Sleutel Mike, Sonani Ravi R, Miller Jessalyn G, Wang Fengbin, Socorro Andres Gonzalez, Chen Yang, Martin Reece, Demeler Borries, Rudolph Michael J, Alva Vikram, Remaut Han, Egelman Edward H, Conticello Vincent P
Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium.
Structural and Molecular Microbiology, VIB-VUB Center for Structural Biology, Brussels, Belgium.
bioRxiv. 2024 Dec 30:2024.12.30.630787. doi: 10.1101/2024.12.30.630787.
Cannulae are tubular protein filaments that accumulate on the extracellular surface of the hyperthermophilic archaeon during cell division. Cannulae have been postulated to act as a primitive extracellular matrix through which cells could communicate or exchange material, although their native biological function remains obscure. Here, we report cryoEM structural analyses of cannulae and of protein assemblies derived from recombinant cannula-like proteins. Three-dimensional reconstructions of cannulae revealed that the structural interactions between protomers in the native and recombinant filaments were based on donor strand complementation, a form of non-covalent polymerization in which a donor β-strand from one subunit is inserted into an acceptor groove in a β-sheet of a neighboring subunit. Donor strand complementation in cannulae is reinforced through calcium ion coordination at the interfaces between structural subunits in the respective assemblies. While donor strand complementation occurs during the assembly of chaperone-usher pili, this process requires the participation of accessory proteins that are localized in the outer membrane. In contrast, we demonstrate that calcium ions can induce assembly of cannulae in the absence of other co-factors. Crystallographic analysis of a recombinant cannula-like protein monomer provided evidence that calcium ion binding primes the precursor for donor strand invasion through unblocking of the acceptor groove. Bioinformatic analysis suggested that structurally homologous cannula-like proteins occurred within the genomes of other hyperthermophilic archaea and were encompassed within the TasA superfamily of biomatrix proteins. CryoEM structural analyses of tubular filaments derived from assembly of a recombinant cannula-like protein from an uncultured species revealed a common mode of assembly to the cannulae, in which donor strand complementation and calcium ion binding stabilized longitudinal and lateral assembly in tubular 2D sheets.
菌毛是在嗜热古菌细胞分裂过程中积累在细胞外表面的管状蛋白质细丝。尽管菌毛的天然生物学功能仍不清楚，但有人推测它可作为一种原始的细胞外基质，细胞可通过它进行通讯或交换物质。在此，我们报告了菌毛以及源自重组类菌毛蛋白的蛋白质组装体的冷冻电镜结构分析。菌毛的三维重建显示，天然和重组细丝中单体之间的结构相互作用基于供体链互补，这是一种非共价聚合形式，其中一个亚基的供体β链插入相邻亚基β片层中的受体凹槽。菌毛中的供体链互补通过各自组装体中结构亚基之间界面处的钙离子配位得到加强。虽然供体链互补发生在伴侣-usher菌毛的组装过程中，但这个过程需要定位在外膜中的辅助蛋白的参与。相比之下，我们证明钙离子可以在没有其他辅助因子的情况下诱导菌毛的组装。对重组类菌毛蛋白单体的晶体学分析提供了证据，表明钙离子结合通过解开受体凹槽来启动供体链入侵的前体。生物信息学分析表明，结构同源的类菌毛蛋白存在于其他嗜热古菌的基因组中，并包含在生物基质蛋白的TasA超家族中。对源自未培养物种的重组类菌毛蛋白组装的管状细丝的冷冻电镜结构分析揭示了与菌毛的一种共同组装模式，其中供体链互补和钙离子结合稳定了管状二维片层中的纵向和横向组装。
