Michael DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, ON, L8S 4K1, Canada.
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, L8S 4K1, Canada.
Nat Commun. 2024 Nov 21;15(1):10108. doi: 10.1038/s41467-024-54509-2.
Type VI secretion systems (T6SSs) are macromolecular assemblies that deliver toxic effector proteins between adjacent bacteria. These effectors span a wide range of protein families that all lack canonical signal sequences that would target them for export. Consequently, it remains incompletely understood how conserved structural components of the T6SS apparatus recognize a diverse repertoire of effectors. Here, we characterize a widespread family of adaptor proteins, containing the domain of unknown function DUF4123, that enable the recognition and export of evolutionarily unrelated effectors. By examining two nearly identical paralogs of the conserved T6SS spike protein, VgrG, we demonstrate that each spike protein exports a structurally unique effector. We further show that the recruitment of each effector to its respective spike protein requires a cognate adaptor protein. Protein-protein interaction experiments demonstrate that these adaptor proteins specifically tether an effector to a structurally conserved but sequence divergent helix-turn-helix motif found at the C-terminus of its cognate VgrG. Using structural predictions and mutagenesis analyses, we elucidate the molecular contacts required for these interactions and discover that these adaptor proteins contain a structurally conserved N-terminal lobe that has evolved to bind VgrG helix-turn-helix motifs and a structurally variable C-terminal lobe that recognizes diverse effector families. Overall, our work provides molecular insight into a mechanism by which conserved T6SS components recognize structurally diverse effectors.
VI 型分泌系统（T6SS）是一种将毒性效应蛋白在相邻细菌之间传递的大分子组装体。这些效应蛋白跨越了广泛的蛋白质家族，它们都缺乏将其靶向输出的经典信号序列。因此，T6SS 装置的保守结构成分如何识别多样化的效应子仍然不完全清楚。在这里，我们描述了一个广泛存在的衔接蛋白家族，其中包含结构未知功能域 DUF4123，该家族能够识别和输出进化上无关的效应子。通过检查保守的 T6SS 刺突蛋白 VgrG 的两个几乎相同的直系同源物，我们证明每个刺突蛋白都能输出一种结构独特的效应子。我们进一步表明，每个效应子被招募到其相应的刺突蛋白需要一个同源衔接蛋白。蛋白质-蛋白质相互作用实验表明，这些衔接蛋白特异性地将效应子与结构保守但序列不同的螺旋-转角-螺旋基序连接起来，该基序位于其同源 VgrG 的 C 末端。通过结构预测和突变分析，我们阐明了这些相互作用所需的分子接触，并发现这些衔接蛋白包含一个结构保守的 N 端结构域，该结构域进化为结合 VgrG 螺旋-转角-螺旋基序，以及一个结构可变的 C 端结构域，该结构域识别多样化的效应子家族。总的来说，我们的工作提供了分子见解，揭示了保守的 T6SS 成分识别结构多样化效应子的机制。
