Gate-controlled proton diffusion and protonation-induced ratchet motion in the stator of the bacterial flagellar motor

Abstract

The proton permeation process of the stator complex MotA/B in the flagellar motor of Escherichia coli was investigated. The atomic model structure of the transmembrane part of MotA/B was constructed based on the previously published disulfide cross-linking and tryptophan scanning mutations. The dynamic permeation of hydronium/ sodium ions and water molecule through the channel formed in MotA/B was observed using a steered molecular dynamics simulation. During the simulation, Leu46 of MotB acts as the gate for hydronium ion permeation, which induced the formation of water wire that may mediate the proton transfer to Asp32 on MotB. Free energy profiles for permeation were calculated by umbrella sampling. The free energy barrier for H 3 O + permeation was consistent with the proton transfer rate deduced from the flagellar rotational speed and number of protons per rotation, which suggests that the gating is the rate-limiting step. Structure and dynamics of the MotA/B with nonprotonated and protonated Asp32, Val43Met, and Val43Leu mutants in MotB were investigated using molecular dynamics simulation. A narrowing of the channel was observed in the mutants, which is consistent with the size-dependent ion selectivity. In MotA/B with the nonprotonated Asp32, the A3 segment in MotA maintained a kink whereas the protonation induced a straighter shape. Assuming that the cytoplasmic domain not included in the atomic model moves as a rigid body, the protonation/deprotonation of Asp32 is inferred to induce a ratchet motion of the cytoplasmic domain, which may be correlated to the motion of the flagellar rotor.proton transfer | bacterial flagellar motor | channel gating | ratchet motion | molecular dynamics B acterial flagella are multifuel engines that convert ion motive force to molecular motor rotation. Escherichia coli has a few proton-driven flagellar motors with stators (protein MotA/B complex) in the inner membrane that act as proton channels (1-5). In addition, Vibrio alginolyticus has a polar flagellum powered by sodium ions (6). Bacillus alcalophilus has motors driven by rubidium (Rb + ), potassium (K + ), and sodium ions (Na + ) that can be converted to Na + -driven motors by a single mutation (7).The proton transfer mechanism in membrane proteins is associated with water wire and/or a hydrogen bond chain (HBC) (8, 9). The water wire comprises water molecules aligned in a protein channel, where protons are transferred by hopping along the wire. Protons are conducted through the hydrogen bonds formed by the polar amino acid residues and water molecules along the proton transfer pathway in the HBC. Protons can also be transferred by diffusion of hydronium ions (H 3 O + ). The diffusion distance in a hydrophilic environment is short in a liquid (the lifetime in water is ca. 1 ps) (10, 11), but it should be longer in a more hydrophobic environment. H 3 O + forms a hydrogen bond (H bond) with the nearest neighbor water molecules and the carbonyl groups, and proton hopping along the H bonds is faster than diffusion of Na ...

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

“…3B) (Che et al, 2008). This is supported by the MD-simulated model structure (Nishihara and Kitao, 2015). 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.…”

“…Limited proteolysis experiments have revealed that a mutation mimicking the state where H + is bound to Asp-32 in E. coli MotB-TM induces conformational changes of MotA C , suggesting that H + translocation through the H + channel is coupled with cyclic conformational changes of MotA C , which may be responsible for flagellar motor rotation (Kojima and Blair, 2001). This is supported by MD simulations of the transmembrane H + channel domain of the MotAB complex (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.…”

“…An atomic model of the transmembrane H + channel of the E. coli MotAB complex has been built by molecular dynamics (MD) simulation based on results obtained by disulfide crosslinking experiments and tryptophan scanning mutagenesis ( Fig. 3A) (Nishihara and Kitao, 2015). MotB forms a homo-dimer in the MotAB complex (Braun and Blair, 2001).…”

“…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).…”

“…The transmembrane ion channel complex is divided into at least three structural parts: a cytoplasmic domain, a transmembrane ion channel and a PGB domain Nishihara and Kitao, 2015;Takekawa et al, 2016). A highly flexible linker connects the transmembrane ion channel and the PGB domain (Terahara et al, 2017a).…”