Oligomerization of EpsE coordinates residues from multiple subunits to facilitate ATPase activity.

EpsE is an ATPase that powers transport of cholera toxin and hydrolytic enzymes through the Type II secretion (T2S) apparatus in the gram-negative bacterium, Vibrio cholerae. On the basis of structures of homologous Type II/IV secretion ATPases and our biochemical data, we believe that EpsE is active as an oligomer, likely a hexamer, and the binding, hydrolysis, and release of nucleotide cause EpsE to undergo dynamic structural changes, thus converting chemical energy to mechanical work, ultimately resulting in extracellular secretion. The conformational changes that occur as a consequence of nucleotide binding would realign conserved arginines (Arg(210), Arg(225), Arg(320), Arg(324), Arg(336), and Arg(369)) from adjoining domains and subunits to complete the active site around the bound nucleotide. Our data suggest that these arginines are essential for ATP hydrolysis, although their roles in shaping the active site of EpsE are varied. Specifically, we have shown that replacements of these arginine residues abrogate the T2S process due to a reduction of ATPase activity yet do not have any measurable effect on nucleotide binding or oligomerization of EpsE. We have further demonstrated that point mutations in the EpsE intersubunit interface also reduce ATPase activity without disrupting oligomerization, strengthening the idea that residues from multiple subunits must precisely interact in order for EpsE to be sufficiently active to support T2S. Our findings suggest that the action of EpsE is similar to that of other Type II/IV secretion ATPase family members, and thus these results may be widely applicable to the family as a whole.