ATPase activity and multimer formation of PilQ protein are required for thin pilus biogenesis in plasmid R64

Plasmid R64 pilQ gene is essential for the formation of thin pilus, a type IV pilus. The pilQ product contains NTP binding motifs and belongs to the P ulE-VirB11 family of NTPases, The pilQ gene was overexpressed with an N-ter minal His tag, and PilQ protein was purified. Purified His tag PilQ protein displayed ATPase activity with a V-max of 0.71 nmol/min/mg of protein and a K-m of 0.26 mM at pH 6.5. By gel filtration chromatography, PilQ protein was eluted at the position corresponding to 460 kDa, suggesting that PilQ p rotein forms a homooctamer, To analyze the relationship between structure a nd function of PilQ protein, amino acid substitutions were introduced withi n several conserved motifs, Among 11 missense mutants, 7 mutants exhibited various levels of reduced DNA transfer frequencies in liquid matings, Four mutant genes (T234I, K238Q, D263N, and H328A) were overexpressed with a His tag, The purified mutant PilQ proteins contained various levels of reduced ATPase activity. Three mutant PilQ proteins formed stable multimers simila r to wild-type PilQ, whereas the PilQ D263N multimer was unstable. PilQ D26 3N monomer exhibited low ATPase activity, while PilQ D263N multimer did not . These results indicate that ATPase activity of the PilQ multimer is essen tial for R64 thin pilus biogenesis.