Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom.
Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom
Proc Natl Acad Sci U S A. 2018 Mar 27;115(13):3458-3463. doi: 10.1073/pnas.1716424115. Epub 2018 Feb 13.
Low copy-number plasmid pLS32 of subsp. contains a partitioning system that ensures segregation of plasmid copies during cell division. The partitioning locus comprises actin-like protein AlfA, adaptor protein AlfB, and the centromeric sequence Similar to the ParMRC partitioning system from plasmid R1, AlfA filaments form actin-like double helical filaments that arrange into an antiparallel bipolar spindle, which attaches its growing ends to sister plasmids through interactions with AlfB and Because, compared with ParM and other actin-like proteins, AlfA is highly diverged in sequence, we determined the atomic structure of nonbundling AlfA filaments to 3.4-Å resolution by cryo-EM. The structure reveals how the deletion of subdomain IIB of the canonical actin fold has been accommodated by unique longitudinal and lateral contacts, while still enabling formation of left-handed, double helical, polar and staggered filaments that are architecturally similar to ParM. Through cryo-EM reconstruction of bundling AlfA filaments, we obtained a pseudoatomic model of AlfA doublets: the assembly of two filaments. The filaments are antiparallel, as required by the segregation mechanism, and exactly antiphasic with near eightfold helical symmetry, to enable efficient doublet formation. The structure of AlfA filaments and doublets shows, in atomic detail, how deletion of an entire domain of the actin fold is compensated by changes to all interfaces so that the required properties of polymerization, nucleotide hydrolysis, and antiparallel doublet formation are retained to fulfill the system's biological raison d'être.
亚种的低拷贝数质粒 pLS32 包含一个分配系统，可确保质粒拷贝在细胞分裂期间进行分离。分配基因座包括肌动蛋白样蛋白 AlfA、衔接蛋白 AlfB 和着丝粒序列。类似于质粒 R1 的 ParMRC 分配系统，AlfA 丝形成类似于肌动蛋白的双螺旋丝，排列成反平行双极纺锤体，通过与 AlfB 的相互作用将其生长末端附着到姐妹质粒上。由于与 ParM 和其他肌动蛋白样蛋白相比，AlfA 在序列上高度分化，我们通过 cryo-EM 确定了无束状 AlfA 丝的原子结构，分辨率为 3.4-Å。该结构揭示了如何通过独特的纵向和横向接触来适应典型肌动蛋白折叠的亚结构域 IIB 的缺失，同时仍然能够形成左手、双螺旋、极性和交错的丝，其结构与 ParM 相似。通过对捆绑 AlfA 丝的 cryo-EM 重建，我们获得了 AlfA 二聚体的伪原子模型：两条丝的组装。这些丝是反平行的，这是分离机制所要求的，并且几乎具有八倍螺旋对称性的反相，从而能够有效地形成二聚体。AlfA 丝和二聚体的结构以原子细节显示了如何通过改变所有界面来补偿肌动蛋白折叠的整个结构域的缺失，从而保留聚合、核苷酸水解和反平行二聚体形成所需的性质，以满足系统的生物学存在理由。
