Structure and function of protein disulfide isomerase

许多分泌性蛋白质中二硫键的形成是其折叠和成熟的关键步骤，而蛋白质二硫键异构酶(PDI)是形成二硫键所必需的关键酶。它氧化新合成的蛋白质中的两个半胱氨酸残基形成二硫键，并催化含有三个或更多半胱氨酸残基的蛋白质中错误形成的二硫键异构化。PDI的初步晶体结构表明，两个催化活性(a和a‘)和两个非活性(b和b’)硫氧还蛋白结构域以字母“U”的形式排列。A和a‘结构域中的活性中心位于“U”结构域的两端，在它们之间有一个大的底物结合裂隙，b和b’结构域构成“U”结构域的底部。随后的较低分辨率结构显示了大规模的构象变化。通过生化、生物物理和结构研究的结合，我们将研究结构域运动对PDI催化活性的贡献，分析酶与不同底物及其氧化还原伙伴Ero1的相互作用，对哺乳动物PDI进行结构表征，以合理解释物种内和物种之间催化活性结构域的差异，并确定抑制剂的结构，以进一步完善催化机理，并作为合理药物设计的起点。

The formation of disulfide bonds in many secretory proteins represents an essential step in their folding and maturation, and protein disulfide isomerase (PDI) is the key enzyme required for disulfide bond formation. It oxidizes two cysteine residues in newly synthesized proteins to form a disufide bridge and also catalyzes the isomerization of incorrectly formed disulfide linkages in proteins containing three or more cysteine residues. Our initial crystal structure of PDI revealed that the two catalytically competent (a and a’) and two inactive (b and b’) thioredoxin-like domains are arranged in the shape of the letter “U”. The active sites in the a and a’ domains are located on opposite ends of the “U” with a large substratebinding cleft located between them, and the b and b’ domains form the base of the “U”. A subsequent lower resolution structure demonstrated large-scale conformational changes. Through a combination of biochemical, biophysical and structural studies we will study the contribution of domain motions to the catalytic activity of PDI, analyze the interaction of the enzyme with different substrates and its redox partner Ero1, structurally characterize mammalian PDI to rationalize variations of the catalytically active domains in and between species, and we will also determine structures with inhibitors to further refine the catalytic mechanism and as a starting point for rational drug design.