TY - JOUR
T1 - Role of a Conserved Prolyl Residue (Pro173) of MotA in the Mechanochemical Reaction Cycle of the Proton-Driven Flagellar Motor of Salmonella
AU - Nakamura, Shuichi
AU - Morimoto, Yusuke V.
AU - Kami-ike, Nobunori
AU - Minamino, Tohru
AU - Namba, Keiichi
N1 - Funding Information:
We thank J. Rothman for providing the pHluorin probe, S. Sugiyama for providing pKSS13, and Y. Sowa for helpful discussion. S.N. is a research fellow of the Japan Society for the Promotion of Science. This work has been supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to S.N., T.M., and K.N.
PY - 2009/10/23
Y1 - 2009/10/23
N2 - The MotA/B complex acts as the stator of the proton-driven bacterial flagellar motor. Proton translocation through the stator complex is efficiently coupled with torque generation by the stator-rotor interactions. In Salmonella enterica serovar Typhimurium, the highly conserved Pro173 residue of MotA is close to the absolutely conserved Asp33 residue of MotB, which is believed to be a proton-binding site. Pro173 is postulated to be involved in coupling proton influx to torque generation. However, it remains unknown what critical function Pro173 carries out. Here, we characterize the motility and the torque-speed relation of the flagellar motor of the slow motile motA(P173A) mutant of Salmonella. Stall torque produced by the mutant motor was at the wild-type level, indicating that neither the number of stators in the motor nor the rotor-stator interaction is affected by the P173A substitution. In agreement with this, the motA(P173A) allele exerted a strong dominant-negative effect on wild-type motility. In contrast, high-speed rotation at low load was significantly impaired by the mutation, suggesting that the maximum rate of torque generation cycle is severely limited. Simulation of the torque-speed curve by a simple kinetic model indicated that the mutation reduces the rate of conformational changes of the MotA/B complex that switches the exposure of Asp33 to the outside and the inside of the cell, thereby slowing down the mechanochemical reaction cycle. Based on these results, we propose that Pro173 plays an important role in facilitating the conformational dynamics of the stator complex for rapid proton translocation and torque generation cycle.
AB - The MotA/B complex acts as the stator of the proton-driven bacterial flagellar motor. Proton translocation through the stator complex is efficiently coupled with torque generation by the stator-rotor interactions. In Salmonella enterica serovar Typhimurium, the highly conserved Pro173 residue of MotA is close to the absolutely conserved Asp33 residue of MotB, which is believed to be a proton-binding site. Pro173 is postulated to be involved in coupling proton influx to torque generation. However, it remains unknown what critical function Pro173 carries out. Here, we characterize the motility and the torque-speed relation of the flagellar motor of the slow motile motA(P173A) mutant of Salmonella. Stall torque produced by the mutant motor was at the wild-type level, indicating that neither the number of stators in the motor nor the rotor-stator interaction is affected by the P173A substitution. In agreement with this, the motA(P173A) allele exerted a strong dominant-negative effect on wild-type motility. In contrast, high-speed rotation at low load was significantly impaired by the mutation, suggesting that the maximum rate of torque generation cycle is severely limited. Simulation of the torque-speed curve by a simple kinetic model indicated that the mutation reduces the rate of conformational changes of the MotA/B complex that switches the exposure of Asp33 to the outside and the inside of the cell, thereby slowing down the mechanochemical reaction cycle. Based on these results, we propose that Pro173 plays an important role in facilitating the conformational dynamics of the stator complex for rapid proton translocation and torque generation cycle.
KW - bacterial flagellar motor
KW - mechanochemical reaction cycle
KW - proton translocation
KW - stator complex
KW - torque generation
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U2 - 10.1016/j.jmb.2009.08.022
DO - 10.1016/j.jmb.2009.08.022
M3 - Article
C2 - 19683537
AN - SCOPUS:70349431086
VL - 393
SP - 300
EP - 307
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 2
ER -