Function of Proline Residues of MotA in Torque Generation by the Flagellar Motor of Escherichia coli

Abstract

Bacterial flagellar motors obtain energy for rotation from the membrane gradient of protons or, in some species, sodium ions. The molecular mechanism of flagellar rotation is not understood. MotA and MotB are integral membrane proteins that function in proton conduction and are believed to form the stator of the motor. Previous mutational studies identified two conserved proline residues in MotA (Pro 173 and Pro 222 in the protein from Escherichia coli) and a conserved aspartic acid residue in MotB (Asp 32) that are important for function. Asp 32 of MotB probably forms part of the proton path through the motor. To learn more about the roles of the conserved proline residues of MotA, we examined motor function in Pro 173 and Pro 222 mutants, making measurements of torque at high load, speed at low and intermediate loads, and solvent-isotope effects (D2O versus H2O). Proton conduction by wild-type and mutant MotA-MotB channels was also assayed, by a growth defect that occurs upon overexpression. Several different mutations of Pro 173 reduced the torque of the motor under high load, and a few prevented motor rotation but still allowed proton flow through the MotA-MotB channels. These and other properties of the mutants suggest that Pro 173 has a pivotal role in coupling proton flow to motor rotation and is positioned in the channel near Asp 32 of MotB. Replacements of Pro 222 abolished function in all assays and were strongly dominant. Certain Pro 222 mutant proteins prevented swimming almost completely when expressed at moderate levels in wild-type cells. This dominance might be caused by rotor-stator jamming, because it was weaker when FliG carried a mutation believed to increase rotor-stator clearance. We propose a mechanism for torque generation, in which specific functions are suggested for the proline residues of MotA and Asp32 of MotB.

Control of speed modulation (chemokinesis) in the unidirectional rotary motor of Sinorhizobium meliloti

“…A comprehensive analysis of extragenic suppressors (located in the motA and fliG genes) led these authors to conclude that the suppressor mutations 'restore motility by introducing compensatory realignments in MotA and FliG'. In their model of flagellar motor mechanics and motor function, Braun et al (1999) proposed that electrostatic interaction at the stator-rotor interface could participate in driving rotor movement. This interpretation is consistent with our prediction that torque is generated by electrostatic force transmission from the stator to the rotor (Fig.…”

The TolQ–TolR proteins energize TolA and share homologies with the flagellar motor proteins MotA–MotB

“…This 'pore' involves, in particular, a conserved aspartate residue of MotB (Asp-32) (Zhou et al, 1998b). Two conserved proline residues of MotA (Pro-173 and Pro-222) (Braun et al, 1999) are also particularly important in torque generation. On the basis of these studies, a model has been proposed (Braun et al, 1999) in which the first proline senses a conformational state and gates proton uptake by the aspartate residue from the periplasm.…”

“…Two conserved proline residues of MotA (Pro-173 and Pro-222) (Braun et al, 1999) are also particularly important in torque generation. On the basis of these studies, a model has been proposed (Braun et al, 1999) in which the first proline senses a conformational state and gates proton uptake by the aspartate residue from the periplasm. The proton uptake causes a change that either permits (via a Brownian ratchet) or drives rotation, a step involving the second proline residue.…”

Autonomous control mechanism of stator assembly in the bacterial flagellar motor in response to changes in the environment

“…A highly conserved Pro-173 residue of E. coli MotA is in very close proximity to Asp-32 of MotB in the H + channel ( Fig. 3A) (Braun et al, 1999;Kim et al, 2008). This Pro-173 residue is postulated to be required for rapid conformational changes of the H + channel induced by the binding of H + to Asp-32 when the motor speed is increased with a decrease in external load (Nakamura et al, 2009b;Nishihara and Kitao, 2015).…”

“…Two conserved proline residues of MotA, Pro-173 and Pro-222, are postulated to be involved in such conformational changes of MotA C coupled with the protonation-deprotonation cycle of Asp-32 in the H + channel ( Fig. 2C) (Braun et al, 1999;Kojima and Blair, 2001).…”