Identifying peptides and sites involved in FtsZ self-assembly in Escherichia coli through in vivo protein photocrosslinking of FtsZ residues mediated by the unnatural amino acid Bpa (p-benzoyl-L-phenylalanine), followed by tandem mass spectrometry analysis of purified FtsZ crosslinking dimers.

Bacterial cell division hinges on the Z-ring, an architecture built from the dynamical assembly and disassembly of FtsZ proteins. This delicate balance ensures not only apparent stability, but also continuous remodeling, both of which are required for Z-ring functioning. However, the molecular nature of such subcellular structures remains elusive. In searching for elusive FtsZ self-assembly interfaces involved in assembling the dynamic Z-ring in bacterial cells, we performed in vivo protein photocrosslinking for FtsZ residues as mediated by the unnatural amino acid Bpa and found some N-IDR and N-domain residues were involved in FtsZ self-assembly interactions. To identify counterpart surfaces with which the N-IDR and N-domain interacted when FtsZ self-assembled, we characterized FtsZ peptides and residues that were crosslinked to Bpa residues introduced into these regions by performing tandem mass spectrometry on purified FtsZ crosslinking dimers. Surprisingly, we observed that Bpa residues introduced to the N-IDR (e.g., at T8 or D10) crosslinked with residues in the N-domain, while Bpa residues introduced to the N-domain (e.g., at F40, K51, Q56 or S62) crosslinked with residues in the N-IDR. These crosslinking results strongly suggested that the N-IDR of one subunit interacted with the N-domain of a neighbouring subunit and vice versa when FtsZ self-assembled in cells.

细菌细胞分裂核心依托于Z环（Z-ring）——这一由FtsZ蛋白（FtsZ）动态组装与解聚形成的亚细胞结构。这种精妙的动态平衡不仅维持了Z环的表观稳定性，同时保障了其持续重塑，而这两点均为Z环行使功能所必需。然而，这类亚细胞结构的分子本质至今仍未明晰。为探寻参与细菌细胞内动态Z环组装的FtsZ自组装界面，我们借助非天然氨基酸Bpa（unnatural amino acid Bpa）介导的体内蛋白质光交联技术，针对FtsZ残基开展实验，结果发现部分N端内在无序区域（N-IDR）与N结构域（N-domain）残基参与了FtsZ的自组装相互作用。为明确FtsZ自组装时与N-IDR及N结构域发生相互作用的对应结合表面，我们通过对纯化获得的FtsZ交联二聚体进行串联质谱分析，鉴定了与这些区域内引入的Bpa残基发生交联的FtsZ肽段与残基。令人意外的是，我们发现引入至N-IDR的Bpa残基（如T8或D10位点）可与N结构域内的残基发生交联；反之，引入至N结构域的Bpa残基（如F40、K51、Q56或S62位点）亦可与N-IDR内的残基发生交联。上述交联实验结果强有力地表明，当FtsZ在细胞内发生自组装时，单个亚基的N-IDR可与相邻亚基的N结构域发生相互作用，反之亦然。