Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología (IBT), Universidad Nacional Autónoma de México (UNAM), Avenida Universidad 2001, Colonia Chamilpa, 62210 Cuernavaca, MOR, Mexico.
Laboratorio de Estructura Biomolecular, Centro de Investigación en Alimentación y Desarrollo A.C. (CIAD), Carretera a Ejido La Victoria Km 0.6, PO Box 1735, 83304 Hermosillo, SON, Mexico.
Acta Crystallogr D Struct Biol. 2017 Apr 1;73(Pt 4):326-339. doi: 10.1107/S2059798317002066. Epub 2017 Mar 31.
The quaternary structure of the redox protein thioredoxin (Trx) has been debated. For bacterial Trx, there is no question regarding its monomeric state. In humans and other eukaryotes, the presence of a cysteine residue at the crystallographic symmetry axis points to the relevance of dimer formation in solution and in vivo. Crystallographic data for shrimp thioredoxin (LvTrx) obtained under different redox conditions reveal a dimeric arrangement mediated by a disulfide bond through residue Cys73 and other hydrophobic interactions located in the crystallographic interface, as reported for human Trx. Through the analysis of five mutants located at the crystallographic interface, this study provides structural and biochemical evidence for the existence in solution of monomeric and dimeric populations of wild-type LvTrx and five mutants. Based on the results of biochemical assays, SAXS studies and the crystallographic structures of three of the studied mutants (Cys73Ser, Asp60Ser and Trp31Ala), it is clear that the Cys73 residue is essential for dimerization. However, its mutation to Ser produces an enzyme which has similar redox activity in vitro to the wild type. A putative regulatory function of dimerization is proposed based on structural analysis. Nonetheless, the biological role of LvTrx dimerization needs to be experimentally unveiled. Additionally, the findings of this work reopen the discussion regarding the existence of similar behaviour in human thioredoxin, which shares a Cys at position 73 with LvTrx, a structural feature that is also present in some Trxs from vertebrates and crustaceans.
氧化还原蛋白硫氧还蛋白（Trx）的四级结构一直存在争议。对于细菌 Trx，其单体状态是毫无疑问的。在人类和其他真核生物中，晶体学对称轴上半胱氨酸残基的存在表明二聚体在溶液中和体内形成的相关性。在不同氧化还原条件下获得的虾硫氧还蛋白（LvTrx）的晶体学数据揭示了通过残基 Cys73 形成二硫键和其他位于晶体学界面的疏水性相互作用介导的二聚体排列，这与人类 Trx 报道的一致。通过对位于晶体学界面的五个突变体的分析，本研究提供了结构和生化证据，证明野生型 LvTrx 和五个突变体在溶液中存在单体和二聚体两种形式。基于生化分析、SAXS 研究以及三种研究突变体（Cys73Ser、Asp60Ser 和 Trp31Ala）的晶体学结构的结果，显然 Cys73 残基对于二聚化是必不可少的。然而，将其突变为丝氨酸会产生一种在体外具有与野生型相似氧化还原活性的酶。根据结构分析，提出了二聚化的潜在调节功能。尽管如此，LvTrx 二聚化的生物学作用仍需要通过实验来揭示。此外，这项工作的发现重新引发了关于人类硫氧还蛋白中存在类似行为的讨论，人类硫氧还蛋白与 LvTrx 在第 73 位共享一个半胱氨酸，这一结构特征也存在于一些脊椎动物和甲壳类动物的 Trx 中。
