肠道菌抗酸性分子伴侣HdeA和HdeB的结构、动力学和活化的分子机理研究

Before infecting the intestinal mucosa, all enteric pathogens have to pass through the highly acidic environment of their hosts' stomach. In the periplasm of Gram-negative bacteria, there are two acid resistance chaperones HdeA and HdeB. Both are well folded and form homo-dimers under neutral pH conditions. When the environmental pH decreases, the homo-dimers dissociate, while at the same time the proteins undergo partial unfolding process. The hydrophobic regions of HdeA/B expose and bind to target proteins to prevent them from denaturation and precipitation. Up to date, there has been significant biochemical research progress on HdeA/B function, and the crystal structures of both proteins have been determined at neutral pH. However, the structures of inactive HdeA/B proteins are not sufficient to elucidate the their functional mechanisms as acid resistance chaperones. The acid activated HdeA/B proteins exhibit a partially unfolded conformation, making them extremely difficult to be investigated by X-ray crystallization method. In this project, we aim to study the structures, dynamics and interactions with substrate proteins of HdeA and HdeB under different pH conditions by using advanced solution NMR technique in combination with other modern biochemical methods, and to provide structural basis for elucidating the molecular mechanisms of their acid resistance chaperone functions.

寄生于哺乳动物肠道内的肠道细菌在进入宿主体内时，都会经过胃部这个低pH环境。革兰氏阴性菌的周质空间中存在两个抗酸分子伴侣HdeA和HdeB蛋白，它们在中性条件下具有良好折叠并形成同源二聚体；当环境pH降低时，它们向单体解离，同时发生部分去折叠；推测其暴露出来的疏水区域与底物蛋白结合，使之免于变性和聚沉。目前，对HdeA/B蛋白已经积累了许多生化和功能数据，二者在中性条件下（非活性状态）的高分辨率晶体结构也分别得到了解析。然而，非活性状态下结构方面的研究并不足以阐明其作为分子伴侣的机理。酸性条件下活化的HdeA/B蛋白具有部分去折叠的构象，因此进行结构方面的研究非常困难。本课题将发挥液相核磁共振技术的特有优势，并结合其它现代生化手段，对大肠杆菌的HdeA和HdeB蛋白进行不同pH条件下的溶液结构、动力学以及和底物蛋白相互作用的研究，深入阐明HdeA/B蛋白发挥抗酸分子伴侣功能的分子机理。

抗酸分子伴侣HdeA和HdeB是一类条件性去折叠蛋白质。它们位于革兰氏阴性细菌的周质空间内，在细菌遭遇酸性胁迫的情况下发挥分子伴侣活性，保护周质空间内的大多数蛋白质免于酸引起的变性和聚沉。HdeA在中性条件下处于良好折叠的无活性状态，当环境pH降到2左右时处于去折叠的活性状态，具有最高的分子伴侣活性；而与之同源的HdeB蛋白则在近中性条件下，即pH4左右的环境，即发挥分子伴侣功能。本课题主要针对HdeA和HdeB的活化机理以及二者功能活性上差异，应用液相核磁共振技术研究这两个蛋白质在不同pH条件下的结构和动力学变化，以深入阐明它们发挥功能的分子机理。我们完成了对HdeA和HdeB蛋白高分辨率的二聚体溶液结构解析，并发现了部分结构区域的运动特性随pH降低有明显的改变，从分子水平上阐明了二者的活化机制。此外，对HdeA与天然底物蛋白之间的相互作用模式进行了初步的研究，提出了HdeA发挥分子伴侣功能的两亲分子模型，为未来进一步研究HdeA和HdeB和底物蛋白形成的复合物结构奠定了基础。