白激酶调节的新结构类别。