Laboratório de Biologia Molecular Estrutural, Departamento de Bioquímica, CCB, Universidade Federal de Santa Catarina, Florianópolis 88040-900, Brazil.
São Carlos Institute of Physics, University of São Paulo, São Carlos, Brazil.
Biochim Biophys Acta Proteins Proteom. 2022 Mar 1;1870(3):140754. doi: 10.1016/j.bbapap.2022.140754. Epub 2022 Jan 5.
Protein tyrosine phosphatases (PTPs) are key virulence factors in pathogenic bacteria, consequently, they have become important targets for new approaches against these pathogens, especially in the fight against antibiotic resistance. Among these targets of interest YopH (Yersinia outer protein H) from virulent species of Yersinia is an example. PTPs can be reversibly inhibited by nitric oxide (NO) since the oxidative modification of cysteine residues may influence the protein structure and catalytic activity. We therefore investigated the effects of NO on the structure and enzymatic activity of Yersinia enterocolitica YopH in vitro. Through phosphatase activity assays, we observe that in the presence of NO YopH activity was inhibited by 50%, and that this oxidative modification is partially reversible in the presence of DTT. Furthermore, YopH S-nitrosylation was clearly confirmed by a biotin switch assay, high resolution mass spectrometry (MS) and X-ray crystallography approaches. The crystal structure confirmed the S-nitrosylation of the catalytic cysteine residue, Cys403, while the MS data provide evidence that Cys221 and Cys234 might also be modified by NO. Interestingly, circular dichroism spectroscopy shows that the S-nitrosylation affects secondary structure of wild type YopH, though to a lesser extent on the catalytic cysteine to serine YopH mutant. The data obtained demonstrate that S-nitrosylation inhibits the catalytic activity of YopH, with effects beyond the catalytic cysteine. These findings are helpful for designing effective YopH inhibitors and potential therapeutic strategies to fight this pathogen or others that use similar mechanisms to interfere in the signal transduction pathways of their hosts.
蛋白质酪氨酸磷酸酶（PTPs）是病原菌的关键毒力因子，因此，它们已成为针对这些病原体的新方法的重要靶点，尤其是在对抗抗生素耐药性方面。在这些感兴趣的靶标中，来自致病性耶尔森氏菌的 YopH（耶尔森氏菌外蛋白 H）就是一个例子。由于半胱氨酸残基的氧化修饰可能影响蛋白质结构和催化活性，因此 PTPs 可以被一氧化氮（NO）可逆抑制。因此，我们研究了 NO 对体外肠侵袭性大肠杆菌 YopH 结构和酶活性的影响。通过磷酸酶活性测定，我们观察到在 NO 的存在下，YopH 的活性被抑制了 50%，并且在 DTT 的存在下，这种氧化修饰是部分可逆的。此外，通过生物素转移测定、高分辨率质谱（MS）和 X 射线晶体学方法，明确证实了 YopH 的 S-亚硝基化。晶体结构证实了催化半胱氨酸残基 Cys403 的 S-亚硝基化，而 MS 数据提供了证据表明 Cys221 和 Cys234 也可能被 NO 修饰。有趣的是，圆二色性光谱显示 S-亚硝基化会影响野生型 YopH 的二级结构，但对催化半胱氨酸到丝氨酸的 YopH 突变体的影响较小。获得的数据表明，S-亚硝基化抑制了 YopH 的催化活性，其作用超出了催化半胱氨酸。这些发现有助于设计有效的 YopH 抑制剂和潜在的治疗策略，以对抗这种病原体或其他使用类似机制干扰其宿主信号转导途径的病原体。
