Oligomerization and activation of the FliI ATPase central to bacterial flagellum assembly

Abstract

SummaryFliI is the peripheral membrane ATPase pivotal to the type III protein export mechanism underlying the assembly of the bacterial flagellum. Gel filtration and multiangle light scattering showed that purified soluble native FliI protein was in a monomeric state but, in the presence of ATP, FliI showed a propensity to oligomerize. Electron microscopy revealed that FliI assembles to a ring structure, the yield of which was increased by the presence of a non-hydrolysable ATP analogue. Single particle analysis of the resulting electron micrograph images, to which no symmetry was applied, showed that the FliI ring structure has sixfold symmetry and an external diameter of ª ª ª ª 10 nm. The oligomeric ring has a central cavity of 2.5-3.0 nm, which is comparable to the known diameter of the flagellar export channel into which export substrates feed. Enzymatic activity of the FliI ATPase showed positive co-operativity, establishing that oligomerization and enzyme activity are coupled. Escherichia coli phospholipids increased enzyme co-operativity, and in vitro cross-linking demonstrated that they promoted FliI multimerization. The data reveal central facets of the structure and action of the flagellar assembly ATPase and, by extension, the homologous ATPases of virulence-related type III export systems.

Structural similarity between the flagellar type III ATPase FliI and F 1 -ATPase subunits

“…In contrast, the FliI hexamer is unstable, and the subunit assembly is controlled by external factors. ATP and phospholipids affect the hexamer formation of FliI (11,13). Oligomerization of FliI is also strongly controlled by a small number of N-terminal residues, and variants of FliI missing those N-terminal residues cannot form the hexamer ring (13, 15), although they still retain a low-level ATPase activity (13).…”

“…FliI is a member of the Walker-type ATPase family (10), and it is thought to form a ring-shaped hexamer for protein export (11)(12)(13)(14). FliI has significant similarity in its primary sequence to the ␣ and ␤ subunits of F 0 F 1 -ATPsynthase (14,15).…”

“…FliI has significant similarity in its primary sequence to the ␣ and ␤ subunits of F 0 F 1 -ATPsynthase (14,15). Although F 1 -ATPase requires both the ␣ and ␤ subunits to form the ␣ 3 ␤ 3 hexameric ring to exert its ATPase activity (16), FliI can self-assemble into a hexameric ring in solution, and it shows a cooperative increase in ATPase activity in the presence of ATP and phospholipids (11,13). Therefore, it is thought that FliI also forms a hexameric ring when it docks to the FlhA-FlhB platform of the flagellar export apparatus (13).…”

Distinct roles of the FliI ATPase and proton motive force in bacterial flagellar protein export

“…FliI shows extensive similarity to the a/b subunits of proton-driven F 0 F 1 -ATPase for its entire molecular structure 6 , although sequence similarity is limited to their respective ATPase domains 7,8 . Unlike F 1 -ATPase, however, which forms the a 3 b 3 hexameric ring, FliI self-assembles into a homohexamer 9,10 . When FliI oligomerization is suppressed by a small deletion in its amino-terminal region, flagellar protein export does not occur efficiently, suggesting that FliI hexamerizes on docking to the export gate made of the six integral membrane components, for which the cytoplasmic domains of FlhA and FlhB are thought to form the docking platform 11 .…”

The type III secretion injectisome, a complex nanomachine for intracellular ‘toxin’ delivery

“…This structure compares to that of the flagellar ATPase FliI. As for HrcN, its oligomerization and enzyme activity are coupled (Claret et al, 2003). Recently, the structure of the catalytic domain from EscN (50% identical to YscN) from EPEC was solved by crystallography at 1.8 Å resolution.…”