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, Bethlehem, Pennsylvania, USA.
Department of Microbiology, Cornell University, Ithaca, New York, USA.
J Biol Chem. 2025 Jul 14;301(8):110480. doi: 10.1016/j.jbc.2025.110480.
The transition metal copper (Cu) is biologically essential across all three domains of life. Several Cu-dependent proteins and enzymes produced by the Gram-positive bacterium Bacillus subtilis have been characterized. However, many questions remain about how Cu is recognized and trafficked to metalate cuproproteins. The ycnKJI operon in B. subtilis encodes a suite of proteins implicated in Cu uptake and regulation, including the Cu-binding protein YcnI and the putative Cu 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 Cu limitation. Together, these data suggest that the Cu(II)-binding sites in both YcnI and YcnJ may 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 Cu that enters the cytoplasm and allowing Cu(II) to be stored for later import on 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 Cu usage.
过渡金属铜（Cu）在生命的所有三个领域中都是生物学所必需的。革兰氏阳性细菌枯草芽孢杆菌产生的几种依赖铜的蛋白质和酶已得到表征。然而，关于铜如何被识别并转运至金属化铜蛋白仍存在许多问题。枯草芽孢杆菌中的ycnKJI操纵子编码了一系列与铜摄取和调节有关的蛋白质，包括铜结合蛋白YcnI和假定的铜转运蛋白YcnJ。在这里，我们证明YcnJ中的一个细胞外结构域（YcnJ）使用组氨酸支架基序以1:1的化学计量比与Cu（II）高亲和力结合。生化结果表明YcnJ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生长缺陷。这些数据共同表明，YcnI和YcnJ中的Cu（II）结合位点可能有助于在铜限制条件下的有效摄取。我们的结果支持一个模型，其中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后续摄取。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。
