Koivunen P, Pirneskoski A, Karvonen P, Ljung J, Helaakoski T, Notbohm H, Kivirikko K I
Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry, University of Oulu, Kajaanintie 52A, FIN-90220 Oulu, Finland.
EMBO J. 1999 Jan 4;18(1):65-74. doi: 10.1093/emboj/18.1.65.
Protein disulfide isomerase (PDI) is a multifunctional polypeptide that acts as a subunit in the animal prolyl 4-hydroxylases and the microsomal triglyceride transfer protein, and as a chaperone that binds various peptides and assists their folding. We report here that deletion of PDI sequences corresponding to the entire C-terminal domain c, previously thought to be critical for chaperone activity, had no inhibitory effect on the assembly of recombinant prolyl 4-hydroxylase in insect cells or on the in vitro chaperone activity or disulfide isomerase activity of purified PDI. However, partially overlapping critical regions for all these functions were identified at the C-terminal end of the preceding thioredoxin-like domain a'. Point mutations introduced into this region identified several residues as critical for prolyl 4-hydroxylase assembly. Circular dichroism spectra of three mutants suggested that two of these mutations may have caused only local alterations, whereas one of them may have led to more extensive structural changes. The critical region identified here corresponds to the C-terminal alpha helix of domain a', but this is not the only critical region for any of these functions.
蛋白质二硫键异构酶（PDI）是一种多功能多肽，它在动物脯氨酰4-羟化酶和微粒体甘油三酯转移蛋白中作为亚基发挥作用，并且作为一种伴侣蛋白，能结合各种肽并协助其折叠。我们在此报告，删除与整个C末端结构域c相对应的PDI序列（此前认为该结构域对伴侣蛋白活性至关重要），对昆虫细胞中重组脯氨酰4-羟化酶的组装、纯化的PDI的体外伴侣蛋白活性或二硫键异构酶活性均无抑制作用。然而，在前面类似硫氧还蛋白的结构域a'的C末端鉴定出了所有这些功能的部分重叠的关键区域。引入该区域的点突变确定了几个对脯氨酰4-羟化酶组装至关重要的残基。三个突变体的圆二色光谱表明，其中两个突变可能仅引起局部改变，而其中一个突变可能导致更广泛的结构变化。此处鉴定出的关键区域对应于结构域a'的C末端α螺旋，但这并非这些功能中任何一项的唯一关键区域。
