冷冻电镜技术解析AMP激活的蛋白激酶结构及构象变化

AMP-activated protein kinase (AMPK) acts as a highly conserved central energy sensor in eukaryotes. It regulates carbohydrates, lipid and many other metabolic pathways in vivo. As its dysfunction can lead to diabetes, obesity, cancer and other metabolic disorders, AMPK has become a popular drug target for treatment of these diseases. Undoubtedly, structural insights into AMPK molecule can be extraordinarily meaningful for such drug design purpose. While our understanding of the structural aspects of AMPK has been largely facilitated in recent years mainly by tough X-ray crystallographic studies, the comprehensive conformational changes between full-length inactive AMPK and active AMPK molecules still require further investigation. Particularly, the KD-AID and the CBM domain may undergo significant conformational changes and/or translocation in AMPK activation process according to our knowledge, which deserve more efforts to be better elucidated. This project will resolve high resolution cryo-EM structures of inactive and active AMPK in different functional states in solution, illustrating the conformational changes occur in full-length AMPK in the process of allosteric activation, especially the changes of KD-AID and CBM domain. Our study will promote the understanding of the molecular mechanism underlying AMPK allosteric activation and benefit target drug development in future.

AMP激活的蛋白激酶（AMP-activated protein kinase, AMPK）是真核细胞中高度保守的能量感应与代谢调节蛋白。作为能量代谢中心分子，它参与调节糖代谢、脂肪酸代谢等多条代谢途径。其功能失调，可致糖尿病、肥胖症、癌症等疾病，AMPK已成为治疗相关疾病的药物靶点。AMPK结构研究对深入理解其发挥功能的分子机制及以AMPK为靶点的药物设计具有重要意义。已解析的AMPK结构以晶体结构为主，但现有的结构信息不能完全阐明AMPK变构激活过程中整体构架水平发生的构象变化，特别是其KD-AID及CBM结构域的分子内运动。本项目拟通过冷冻电镜技术解析AMPK近生理溶液态的高分辨三维结构及不同功能态的构象，重点关注KD-AID及CBM的构象变化及分子内转移，从整体分子水平揭示AMPK激活过程中的构象变化，推进对AMPK发挥酶活的分子机制的认识，为相关药物设计提供结构依据。

AMP激活的蛋白激酶（AMP-activated protein kinase, AMPK）是真核生物中调节机体能量代谢平衡的关键蛋白分子，与癌症等多种代谢疾病相关，是重要的药物靶点。理解AMPK的催化激活原理、活性调节机制、底物特异性及其调控代谢活动的机制，具有重要意义。基于此，本项目对AMPK的结构与功能展开了以下研究。首先，我们通过电镜单颗粒技术，解析不同活性状态下AMPK蛋白的结构，以揭示AMPK 变构激活过程中的构象变化机制。我们取得了蛋白表达纯化、电镜样品制备、三维重构等重要进展，发现磷酸化AMPK与非磷酸化AMPK之间存在显著的结构差异，揭示了磷酸化修饰可通过改变AMPK的结构而调节其活性的分子机制。其次，我们发现AMPK各亚型在人体中具有组织特异性和底物特异性，据此我们纯化了几种具有组织特异性表达的AMPK蛋白亚型及其底物蛋白，并制备了AMPK与其相互作用分子形成蛋白复合物的样品，用于结构解析。此外，我们研究发现AMPK PRKAA2亚型在肝癌组织中高表达，且与患者较差的预后相关；PRKAA2可通过调节肝癌免疫微环境及细胞通讯促进肿瘤进展；PRKAA2与多种化疗药物的敏感性显著相关，暗示PRKAA2在个性化治疗中具有重要的指导意义。本项目为深入探索AMPK发挥功能的分子机制及开发靶向AMPK的治疗策略奠定了理论基础。