Department of Molecular Enzymology, Göttingen Center of Molecular Biosciences, Georg August University Göttingen, Göttingen, Germany.
Department of Structural Dynamics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Nature. 2021 May;593(7859):460-464. doi: 10.1038/s41586-021-03513-3. Epub 2021 May 5.
Disulfide bonds between cysteine residues are important post-translational modifications in proteins that have critical roles for protein structure and stability, as redox-active catalytic groups in enzymes or allosteric redox switches that govern protein function. In addition to forming disulfide bridges, cysteine residues are susceptible to oxidation by reactive oxygen species, and are thus central not only to the scavenging of these but also to cellular signalling and communication in biological as well as pathological contexts. Oxidized cysteine species are highly reactive and may form covalent conjugates with, for example, tyrosines in the active sites of some redox enzymes. However, to our knowledge, regulatory switches with covalent crosslinks other than disulfides have not previously been demonstrated. Here we report the discovery of a covalent crosslink between a cysteine and a lysine residue with a NOS bridge that serves as an allosteric redox switch in the transaldolase enzyme of Neisseria gonorrhoeae, the pathogen that causes gonorrhoea. X-ray structure analysis of the protein in the oxidized and reduced state reveals a loaded-spring mechanism that involves a structural relaxation upon redox activation, which is propagated from the allosteric redox switch at the protein surface to the active site in the protein interior. This relaxation leads to a reconfiguration of key catalytic residues and elicits an increase in enzymatic activity of several orders of magnitude. The redox switch is highly conserved in related transaldolases from other members of the Neisseriaceae; for example, it is present in the transaldolase of Neisseria meningitides (a pathogen that is the primary cause of meningitis and septicaemia in children). We surveyed the Protein Data Bank and found that the NOS bridge exists in diverse protein families across all domains of life (including Homo sapiens) and that it is often located at catalytic or regulatory hotspots. Our findings will inform strategies for the design of proteins and peptides, as well as the development of new classes of drugs and antibodies that target the lysine-cysteine redox switch.
半胱氨酸残基之间的二硫键是蛋白质翻译后的重要修饰，对蛋白质结构和稳定性具有关键作用，因为它们可以作为酶的氧化还原活性催化基团或调控蛋白质功能的变构氧化还原开关。除了形成二硫键外，半胱氨酸残基还容易受到活性氧物种的氧化，因此不仅是清除这些物质的核心，而且在生物和病理环境中也是细胞信号转导和通讯的核心。氧化的半胱氨酸物种具有很高的反应性，可能与某些氧化还原酶的活性部位中的酪氨酸形成共价结合物。然而，据我们所知，以前尚未证明除二硫键以外的共价交联具有调控开关的作用。在这里，我们报告了在淋病奈瑟菌（引起淋病的病原体）的转醛醇酶中发现了一种半胱氨酸和赖氨酸残基之间的共价交联，其带有一个 NOS 桥，充当变构氧化还原开关。对氧化和还原状态下的蛋白质进行的 X 射线结构分析揭示了一种加载弹簧机制，该机制涉及氧化还原激活时的结构松弛，这种松弛从蛋白质表面的变构氧化还原开关传播到蛋白质内部的活性部位。这种松弛导致关键催化残基的重新配置，并引发酶活性增加几个数量级。该氧化还原开关在来自奈瑟菌科的其他相关转醛醇酶中高度保守；例如，它存在于脑膜炎奈瑟菌（导致儿童脑膜炎和败血症的主要病原体）的转醛醇酶中。我们对蛋白质数据库进行了调查，发现 NOS 桥存在于生命所有领域（包括人类）的不同蛋白质家族中，并且它通常位于催化或调节热点。我们的发现将为蛋白质和肽的设计以及针对赖氨酸-半胱氨酸氧化还原开关的新药和抗体的开发提供信息。
