Deciphering the initiation pathway of CagT4SS assembly by in vitro reconstitution of subcomplexes

Cag type IV secretion system (CagT4SS) mediates the translocation of diverse substrates and is critical for the Helicobacter pylori pathogenesis. Its assembly is initiated by the central cylinder comprising CagX, CagY and CagM, however, the order and detailed mechanism remain unclear. Here, we characterize their respective self-oligomerizing properties and in vitro reconstitute subcomplexes that recapitulate features of the native nanomachine. By integrating multiple biophysical analyses, the structures, binding kinetics and conformational dynamics of assembly intermediates are shown. The assembly begins with self-oligomerization of CagX, followed by CagY association to form a stable periplasmic ring complex (PRC). CagM is subsequently coupled with CagX through multimerization of dimers, with CagY enhancing both the stability and cooperativity of its association. Our findings show the spatial and temporal nature of the assembly process, in particular the significance of PRC substructure, providing mechanistic insights into the biogenesis of bacterial T4SSs beyond static models.