Interaction between Na+ ion and carboxylates of the PomA-PomB stator unit studied by ATR-FTIR spectroscopy.
Bacterial flagellar motors are molecular machines powered by the electrochemical potential gradient of specific ions across the membrane. The PomA-PomB stator complex of Vibrio alginolyticus couples Na(+) influx to torque generation in this supramolecular motor, but little is known about how Na(+) associates with the PomA-PomB complex in the energy conversion process. Here, by means of attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy, we directly observed binding of Na(+) to carboxylates in the PomA-PomB complex, including the functionally essential residue Asp24. The Na(+) affinity of Asp24 is estimated to be approximately 85 mM, close to the apparent K(m) value from the swimming motility of the cells (78 mM). At least two other carboxylates are shown to be capable of interacting with Na(+), but with somewhat lower affinities. We conclude that Asp24 and at least two other carboxylates constitute Na(+) interaction sites in the PomA-PomB complex. This work reveals features of the Na(+) pathway in the PomA-PomB Na(+) channel by using vibrational spectroscopy.
Sudo
Yuki
Y
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.
Kitade
Yuya
Y
Furutani
Yuji
Y
Kojima
Masaru
M
Kojima
Seiji
S
Homma
Michio
M
Kandori
Hideki
H
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002264:Carboxylic Acids / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005407:Flagella / Q000235:genetics / Q000378:metabolism; D020409:Molecular Motor Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D012964:Sodium / Q000378:metabolism*; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism*; D012965:Sodium Chloride / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D044149:Vibrio alginolyticus / Q000235:genetics / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002264:Carboxylic Acids; D020409:Molecular Motor Proteins; C103352:PomA protein, bacteria; C107541:PomB protein, Vibrio; D015222:Sodium Channels; D012965:Sodium Chloride; D012964:Sodium