The CIAO3 protein operates at a crossroad of the cytosolic iron–sulfur protein assembly (CIA) machinery. Although the functional role of CIAO3 has been recently characterized, a description of its interaction network is still not complete. Literature data suggested that CIAO3 interacts individually with CIA2A and CIAO1 protein, with the latter two interacting each other. However, no experimental data are available yet showing the formation of a possible ternary complex composed by CIAO3, CIAO1, and CIA2A. This work shows, for the first time, via size exclusion chromatography coupled with multiangle light scattering, UV–vis absorption and electron paramagnetic resonance (EPR) spectroscopies, the formation of a stable, [4Fe-4S]-bound, complex, composed by CIAO3 and the hetero-CIA2A–CIAO1 complex. Moreover, site-directed mutagenesis data suggested a structural role for the C-terminal [4Fe-4S] cluster of the CIAO3 protein. These findings can provide solid bases for further investigation of the molecular mechanisms involving these CIA machinery proteins.

中文翻译：

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CIAO3蛋白与人类胞质铁硫簇组装机械的两个关键参与者形成了稳定的三元复合物。

CIAO3蛋白在胞质铁硫蛋白组装（CIA）机制的十字路口运行。尽管最近已对CIAO3的功能作用进行了描述，但对其交互网络的描述仍不完整。文献数据表明CIAO3与CIA2A和CIAO1蛋白分别相互作用，而后两个相互作用。但是，尚无实验数据显示由CIAO3，CIAO1和CIA2A组成的可能的三元复合物的形成。这项工作首次显示了通过尺寸排阻色谱结合多角度光散射，UV-vis吸收和电子顺磁共振（EPR）光谱学，形成了稳定的，[4Fe-4S]结合的复合物，由CIAO3和杂种CIA2A–CIAO1复合体。此外，定点诱变数据表明CIAO3蛋白的C端[4Fe-4S]簇具有结构性作用。这些发现可为进一步研究涉及这些CIA机制蛋白的分子机制提供坚实的基础。