Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
J Biol Chem. 2010 Mar 12;285(11):7847-51. doi: 10.1074/jbc.C109.084921. Epub 2010 Jan 27.
The folding of proinsulin, the single-chain precursor of insulin, ensures native disulfide pairing in pancreatic beta-cells. Mutations that impair folding cause neonatal diabetes mellitus. Although the classical structure of insulin is well established, proinsulin is refractory to crystallization. Here, we employ heteronuclear NMR spectroscopy to characterize a monomeric analogue. Proinsulin contains a native-like insulin moiety (A- and B-domains); the tethered connecting (C) domain (as probed by {(1)H}-(15)N nuclear Overhauser enhancements) is progressively less ordered. Although the BC junction is flexible, residues near the CA junction exhibit alpha-helical-like features. Relative to canonical alpha-helices, however, segmental (13)C(alpha/beta) chemical shifts are attenuated, suggesting that this junction and contiguous A-chain residues are molten. We propose that flexibility at each C-domain junction facilitates prohormone processing. Studies of protease SPC3 (PC1/3) suggest that C-domain sequences contribute to cleavage site selection. The structure of proinsulin provides a foundation for studies of insulin biosynthesis and its impairment in monogenic forms of diabetes mellitus.
胰岛素原是胰岛素的单链前体，它的折叠确保了胰腺β细胞中天然二硫键的配对。折叠缺陷导致的突变会引起新生儿糖尿病。尽管经典的胰岛素结构已经确立，但胰岛素原很难结晶。在这里，我们采用异核 NMR 光谱学来表征一种单体类似物。胰岛素原包含一个类似天然的胰岛素部分（A 域和 B 域）；连接（C）域（通过 {(1)H}-(15)N 核 Overhauser 增强来探测）的有序性逐渐降低。尽管 BC 连接处具有柔韧性，但 CA 连接处附近的残基表现出类似α-螺旋的特征。然而，与典型的α-螺旋相比，片段化的 (13)C(alpha/beta)化学位移被削弱，这表明该连接处和相邻的 A 链残基是熔融的。我们提出，每个 C 域连接处的灵活性有助于前激素的加工。蛋白酶 SPC3（PC1/3）的研究表明，C 域序列有助于切割位点的选择。胰岛素原的结构为胰岛素生物合成及其在单基因形式糖尿病中的缺陷的研究提供了基础。
