Stabilization of a Cu-binding site by a highly conserved tryptophan residue
Stabilization of a Cu-binding site by a highly conserved tryptophan residue
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DOI：
10.1016/j.jinorgbio.2024.112501
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发表时间：
2024-02-10
影响因子：
3.9
通讯作者：
Fisher，Oriana S.
中科院分区：
文献类型：
--
作者：
Silva，Yuri Rafael de Oliveira;Zheng，Dia;Fisher，Oriana S.
铜是几乎所有活着的生物体的基本辅因子。在我们对革兰氏阳性细菌如何进口铜并维持体内平衡的知识方面，仍然存在许多空白。为了更好地了解这些过程是如何工作的，我们这里重点研究它们负责调节革兰氏阳性杆菌枯草芽孢杆菌中铜水平的cnKJI操纵子。该操纵子编码三种铜相关蛋白：铜依赖转录抑制蛋白(YcnK)、铜进口蛋白(YcnJ)和功能未知的铜结合蛋白(YcnI)。我们先前发现YcnI的细胞外未知功能域1775(DUF1775)含有一个单组氨酸支撑基序，该基序与单个铜(II)离子配位。铜(II)结合部位含有高度保守的色氨酸残基。在这里，我们研究了色氨酸的作用以及蛋白质与铜的其他氧化态相互作用的能力。我们发现YcnI在氧化的Cu(II)状态下表现出强烈的结合铜的偏好，保守的色氨酸残基对相互作用并不是必需的。我们测定了色氨酸变异体的结构，分辨率为1.95，表明色氨酸是稳定金属结合作用所必需的，并发现该变异体对铜(II)的亲和力弱于野生型蛋白。总之，这些数据为DUF1775结构域家族提供了进一步的见解，并揭示了保守的色氨酸残基的作用。
Copper serves as an essential cofactor for nearly all living organisms. There are still many gaps remaining in our knowledge of how Gram-positive bacteria import copper and maintain homeostasis. To obtain a better understanding of how these processes work, here we focus on theycnKJIoperon responsible for regulating copper levels in the Gram-positive bacteriumBacillus subtilis. This operon encodes three Cu-related proteins: a copper-dependent transcriptional repressor (YcnK), a putative copper importer (YcnJ), and a copper-binding protein of unknown function (YcnI). We previously found that YcnI's extracellularDomain ofUnknownFunction 1775 (DUF1775) houses a monohistidine brace motif that coordinates a single Cu(II) ion. The Cu(II) binding site includes a highly conserved tryptophan residue. Here, we investigate the role of that tryptophan and the ability of the protein to interact with other oxidation states of Cu. We find that YcnI exhibits strong preference for binding Cu in the oxidized Cu(II) state, and that the conserved tryptophan residue is not essential for the interaction. We determine the structure of a tryptophan variant to 1.95 Å resolution that indicates that the tryptophan is needed to stabilize the metal binding interaction, and find that this variant has weaker affinity for Cu(II) than the wild-type protein. Together, these data provide further insights into the DUF1775 domain family and reveal the role of the conserved tryptophan residue.