Institute of Chemistry/Organic and Bioorganic Chemistry, University of Graz, Graz, Austria.
Institute of Molecular Biosciences, University of Graz, Graz, Austria.
Mol Microbiol. 2021 Jun;115(6):1277-1291. doi: 10.1111/mmi.14673. Epub 2021 Jan 25.
The transmembrane protein ToxR plays a key role in the virulence expression system of Vibrio cholerae. The activity of ToxR is dependent on its periplasmic sensor domain (ToxRp) and on the inner membrane protein ToxS. Herein, we present the Nuclear Magnetic Resonance NMR solution structure of the sensory ToxRp containing an intramolecular disulfide bond. The presented structural and dynamic experiments with reduced and oxidized ToxRp propose an explanation for the increased proteolytic sensitivity of reduced ToxR. Additionally, for the first time, we could identify the formation of a strong heterodimer complex between the periplasmic domains of ToxR and ToxS in solution. NMR interaction studies reveal that binding of ToxS is not dependent on the redox state of ToxR cysteines, and formed complexes are structurally similar. By monitoring the proteolytic cleavage of ToxRp with NMR, we additionally provide a direct evidence of ToxS protective function. Taken together our results suggest that ToxR activity is regulated by its stability which is, on the one hand, dependent on the redox states of its cysteines, influencing the stability of its fold, and on the other hand, on its interaction with ToxS, which binds independent on the cysteines and acts as a protection against proteases.
跨膜蛋白 ToxR 在霍乱弧菌毒力表达系统中起着关键作用。ToxR 的活性依赖于其周质传感器结构域（ToxRp）和内膜蛋白 ToxS。本文介绍了含有分子内二硫键的感应 ToxRp 的核磁共振（NMR）溶液结构。还原和氧化的 ToxRp 的结构和动态实验提出了对还原 ToxR 增加蛋白水解敏感性的解释。此外，我们首次在溶液中鉴定了 ToxR 和 ToxS 的周质结构域之间形成的强异二聚体复合物。NMR 相互作用研究表明，ToxS 的结合不依赖于 ToxR 半胱氨酸的氧化还原状态，形成的复合物在结构上相似。通过 NMR 监测 ToxRp 的蛋白水解切割，我们还提供了 ToxS 保护功能的直接证据。总之，我们的结果表明，ToxR 的活性受到其稳定性的调节，一方面，其半胱氨酸的氧化还原状态影响其折叠的稳定性，另一方面，其与 ToxS 的相互作用，ToxS 独立于半胱氨酸结合并作为对抗蛋白酶的保护。
