Hexamers of the type II secretion ATPase GspE from Vibrio cholerae with increased ATPase activity.

The type II secretion system (T2SS), a multiprotein machinery spanning two membranes in Gram-negative bacteria, is responsible for the secretion of folded proteins from the periplasm across the outer membrane. The critical multidomain T2SS assembly ATPase GspE(EpsE) had not been structurally characterized as a hexamer. Here, four hexamers of Vibrio cholerae GspE(EpsE) are obtained when fused to Hcp1 as an assistant hexamer, as shown with native mass spectrometry. The enzymatic activity of the GspE(EpsE)-Hcp1 fusions is ∼20 times higher than that of a GspE(EpsE) monomer, indicating that increasing the local concentration of GspE(EpsE) by the fusion strategy was successful. Crystal structures of GspE(EpsE)-Hcp1 fusions with different linker lengths reveal regular and elongated hexamers of GspE(EpsE) with major differences in domain orientation within subunits, and in subunit assembly. SAXS studies on GspE(EpsE)-Hcp1 fusions suggest that even further variability in GspE(EpsE) hexamer architecture is likely.