de Oliveira Silva Yuri Rafael, Barnes Grayson, Zheng Dia, Zhitnitsky Daniel, Geathers Samuel J, Peters Stephen C, Szalai Veronika A, Helmann John D, Fisher Oriana S
Department of Chemistry, Lehigh University, 6 E Packer Ave, Bethlehem, PA USA 18015.
Department of Microbiology, Cornell University, Ithaca, NY USA 14853.
bioRxiv. 2025 May 27:2025.05.23.655588. doi: 10.1101/2025.05.23.655588.
The transition metal copper is biologically essential across all three domains of life. Several copper-dependent proteins and enzymes produced by the Gram-positive bacterium have been characterized. However, many questions remain about how copper is recognized and trafficked to metalate cuproproteins. The operon in encodes a suite of proteins implicated in copper uptake and regulation, including the copper-binding protein YcnI and the putative copper importer YcnJ. Here, we demonstrate that one of the extracellular domains within YcnJ (YcnJ) binds Cu(II) in 1:1 stoichiometry with high affinity using a histidine brace motif. Biochemical results reveal that YcnJ and YcnI can exchange Cu(II). Genetic studies reveal that loss of either YcnI or YcnJ, or mutation of the key residues required for Cu(II)-binding, leads to a growth defect under conditions of copper limitation. Together, these data suggest that the Cu(II)-binding sites in both YcnI and YcnJ contribute to efficient import under Cu limited conditions. Our results support a model in which YcnI may sequester Cu(II) from YcnJ, serving a regulatory role to limit the amount of copper that enters the cytoplasm and allowing Cu(II) to be stored for later import in the outer face of the membrane. This transfer of Cu(II) between extracellular domains of membrane-bound proteins represents a potential new paradigm in bacterial copper usage.
过渡金属铜在生命的所有三个域中都是生物学必需的。革兰氏阳性细菌产生的几种铜依赖性蛋白质和酶已得到表征。然而，关于铜如何被识别并运输到金属化铜蛋白中，仍有许多问题。[细菌名称]中的[操纵子名称]操纵子编码了一组与铜摄取和调节有关的蛋白质，包括铜结合蛋白YcnI和假定的铜导入蛋白YcnJ。在这里，我们证明YcnJ中的一个细胞外结构域（YcnJ-ECD）使用组氨酸支架基序以1:1的化学计量比高亲和力结合Cu(II)。生化结果表明YcnJ-ECD和YcnI可以交换Cu(II)。遗传学研究表明，YcnI或YcnJ的缺失，或Cu(II)结合所需关键残基的突变，会导致在铜限制条件下出现生长缺陷。总之，这些数据表明YcnI和YcnJ中的Cu(II)结合位点有助于在铜限制条件下进行有效导入。我们的结果支持一个模型，即YcnI可能从YcnJ中螯合Cu(II)，起到调节作用，限制进入细胞质的铜量，并允许Cu(II)存储在膜外表面以便以后导入。膜结合蛋白细胞外结构域之间的这种Cu(II)转移代表了细菌铜利用的一种潜在新范式。
