College of Plant Protection, Hainan University, Haikou, Hainan Province, China.
Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests (Hainan University), Ministry of Education, Haikou, Hainan Province, China.
BMC Microbiol. 2020 Nov 12;20(1):344. doi: 10.1186/s12866-020-02029-6.
Heat resistance is a common characteristic of harpins, a class of proteins found in Gram-negative bacteria, which may be related to the stability of coiled-coil (CC) structure. The CC structure is a ubiquitous protein folding and assembly motif made of α-helices wrapping around each other forming a supercoil. Specifically, whether the stability of the CC structure near to N-terminus of four selected harpin proteins from Xanthomonas (hereafter referred to as Hpa1) would influence their characteristics of heat resistance was investigated. We used bioinformatics approach to predict the structure of Hpa1, used the performance of hypersensitive response (HR)-induction activity of Hpa1 and circular dichroism (CD) spectral analyses to detect the relationship between the stability of the CC structure of Hpa1 and heat resistance.
Each of four-selected Hpa1 has two α-helical regions with one in their N-terminus that could form CC structure, and the other in their C-terminus that could not. And the important amino acid residues involved in the CC motifs are located on helices present on the surface of these proteins, indicating they may engage in the formation of oligo mericaggregates, which may be responsible for HR elicitation by harpins and their high thermal stability. Increased or decreased the probability of forming a CC could either induce a stronger HR response or eliminate the ability to induce HR in tobacco after high temperature treatment. In addition, although the four Hpa1 mutants had little effect on the induction of HR by Hpa1, its thermal stability was significantly decreased. The α-helical content increased with increasing temperature, and the secondary structures of Hpa1 became almost entirely α-helices when the temperature reached 200 °C. Moreover, the stability of the CC structure near to N-terminus was found to be positively correlated with the heat resistance of Hpa1.
The stability of the CC structure might sever as an inner drive for mediating the heat resistance of harpin proteins. Our results offer a new insight into the interpretation of the mechanism involved in the heat resistance of harpin protein and provide a theoretical basis for further harpin function investigations and structure modifications.
耐热性是革兰氏阴性细菌中一类蛋白——harpin 蛋白的共同特征，其可能与卷曲螺旋（CC）结构的稳定性有关。CC 结构是一种普遍存在的蛋白质折叠和组装基序，由彼此缠绕的α-螺旋组成超螺旋。具体来说，我们研究了来自黄单胞菌的 4 种 harpin 蛋白（以下简称 Hpa1）近 N 端 CC 结构的稳定性是否会影响它们的耐热特性。我们使用生物信息学方法预测 Hpa1 的结构，利用 Hpa1 的超敏反应（HR）诱导活性和圆二色性（CD）光谱分析来检测 Hpa1 的 CC 结构稳定性与耐热性之间的关系。
所选择的 4 种 Hpa1 中的每一种都有两个α-螺旋区域，其中一个在 N 端，可以形成 CC 结构，另一个在 C 端，不能形成 CC 结构。参与 CC 基序的重要氨基酸残基位于这些蛋白质表面存在的螺旋上，表明它们可能参与形成寡聚体聚集物，这可能是 harpin 诱导 HR 及其高热稳定性的原因。增加或减少形成 CC 的可能性会导致更强的 HR 反应，或者在高温处理后消除 Hpa1 诱导 HR 的能力。此外，尽管 4 种 Hpa1 突变体对 Hpa1 诱导 HR 的影响不大，但它的热稳定性却显著降低。α-螺旋含量随温度升高而增加，当温度达到 200°C 时，Hpa1 的二级结构几乎完全变成α-螺旋。此外，发现近 N 端 CC 结构的稳定性与 Hpa1 的耐热性呈正相关。
CC 结构的稳定性可能是介导 harpin 蛋白耐热性的内在驱动力。我们的研究结果为解释 harpin 蛋白耐热性的机制提供了新的见解，并为进一步研究 harpin 功能和结构修饰提供了理论依据。
