Redox-Dependent Domain Rearrangement of Protein Disulfide Isomerase from a Thermophilic Fungus

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• 作者：Masayoshi Nakasako ; Aya Maeno ; Eiji Kurimoto ; Takushi Harada ; Yoshiki Yamaguchi ; Toshihiko Oka ; Yuki Takayama ; Aya Iwata ; Koichi Kato
• 刊名：Biochemistry
• 出版年：2010
• 出版时间：August 17, 2010
• 年：2010
• 卷：49
• 期：32
• 页码：6953-6962
• 全文大小：1057K
• 年卷期：v.49,no.32(August 17, 2010)
• ISSN：1520-4995

文摘

Protein disulfide isomerase (PDI) acts as folding catalyst and molecular chaperone for disulfide-containing proteins through the formation, breakage, and rearrangement of disulfide bonds. PDI has a modular structure comprising four thioredoxin domains, a, b, b′, and a′, followed by a short segment, c. The a and a′ domains have an active site cysteine pair for the thiol−disulfide exchange reaction, which alters PDI between the reduced and oxidized forms, and the b′ domain provides a primary binding site for substrate proteins. Although the structures and functions of PDI have studied, it is still argued whether the overall conformation of PDI depends on the redox state of the active site cysteine pair. Here, we report redox-dependent conformational and solvation changes of PDI from a thermophilic fungus elucidated by small-angle X-ray scattering (SAXS) analysis. The redox state and secondary structures of PDI were also characterized by nuclear magnetic resonance and circular dichroic spectroscopy, respectively. The oxidized form of PDI showed SAXS differences from the reduced form, and the low-resolution molecular models restored from the SAXS profiles differed between the two forms regarding the positions of the a′−c region relative to the a−b−b′ region. The normal mode analysis of the crystal structure of yeast PDI revealed that the inherent motions of the a−b−b′ and a′−c regions expose the substrate binding surface of the b′ domain. The apparent molecular weight of the oxidized form estimated from SAXS was 1.1 times larger than that of the reduced form, whereas the radius of gyration (ca. 33 Å) was nearly independent of the redox state. These results suggest that the conformation of PDI is controlled by the redox state of the active site cysteine residues in the a and a′ domains and that the conformational alternation accompanies solvation changes in the active site cleft formed by the a, b, b′, and a′ domains. On the basis of the results presented here, we propose a mechanism explaining the observed redox-dependent conformational and solvation changes of PDI.