Characterization of the periplasmic domain of MotB and implications for its role in the stator assembly of the bacterial flagellar motor.
MotA and MotB are integral membrane proteins that form the stator complex of the proton-driven bacterial flagellar motor. The stator complex functions as a proton channel and couples proton flow with torque generation. The stator must be anchored to an appropriate place on the motor, and this is believed to occur through a putative peptidoglycan-binding (PGB) motif within the C-terminal periplasmic domain of MotB. In this study, we constructed and characterized an N-terminally truncated variant of Salmonella enterica serovar Typhimurium MotB consisting of residues 78 through 309 (MotB(C)). MotB(C) significantly inhibited the motility of wild-type cells when exported into the periplasm. Some point mutations in the PGB motif enhanced the motility inhibition, while an in-frame deletion variant, MotB(C)(Delta197-210), showed a significantly reduced inhibitory effect. Wild-type MotB(C) and its point mutant variants formed a stable homodimer, while the deletion variant was monomeric. A small amount of MotB was coisolated only with the secreted form of MotB(C)-His(6) by Ni-nitrilotriacetic acid affinity chromatography, suggesting that the motility inhibition results from MotB-MotB(C) heterodimer formation in the periplasm. However, the monomeric mutant variant MotB(C)(Delta197-210) did not bind to MotB, suggesting that MotB(C) is directly involved in stator assembly. We propose that the MotB(C) dimer domain plays an important role in targeting and stable anchoring of the MotA/MotB complex to putative stator-binding sites of the motor.
Kojima
Seiji
S
Dynamic NanoMachine Project, ICORP, JST, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan. 4seiji@bunshi4.bio.nagoya-u.ac.jp
Furukawa
Yukio
Y
Matsunami
Hideyuki
H
Minamino
Tohru
T
Namba
Keiichi
K
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002465:Cell Movement / Q000235:genetics; D004252:DNA Mutational Analysis; D019281:Dimerization; D005407:Flagella / Q000378:metabolism*; D018628:Gene Dosage; D008969:Molecular Sequence Data; D019897:Periplasm / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012486:Salmonella typhimurium / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D017384:Sequence Deletion
D001426:Bacterial Proteins; C063010:MotA protein, Bacteria; C064779:MotB protein, Bacteria