Mezhyrova Julija, Martin Janosch, Börnsen Clara, Dötsch Volker, Frangakis Achilleas Stefanos, Morgner Nina, Bernhard Frank
Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Frankfurt am Main 60438, Germany.
Institute of Physical and Theoretical Chemistry, Goethe University, Frankfurt am Main 60438, Germany.
Microbiome Res Rep. 2023 Jul 20;2(4):28. doi: 10.20517/mrr.2023.28. eCollection 2023.
The peptide MS2-L represents toxins of the ssRNA Leviviridae phage family and consists of a predicted N-terminal soluble domain followed by a transmembrane domain. MS2-L mediates bacterial cell lysis through the formation of large lesions in the cell envelope, but further details of this mechanism as a prerequisite for applied bioengineering studies are lacking. The chaperone DnaJ is proposed to modulate MS2-L activity, whereas other cellular targets of MS2-L are unknown. Here, we provide a combined and overexpression approach to reveal molecular insights into MS2-L action and its interaction with DnaJ. Full-length MS2-L and truncated derivatives were synthesized cell-free and co-translationally inserted into nanodiscs or solubilized in detergent micelles. By native liquid bead ion desorption mass spectrometry, we demonstrate that MS2-L assembles into high oligomeric states after membrane insertion. Oligomerization is directed by the transmembrane domain and is impaired in detergent environments. Studies with truncated MS2-L derivatives provide evidence that the soluble domain acts as a modulator of oligomer formation. DnaJ strongly interacts with MS2-L in membranes as well as in detergent environments. However, this interaction affects neither the MS2-L membrane insertion efficiency nor its oligomerization in nanodisc membranes. In accordance with the in vitro data, the assembly of MS2-L derivatives into large membrane located clusters was monitored by overexpression of corresponding fusions with fluorescent monitors in cells. Analysis by cryo-electron microscopy indicates that lesion formation is initiated in the outer membrane, followed by disruption of the peptidoglycan layer and disintegration of the inner membrane. MS2-L forms oligomeric complexes similar to the related phage toxin ΦX174-E. The oligomeric interface of both peptides is located within their transmembrane domains. We propose a potential function of the higher-order assembly of small phage toxins in membrane disintegration and cell lysis.
肽MS2-L代表单链RNA微小噬菌体科噬菌体家族的毒素，由一个预测的N端可溶性结构域和一个跨膜结构域组成。MS2-L通过在细胞膜上形成大的损伤来介导细菌细胞裂解，但作为应用生物工程研究前提的这一机制的更多细节尚不清楚。伴侣蛋白DnaJ被认为可调节MS2-L的活性，而MS2-L的其他细胞靶点尚不清楚。在此，我们提供了一种组合和过表达方法，以揭示MS2-L作用及其与DnaJ相互作用的分子见解。全长MS2-L和截短衍生物在无细胞体系中合成，并在共翻译过程中插入纳米圆盘或溶解在去污剂胶束中。通过原生液体珠离子解吸质谱，我们证明MS2-L在膜插入后组装成高寡聚状态。寡聚化由跨膜结构域引导，在去污剂环境中会受到损害。对截短的MS2-L衍生物的研究提供了证据，表明可溶性结构域作为寡聚体形成的调节剂。DnaJ在膜以及去污剂环境中都与MS2-L强烈相互作用。然而，这种相互作用既不影响MS2-L的膜插入效率，也不影响其在纳米圆盘膜中的寡聚化。根据体外数据，通过在细胞中过表达与荧光监测器的相应融合物，监测了MS2-L衍生物组装成位于膜上的大聚集体的情况。冷冻电子显微镜分析表明，损伤形成始于外膜，随后是肽聚糖层的破坏和内膜的解体。MS2-L形成与相关噬菌体毒素ΦX174-E相似的寡聚复合物。两种肽的寡聚界面都位于它们的跨膜结构域内。我们提出了小噬菌体毒素高阶组装在膜解体和细胞裂解中的潜在功能。
