Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, 23955-6900 Thuwal, Saudi Arabia.
Department of Chemistry, Technische Universität München, Lichtenbergstr. 4, 85748 Garching, Germany.
Molecules. 2019 Jul 24;24(15):2691. doi: 10.3390/molecules24152691.
In plants, much like in animals, nitric oxide (NO) has been established as an important gaseous signaling molecule. However, contrary to animal systems, NO-sensitive or NO-responsive proteins that bind NO in the form of a sensor or participating in redox reactions have remained elusive. Here, we applied a search term constructed based on conserved and functionally annotated amino acids at the centers of Heme Nitric Oxide/Oxygen (H-NOX) domains in annotated and experimentally-tested gas-binding proteins from lower and higher eukaryotes, in order to identify candidate NO-binding proteins in . The selection of candidate NO-binding proteins identified from the motif search was supported by structural modeling. This approach identified AtLRB3 (At4g01160), a member of the Light Response Bric-a-Brac/Tramtrack/Broad Complex (BTB) family, as a candidate NO-binding protein. AtLRB3 was heterologously expressed and purified, and then tested for NO-response. Spectroscopic data confirmed that AtLRB3 contains a histidine-ligated heme cofactor and importantly, the addition of NO to AtLRB3 yielded absorption characteristics reminiscent of canonical H-NOX proteins. Furthermore, substitution of the heme iron-coordinating histidine at the H-NOX center with a leucine strongly impaired the NO-response. Our finding therefore established AtLRB3 as a NO-interacting protein and future characterizations will focus on resolving the nature of this response.
在植物中，就像在动物中一样，一氧化氮（NO）已被确定为一种重要的气态信号分子。然而，与动物系统相反，能够结合以传感器形式存在的 NO 或参与氧化还原反应的 NO 敏感或 NO 响应蛋白仍然难以捉摸。在这里，我们应用了一种基于在注释和经过实验测试的气体结合蛋白中的 Heme Nitric Oxide/Oxygen（H-NOX）结构域中心的保守和功能注释氨基酸构建的搜索项，以鉴定 在低等和高等真核生物中。 motif 搜索鉴定的候选 NO 结合蛋白的选择得到了结构建模的支持。这种方法鉴定出 AtLRB3（At4g01160）是 Light Response Bric-a-Brac/Tramtrack/Broad Complex（BTB）家族的成员，是候选的 NO 结合蛋白。AtLRB3 被异源表达和纯化，然后进行 NO 反应测试。光谱数据证实 AtLRB3 含有一个组氨酸配位的血红素辅因子，重要的是，向 AtLRB3 中添加 NO 会产生与典型的 H-NOX 蛋白相似的吸收特征。此外，用 H-NOX 中心的组氨酸取代铁配位的血红素强烈削弱了对 NO 的反应。因此，我们的发现确立了 AtLRB3 作为与 NO 相互作用的蛋白质，未来的特征分析将集中于解决这种反应的性质。
