A Bacillus Flagellar Motor That Can Use Both Na+ and K+ as a Coupling Ion Is Converted by a Single Mutation to Use Only Na+

Terahara, Naoya; Sano, Motohiko; Ito, Masahiro

doi:10.1371/journal.pone.0046248

Cited by 63 publications

(64 citation statements)

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“…Terahara et al (7) reported that MotPS of B. alcalophilus is the stator driven by Rb + , K + , and Na + , whereas the Met33Leu mutant (equivalent of residue 43 in E. coli) on MotS selectively uses Na + . H + -driven motors commonly have Val at the equivalent positions of MotB, and Na + -driven motors have Leu instead, such as in V. alginolyticus.…”

Section: Significancementioning

confidence: 99%

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

Nishihara

Kitao

2015

Proc. Natl. Acad. Sci. U.S.A.

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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 ...

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Section: Significancementioning

confidence: 99%

“…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).…”

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confidence: 99%

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

Nishihara

Kitao

2015

Proc. Natl. Acad. Sci. U.S.A.

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“…The motP/motS genes are universally located down-stream of the ccpA gene, which encodes a central regulator of carbon metabolism in Bacillus species and other Gram-positive organisms (Moreno et al, 2001;Ito et al, 2011). As an exception, extremely alkaliphilic Bacillus alcalophilus Vedder 1934 has a MotP/MotS type stator which utilizes Na + , K + and Rb + as the coupling ions for flagellar rotation (Terahara et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Suppressor mutants from MotB-D24E and MotS-D30E in the flagellar stator complex of Bacillus subtilis

Takahashi

Koyama

Ito

2014

J. Gen. Appl. Microbiol.

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The bacterial flagellar motor is mainly energized by either a proton (H + ) or sodium ion (Na + ) motive force and the motor torque is generated by interaction at the rotor-stator interface. MotA/MotB-type stators use H + as the coupling ion, whereas MotP/ MotS-and PomA/PomB-type stators use Na + . Bacillus subtilis employs both H + -coupled MotA/MotB and Na + -coupled MotP/MotS stators, which contribute to the torque required for flagellar rotation. In Escherichia coli, there is a universally conserved Asp-32 residue of MotB that is critical for motility and is a predicted H + -binding site. In B. subtilis, the conserved aspartic acid residue corresponds to Asp-24 of MotB (MotB-D24) and Asp-30 of MotS (MotS-D30). Here we report the isolation of two mutants, MotB-D24E and MotS-D30E, which showed a nonmotile and poorly motile phenotype, respectively. Up-motile mutants were spontaneously isolated from each mutant. We identified a suppressor mutation at MotB-T181A and MotP-L172P, respectively. Mutants MotB-T181A and MotP-L172P showed about 50% motility and a poorly motile phenotype compared to each wild type strain. These suppressor sites were suggested to indirectly affect the structure of the ion influx pathway.

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“…Recently, a nonselective member of this family was characterized from Bacillus alcalophilus (Ns V Ba: nonselective voltage‐gated B. alcalophilus ) (14). This channel exhibits a unique selectivity filter (sequence: TLDSWGSG) which conducts K + as well as Na + ions, an adaptation that allows B. alcalophilus to grow in high‐K + conditions (15). Crystal structures from the full‐length bacterial channels Na V Ab and Na V Rh exhibit voltage sensors in partially and fully activated states (7, 9).…”

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A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel

et al. 2017

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Voltage-gated sodium channels (Na V s) are activated by transiting the voltage sensor from the deactivated to the activated state. The crystal structures of several bacterial Na V s have captured the voltage sensor module (VSM) in an activated state, but structure of the deactivated voltage sensor remains elusive. In this study, we sought to identify peptide toxins stabilizing the deactivated VSM of bacterial Na V s. We screened fractions from several venoms and characterized a cystine knot toxin called JZTx-27 from the venom of tarantula Chilobrachys jingzhao as a high-affinity antagonist of the prokaryotic Na V s nonselective voltage-gated, Bacillus alcalophilus (Ns V Ba) and bacterial sodium channel from Bacillus halodurans (NaChBac) (IC 50 = 112 nM and 30 nM, respectively). JZTx-27 was more efficacious at weaker depolarizing voltages and significantly slowed the activation but accelerated the deactivation of Ns V Ba, whereas the local anesthetic drug lidocaine was shown to antagonize Ns V Ba without affecting channel gating. Mutation analysis confirmed that JZTx-27 bound to S3-4 linker of Ns V Ba, with F98 being the critical residue in determining toxin affinity. All electrophysiological data and in silico analysis suggested that JZTx-27 trapped VSM of Ns V Ba in one of the deactivated states. In mammalian Na V s, JZTx-27 preferably inhibited the inactivation of Na V 1.5 by targeting the fourth transmembrane domain. To our knowledge, this is the first report of peptide antagonist for prokaryotic Na V s. More important, we proposed that JZTx-27 stabilized the Ns V Ba VSM in the deactivated state and may be used as a probe to determine the structure of the deactivated VSM of Na V s.-Tang, C., Zhou, X., Nguyen, P. T., Zhang, Y., Hu, Z., Zhang, C., Yarov-Yarovoy, V. DeCaen, P. G. Liang, S., Liu, Z. A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel. FASEB J. 31, 000-000 (2017). www.fasebj.orgMutations in Na V s cause a variety of diseases of the heart and central and peripheral nervous systems (e.g., long QT syndrome and epilepsy) (1, 2). Many of these mutations are found within the voltage-sensor modules (VSMs) of Na V s, which alter the voltagedependent kinetics of the channel gating (3-5). Understanding structure and function of the voltage sensor of Na V s will provide insight into the molecular basis of electrical signaling in normal and diseased conditions. Eukaryotic Na V s are large proteins with 24 transmembrane segments, making them challenging to study by using crystallographic techniques (6). Several laboratories have crystalized Na V s from bacteria, which are relatively small and homotetrameric (7-10). Four monomers of bacterial Na V s assemble to form a channel. Analogous to the 4 domains (I-IV) of eukaryotic Na V s, each bacterial channel monomer contains a voltage sensor and a pore domain. For prokaryotic Na V s, the first 4 transmembrane segments (S1-4) form a voltage sensor module (VSM) and the last 2 transmembrane segments (S5 and -6) compr...

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A Bacillus Flagellar Motor That Can Use Both Na+ and K+ as a Coupling Ion Is Converted by a Single Mutation to Use Only Na+

Cited by 63 publications

References 51 publications

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

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

Suppressor mutants from MotB-D24E and MotS-D30E in the flagellar stator complex of Bacillus subtilis

A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel

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