Architecture of the Wza-Wzc complex that mediates colanic acid translocation across the cell envelope in Gram-negative bacteria.

Abstract

The Wzx/Wzy-dependent pathway is a highly conserved mechanism for bacterial polysaccharide biosynthesis. This pathway is exemplified by the assembly and export of colanic acid mediated by the Wza-Wzc complex. While recent structural investigations have yielded valuable insights into the molecular basis of colanic acid biosynthesis, the overall architecture of the Wza-Wzc complex and the regulatory mechanism of colanic acid export remain incompletely understood. Here, we report cryo-electron microscopy (cryo-EM) structures of the Wza-Wzc complex from E. coli K12 in two functionally states: the autophosphorylated wild-type (Wza-Wzc^WT) state and the non-phosphorylated (Wza-Wzc^K540M) state. Both structures reveal that Wza and Wzc assemble into a continuous channel spanning the entire bacterial cell envelope, indicating that phosphorylation of Wzc does not disassemble the intact export channel. Relative to Wza-Wzc^K540M, the octameric periplasmic domain of Wzc in Wza-Wzc^WT undergoes a significant conformational rearrangement. This rearrangement reduces constriction region's diameter and confers a negatively charged property to the interior of the Wzc octamer, which is proposed to inhibit colanic acid export. Collectively, these structural findings confirm that the Wza-Wzc complex forms a colanic acid export channel throughout Wzc's phosphorylation-dephosphorylation cycle, advancing our mechanistic understanding of the Wzx/Wzy-dependent polysaccharide biosynthesis pathway in Gram-negative bacteria.