Redox-dependent changes in RsrA, an anti-sigma factor in Streptomyces coelicolor:: Zinc release and disulfide bond formation
Redox-dependent changes in RsrA, an anti-sigma factor in Streptomyces coelicolor:: Zinc release and disulfide bond formation
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DOI：
10.1016/j.jmb.2003.10.065
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发表时间：
2004-01-09
影响因子：
5.6
通讯作者：
Roe, JH
中科院分区：
文献类型：
--
作者：
Bae, JB;Park, JH;Roe, JH
sigma(R) 是一种 sigma 因子，用于转录基因以保护细胞免受产生抗生素的天蓝色链霉菌中的氧化应激。 sigma(R) 的可用性受 RsrA（一种抗 sigma 因子）调节，其 sigma(R) 结合活性通过硫醇-二硫化物交换受环境中氧化还原变化的调节。我们发现还原的 RsrA 含有化学计量量的锌，而氧化形式的锌含量非常少：根据显色锌螯合剂 4-(2-吡啶基)-间苯二酚 (PAR) 的监测，氧化后每 mol RsrA 释放 1 mol 的锌。对多种半胱氨酸和组氨酸取代的几种 RsrA 突变体中锌结合的测量表明，C3、H7、C41 和 C44 作为锌结合位点。突变蛋白的锌结合活性和 sigma(R) 结合活性并不重合，表明锌对于 RsrA 的抗 sigma 活性可能不是绝对必需的。无锌 apo-RsrA 结合 sigma(R) 并在体外抑制 sigma(R) 依赖性转录。与Zn-RsrA相比，通过表面等离子体共振分析测量，apo-RsrA的抗转录活性大约低三倍，其sigma(R)结合亲和力降低大约九倍。 Apo-RsrA 对蛋白酶更敏感，这表明锌可以使 RsrA 保持更紧凑的结构，并针对结合 sigma(R) 进行优化。通过 MALDI-TOF 质谱法测定了形成二硫键的半胱氨酸对，揭示了 C11 与重要半胱氨酸残基 C41 或 44（很可能是 C44）之一之间形成了关键的二硫键。提出了 RsrA 氧化还原调节机制的改进模型。 (C) 2003 Elsevier Ltd. 保留所有权利。
sigma(R) is a sigma factor for transcribing genes to defend cells against oxidative stresses in the antibiotic-producing bacterium Streptomyces coelicolor. The availability of sigma(R) is regulated by RsrA, an anti-sigma factor, whose sigma(R)- binding activity is regulated by redox changes in the environment, via thiol-disulfide exchange. We found that reduced RsrA contains zinc in a stoichiometric amount, whereas oxidized form has very little: 1 mol of zinc per mol of RsrA was released upon oxidation as monitored by a chromogenic Zn-chelator, 4-(2-pyridylazo)-resorcinol (PAR). Measurement of zinc bound in several RsrA mutants of various cysteine and histidine substitutions suggested that C3, H7, C41, and C44 serve as zinc-binding sites. The zinc-binding and sigma(R)-binding activities of mutant proteins did not coincide, suggesting that zinc might not be absolutely required for the anti-sigma activity of RsrA. Zn-free apo-RsrA bound sigma(R) and inhibited sigma(R)-dependent transcription in vitro. Compared with Zn-RsrA, the anti-transcription activity of apo-RsrA was about threefold lower and its sigma(R)-binding affinity decreased by about ninefold when measured by surface plasmon resonance analysis. Apo-RsrA was more sensitive to protease, suggesting that zinc allows RsrA to maintain a more compact structure, optimized for binding sigma(R). The cysteine pairs that form disulfide bonds were determined by MALDI-TOF mass spectrometry, revealing formation of the critical disulfide bond between C11 and one of the essential cysteine residues C41 or 44, most likely C44. An improved model for the mechanism of redox-modulation of RsrA was presented. (C) 2003 Elsevier Ltd. All rights reserved.