The RVB proteins, composed of the conservative paralogs, RVB1 and RVB2, belong to the AAA+ (ATPases Associated with various cellular Activities) protein superfamily and are present in archaea and eukaryotes. The most distinct structural features are their ability to interact with each other forming the RVB1/2 complex and their participation in several macromolecular protein complexes leading them to be involved in many biological processes. We report here the biochemical and biophysical characterization of the Neurospora crassa RVB-1/RVB-2 complex. Chromatographic analyses revealed that the complex (APO) predominantly exists as a dimer in solution although hexamers were also observed. Nucleotides influence the oligomerization state, while ATP favors hexamers formation, ADP favors the formation of multimeric states, likely dodecamers, and the Molecular Dynamics (MD) simulations revealed the contribution of certain amino acid residues in the nucleotide stabilization. The complex binds to dsDNA fragments and exhibits ATPase activity, which is strongly enhanced in the presence of DNA. In addition, both GFP-fused proteins are predominantly nuclear, and their nuclear localization signals (NLS) interact with importin-α (NcIMPα). Our findings show that some properties are specific of the fungus proteins despite of their high identity to orthologous proteins. They are essential proteins in N. crassa, and the phenotypic defects exhibited by the heterokaryotic strains, mainly related to growth and development, indicate N. crassa as a promising organism to investigate additional biological and structural aspects of these proteins.

RVB 蛋白由保守的旁系同源物 RVB1 和 RVB2 组成，属于 AAA+（与各种细胞活性相关的 ATPases A）蛋白超家族，存在于古生菌和真核生物中。最明显的结构特征是它们能够相互相互作用形成 RVB1/2 复合物，以及它们参与几种大分子蛋白质复合物，导致它们参与许多生物过程。我们在这里报告粗糙脉孢菌的生化和生物物理特征RVB-1/RVB-2 复合物。色谱分析表明复合物 (APO) 在溶液中主要以二聚体形式存在，尽管也观察到六聚体。核苷酸影响寡聚化状态，而 ATP 有利于形成六聚体，ADP 有利于形成多聚体状态，可能是十二聚体，分子动力学 (MD) 模拟揭示了某些氨基酸残基在核苷酸稳定化中的作用。该复合物与 dsDNA 片段结合并表现出 ATPase 活性，这种活性在 DNA 存在时会大大增强。此外，两种 GFP 融合蛋白主要是核蛋白，它们的核定位信号 (NLS) 与输入蛋白-α (NcIMPα) 相互作用。我们的研究结果表明，尽管真菌蛋白与直系同源蛋白具有高度同一性，但它们的某些特性是特定于真菌蛋白的。N. crassa和异核菌株表现出的表型缺陷，主要与生长和发育有关，表明N. crassa作为研究这些蛋白质的其他生物学和结构方面的有希望的生物。