Intermolecular cross-linking between the periplasmic Loop3-4 regions of PomA, a component of the Na+-driven flagellar motor of Vibrio alginolyticus

PomA and PomB form a complex that conducts sodium ions and generates the torque for the Na(+)-driven polar flagellar motor of Vibrio alginolyticus. PomA has four transmembrane segments. One periplasmic loop (loop(1-2)) connects segments 1 and 2, and another (loop(3-4)), in which cysteine-scanning mutagenesis had been carried out, connects segments 3 and 4. When PomA with an introduced Cys residue (Cys-PomA) in the C-terminal periplasmic loop (loop(3-4)) was examined without exposure to a reducing reagent, a 43-kDa band was observed, whereas only a 25-kDa band, which corresponds to monomeric PomA, was observed under reducing conditions. The intensity of the 43-kDa band was enhanced in most mutants by the oxidizing reagent CuCl(2). The 43-kDa band was strongest in the P172C mutant. The motility of the P172C mutant was severely reduced, and P172C showed a dominant-negative effect, whereas substitution of Pro with Ala, Ile, or Ser at this position did not affect motility. In the presence of DTT, the ability to swim was partially restored, and the amount of 43-kDa protein was reduced. These results suggest that the disulfide cross-link disturbs the function of PomA. When the mutated Cys residue was modified with N-ethylmaleimide, only the 25-kDa PomA band was labeled, demonstrating that the 43-kDa form is a cross-linked homodimer and suggesting that the loops(3-4) of adjacent subunits of PomA are close to each other in the assembled motor. We propose that this loop region is important for dimer formation and motor function.     

Interaction of PomB with the third transmembrane segment of PomA in the Na+-driven polar flagellum of Vibrio alginolyticus

The marine bacterium Vibrio alginolyticus has four motor components, PomA, PomB, MotX, and MotY, responsible for its Na(+)-driven flagellar rotation. PomA and PomB are integral inner membrane proteins having four and one transmembrane segments (TMs), respectively, which are thought to form an ion channel complex. First, site-directed Cys mutagenesis was systematically performed from Asp-24 to Glu-41 of PomB, and the resulting mutant proteins were examined for susceptibility to a sulfhydryl reagent. Secondly, the Cys substitutions at the periplasmic boundaries of the PomB TM (Ser-38) and PomA TMs (Gly-23, Ser-34, Asp-170, and Ala-178) were combined. Cross-linked products were detected for the combination of PomB-S38C and PomA-D170C mutant proteins. The Cys substitutions in the periplasmic boundaries of PomA TM3 (from Met-169 to Asp-171) and the PomB TM (from Leu-37 to Ser-40) were combined to construct a series of double mutants. Most double mutations reduced the motility, whereas each single Cys substitution slightly affected it. Although the motility of the strain carrying PomA-D170C and PomB-S38C was significantly inhibited, it was recovered by reducing reagent. The strain with this combination showed a lower affinity for Na(+) than the wild-type combination. PomA-D148C and PomB-P16C, which are located at the cytoplasmic boundaries of PomA TM3 and the PomB TM, also formed the cross-linked product. From these lines of evidence, we infer that TM3 of PomA and the TM of PomB are in close proximity over their entire length and that cooperation between these two TMs is required for coupling of Na(+) conduction to flagellar rotation.     

Functional reconstitution of the Na(+)-driven polar flagellar motor component of Vibrio alginolyticus

The bacterial flagellar motor is a molecular machine that couples the influx of specific ions to the generation of the force necessary to drive rotation of the flagellar filament. Four integral membrane proteins, PomA, PomB, MotX, and MotY, have been suggested to be directly involved in torque generation of the Na(+)-driven polar flagellar motor of Vibrio alginolyticus. In the present study, we report the isolation of the functional component of the torque-generating unit. The purified protein complex appears to consist of PomA and PomB and contains neither MotX nor MotY. The PomA/B protein, reconstituted into proteoliposomes, catalyzed (22)Na(+) influx in response to a potassium diffusion potential. Sodium uptake was abolished by the presence of Li(+) ions and phenamil, a sodium channel blocker. This is the first demonstration of a purification and functional reconstitution of the bacterial flagellar motor component involved in torque generation. In addition, this study demonstrates that the Na(+)-driven motor component, PomA and PomB, forms the Na(+)-conducting channel.     

Multimeric structure of PomA, a component of the Na+-driven polar flagellar motor of vibrio alginolyticus

Four integral membrane proteins, PomA, PomB, MotX, and MotY, are thought to be directly involved in torque generation of the Na(+)-driven polar flagellar motor of Vibrio alginolyticus. Our previous study showed that PomA and PomB form a complex, which catalyzes sodium influx in response to a potassium diffusion potential. PomA forms a stable dimer when expressed in a PomB null mutant. To explore the possible functional dependence of PomA domains in adjacent subunits, we prepared a series of PomA dimer fusions containing different combinations of wild-type or mutant subunits. Introduction of the mutation P199L, which completely inactivates flagellar rotation, into either the first or the second half of the dimer abolished motility. The P199L mutation in monomeric PomA also altered the PomA-PomB interaction. PomA dimer with the P199L mutation even in one subunit also had no ability to interact with PomB, indicating that the both subunits in the dimer are required for the functional interaction between PomA and PomB. Flagellar rotation by wild-type PomA dimer was completely inactivated by phenamil, a sodium channel blocker. However, activity was retained in the presence of phenamil when either half of the dimer was replaced with a phenamil-resistant subunit, indicating that both subunits must bind phenamil for motility to be fully inhibited. These observations demonstrate that both halves of the PomA dimer function together to generate the torque for flagellar rotation.     

Deletion analysis of the carboxyl-terminal region of the PomB component of the vibrio alginolyticus polar flagellar motor

The stator of the sodium-driven flagellar motor of Vibrio alginolyticus is a membrane protein complex composed of four PomA and two PomB subunits. PomB has a peptidoglycan-binding motif in the C-terminal region. In this study, four kinds of PomB deletions in the C terminus were constructed. None of the deletion proteins restored motility of the DeltapomB strain. The PomA protein was coisolated with all of the PomB derivatives under detergent-solubilized conditions. Homotypic disulfide cross-linking of all of the deletion derivatives through naturally occurring Cys residues was detected. We conclude that the C-terminal region of PomB is essential for motor function but not for oligomerization of PomB with itself or PomA.     

Roles of charged residues in the C-terminal region of PomA, a stator component of the Na+-driven flagellar motor

Bacterial flagellar motors use specific ion gradients to drive their rotation. It has been suggested that the electrostatic interactions between charged residues of the stator and rotor proteins are important for rotation in Escherichia coli. Mutational studies have indicated that the Na(+)-driven motor of Vibrio alginolyticus may incorporate interactions similar to those of the E. coli motor, but the other electrostatic interactions between the rotor and stator proteins may occur in the Na(+)-driven motor. Thus, we investigated the C-terminal charged residues of the stator protein, PomA, in the Na(+)-driven motor. Three of eight charge-reversing mutations, PomA(K203E), PomA(R215E), and PomA(D220K), did not confer motility either with the motor of V. alginolyticus or with the Na(+)-driven chimeric motor of E. coli. Overproduction of the R215E and D220K mutant proteins but not overproduction of the K203E mutant protein impaired the motility of wild-type V. alginolyticus. The R207E mutant conferred motility with the motor of V. alginolyticus but not with the chimeric motor of E. coli. The motility with the E211K and R232E mutants was similar to that with wild-type PomA in V. alginolyticus but was greatly reduced in E. coli. Suppressor analysis suggested that R215 may participate in PomA-PomA interactions or PomA intramolecular interactions to form the stator complex.     

The Vibrio motor proteins, MotX and MotY, are associated with the basal body of Na-driven flagella and required for stator formation

The four motor proteins PomA, PomB, MotX and MotY, which are believed to be stator proteins, are essential for motility by the Na(+)-driven flagella of Vibrio alginolyticus. When we purified the flagellar basal bodies, MotX and MotY were detected in the basal body, which is the supramolecular complex comprised of the rotor and the bushing, but PomA and PomB were not. By antibody labelling, MotX and MotY were detected around the LP ring. These results indicate that MotX and MotY associate with the basal body. The basal body had a new ring structure beneath the LP ring, which was named the T ring. This structure was changed or lost in the basal body from a DeltamotX or DeltamotY strain. The T ring probably comprises MotX and MotY. In the absence of MotX or MotY, we demonstrated that PomA and PomB were not localized to a cell pole. From the above results, we suggest that MotX and MotY of the T ring are involved in the incorporation and/or stabilization of the PomA/PomB complex in the motor.     

Cell-free synthesis of the torque-generating membrane proteins, PomA and PomB, of the Na+-driven flagellar motor in Vibrio alginolyticus

Flagellar motor proteins, PomA and PomB, are essential for converting the sodium motive force into rotational energy in the Na(+)-driven flagella motor of Vibrio alginolyticus. PomA and PomB, which are cytoplasmic membrane proteins, together comprise the stator complex of the motor and form a Na(+) channel. We tried to synthesize PomA and PomB by using the cell-free protein synthesis system, PURESYSTEM. We succeeded in doing so in the presence of liposomes, and showed an interaction between them using the pull-down assay. It seems likely that the proteins are inserted into liposomes and assembled spontaneously. The N-terminal region of in vitro synthesized PomB appeared to be lost, but this problem was suppressed by fusing GFP to the N-terminus of PomB or by mutagenesis at Pro-11 or Pro-12. A structural change of the N-terminal region of PomB by these modifications may prevent cleavage during protein synthesis in PURESYSTEM. The mutations did not affect the functioning of the motor. Using this system, biochemical analysis of PomA and PomB can be performed easily and efficiently.     

Characterization of PomA mutants defective in the functional assembly of the Na(+)-driven flagellar motor in Vibrio alginolyticus

The polar flagellar motor of Vibrio alginolyticus rotates using Na(+) influx through the stator, which is composed of 2 subunits, PomA and PomB. About a dozen stators dynamically assemble around the rotor, depending on the Na(+) concentration in the surrounding environment. The motor torque is generated by the interaction between the cytoplasmic domain of PomA and the C-terminal region of FliG, a component of the rotor. We had shown previously that mutations of FliG affected the stator assembly around the rotor, which suggested that the PomA-FliG interaction is required for the assembly. In this study, we examined the effects of various mutations mainly in the cytoplasmic domain of PomA on that assembly. All mutant stators examined, which resulted in the loss of motor function, assembled at a lower level than did the wild-type PomA. A His tag pulldown assay showed that some mutations in PomA reduced the PomA-PomB interaction, but other mutations did not. Next, we examined the ion conductivity of the mutants using a mutant stator that lacks the plug domain, PomA/PomB(ΔL)(Δ41-120), which impairs cell growth by overproduction, presumably because a large amount of Na(+) is conducted into the cells. Some PomA mutations suppressed this growth inhibition, suggesting that such mutations reduce Na(+) conductivity, so that the stators could not assemble around the rotor. Only the mutation H136Y did not impair the stator formation and ion conductivity through the stator. We speculate that this particular mutation may affect the PomA-FliG interaction and prevent activation of the stator assembly around the rotor.     

The systematic substitutions around the conserved charged residues of the cytoplasmic loop of Na+-driven flagellar motor component PomA

PomA, a homolog of MotA in the H+-driven flagellar motor, is an essential component for torque generation in the Na+-driven flagellar motor. Previous studies suggested that two charged residues, R90 and E98, which are in the single cytoplasmic loop of MotA, are directly involved in this process. These residues are conserved in PomA of Vibrio alginolyticus as R88 and E96, respectively. To explore the role of these charged residues in the Na+-driven motor, we replaced them with other amino acids. However, unlike in the H+-driven motor, both of the single and the double PomA mutants were functional. Several other positively and negatively charged residues near R88 and E96, namely K89, E97 and E99, were neutralized. Motility was retained in a strain producing the R88A/K89A/E96Q/E97Q/E99Q (AAQQQ) PomA protein. The swimming speed of the AAQQQ strain was as fast as that of the wild-type PomA strain, but the direction of motor rotation was abnormally counterclockwise-biased. We could, however, isolate non-motile or poorly motile mutants when certain charged residues in PomA were reversed or neutralized. The charged residues at positions 88-99 of PomA may not be essential for torque generation in the Na+-driven motor and might play a role in motor function different from that of the equivalent residues of the H+-driven motor.     

Concerted effects of amino acid substitutions in conserved charged residues and other residues in the cytoplasmic domain of PomA, a stator component of Na+-driven flagella

PomA is a membrane protein that is one of the essential components of the sodium-driven flagellar motor in Vibrio species. The cytoplasmic charged residues of Escherichia coli MotA, which is a PomA homolog, are believed to be required for the interaction of MotA with the C-terminal region of FliG. It was previously shown that a PomA variant with neutral substitutions in the conserved charged residues (R88A, K89A, E96Q, E97Q, and E99Q; AAQQQ) was functional. In the present study, five other conserved charged residues were replaced with neutral amino acids in the AAQQQ PomA protein. These additional substitutions did not affect the function of PomA. However, strains expressing the AAQQQ PomA variant with either an L131F or a T132M substitution, neither of which affected motor function alone, exhibited a temperature-sensitive (TS) motility phenotype. The double substitutions R88A or E96Q together with L131F were sufficient for the TS phenotype. The motility of the PomA TS mutants immediately ceased upon a temperature shift from 20 to 42 degrees C and was restored to the original level approximately 10 min after the temperature was returned to 20 degrees C. It is believed that PomA forms a channel complex with PomB. The complex formation of TS PomA and PomB did not seem to be affected by temperature. Suppressor mutations of the TS phenotype were mapped in the cytoplasmic boundaries of the transmembrane segments of PomA. We suggest that the cytoplasmic surface of PomA is changed by the amino acid substitutions and that the interaction of this surface with the FliG C-terminal region is temperature sensitive.     

Assembly of motor proteins, PomA and PomB, in the Na+-driven stator of the flagellar motor

PomA and PomB are transmembrane proteins that form the stator complex in the sodium-driven flagellar motor of Vibrio alginolyticus and are believed to surround the rotor part of the flagellar motor. We constructed and observed green fluorescent protein (GFP) fusions of the stator proteins PomA and PomB in living cells to clarify how stator proteins are assembled and installed into the flagellar motor. We were able to demonstrate that GFP-PomA and GFP-PomB localized to a cell pole dependent on the presence of the polar flagellum. Localization of the GFP-fused stator proteins required their partner subunit, PomA or PomB, and the C-terminal domain of PomB, which has a peptidoglycan-binding motif. Each of the GFP-fused stator proteins was co-isolated with its partner subunit from detergent-solubilized membrane. From these lines of evidence, we have demonstrated that the stator proteins are incorporated into the flagellar motor as a PomA/PomB complex and are fixed to the cell wall via the C-terminal domain of PomB.     

Functional role of a conserved aspartic acid residue in the motor of the Na-driven flagellum from Vibrio cholerae

The flagellar motor consists of a rotor and a stator and couples the flux of cations (H⁺ or Na⁺) to the generation of the torque necessary to drive flagellum rotation. The inner membrane proteins PomA and PomB are stator components of the Na⁺-driven flagellar motor from Vibrio cholerae. Affinity-tagged variants of PomA and PomB were co-expressed in trans in the non-motile V. cholerae pomAB deletion strain to study the role of the conserved D23 in the transmembrane helix of PomB. At pH 9, the D23E variant restored motility to 100% of that observed with wild type PomB, whereas the D23N variant resulted in a non-motile phenotype, indicating that a carboxylic group at position 23 in PomB is important for flagellum rotation. Motility tests at decreasing pH revealed a pronounced decline of flagellar function with a motor complex containing the PomB-D23E variant. It is suggested that the protonation state of the glutamate residue at position 23 determines the performance of the flagellar motor by altering the affinity of Na⁺ to PomB. The conserved aspartate residue in the transmembrane helix of PomB and its H⁺-dependent homologs might act as a ligand for the coupling cation in the flagellar motor.     

Ion-coupling determinants of Na+-driven and H+-driven flagellar motors

The bacterial flagellar motor is a tiny molecular machine that uses a transmembrane flux of H(+) or Na(+) ions to drive flagellar rotation. In proton-driven motors, the membrane proteins MotA and MotB interact via their transmembrane regions to form a proton channel. The sodium-driven motors that power the polar flagellum of Vibrio species contain homologs of MotA and MotB, called PomA and PomB. They require the unique proteins MotX and MotY. In this study, we investigated how ion selectivity is determined in proton and sodium motors. We found that Escherichia coli MotA/B restore motility in DeltapomAB Vibrio alginolyticus. Most hypermotile segregants isolated from this weakly motile strain contain mutations in motB. We constructed proteins in which segments of MotB were fused to complementary portions of PomB. A chimera joining the N terminus of PomB to the periplasmic C terminus of MotB (PotB7(E)) functioned with PomA as the stator of a sodium motor, with or without MotX/Y. This stator (PomA/PotB7(E)) supported sodium-driven motility in motA or motB E.coli cells, and the swimming speed was even higher than with the original stator of E.coli MotA/B. We conclude that the cytoplasmic and transmembrane domains of PomA/B are sufficient for sodium-driven motility. However, MotA expressed with a B subunit containing the N terminus of MotB fused to the periplasmic domain of PomB (MomB7(E)) supported sodium-driven motility in a MotX/Y-dependent fashion. Thus, although the periplasmic domain of PomB is not necessary for sodium-driven motility in a PomA/B motor, it can convert a MotA/B proton motor into a sodium motor.     

Coupling ion specificity of chimeras between H(+)- and Na(+)-driven motor proteins, MotB and PomB, in Vibrio polar flagella

We have shown that a hybrid motor consisting of proton-type Rhodobacter sphaeroides MotA and sodium-type VIBRIO: alginolyticus PomB, MotX and MotY, can work as a sodium-driven motor in VIBRIO: cells. In this study, we tried to substitute the B subunits, which contain a putative ion-binding site in the transmembrane region. Rhodobacter sphaeroides MotB did not work with either MotA or PomA in Vibrio cells. Therefore, we constructed chimeric proteins (MomB), which had N-terminal MotB and C-terminal PomB. MomB proteins, with the entire transmembrane region derived from the H(+)-type MotB, gave rise to an Na(+) motor with MotA. The other two MomB proteins, in which the junction sites were within the transmembrane region, also formed Na(+) motors with PomA, but were changed for Na(+) or Li(+) specificity. These results show that the channel part consisting of the transmembrane regions from the A and B subunits can interchange Na(+)- and H(+)-type subunits and this can affect the ion specificity. This is the first report to have changed the specificity of the coupling ions in a bacterial flagellar motor.     

Functional Transfer of an Essential Aspartate for the Ion-binding Site in the Stator Proteins of the Bacterial Flagellar Motor

Rotation of the bacterial flagellar motor exploits the electrochemical potential of the coupling ion (H⁺ or Na⁺) as its energy source. In the marine bacterium Vibrio alginolyticus, the stator complex is composed of PomA and PomB, and conducts Na⁺ across the cytoplasmic membrane to generate rotation. The transmembrane (TM) region of PomB, which forms the Na⁺-conduction pathway together with TM3 and TM4 of PomA, has a highly conserved aspartate residue (Asp24) that is essential for flagellar rotation. This residue contributes to the Na⁺-binding site. However, it is not clear whether residues other than Asp24 are involved in binding the coupling ion. We examined the possibility that loss of the negative charge of Asp24 can be suppressed by introduction of negatively charged residues in TM3 or TM4 of PomA. The motility defect associated with the D24N substitution in PomB could be rescued only by a N194D substitution in PomA. This result suggests that there must be a negatively charged ion-binding pocket in the stator complex but that the presence of a negatively charged residue at position 24 of PomB is not essential. A tandemly fused PomA dimer containing the N194D mutation either in its N-terminal or C-terminal half with PomB-D24N was functional, suggesting that PomB-D24N can form an ion-binding pocket with either subunit of PomA dimer. The findings obtained in this study provide important clues to the mechanism of ion binding in the stator complex.     

Cloning and characterization of motX, a Vibrio alginolyticus sodium-driven flagellar motor gene.

Four motor proteins, MotX, MotY, PomA, and PomB, have been identified as constituents of the Na(+)-driven flagellum of Vibrio species. In this study, the complete motX gene was cloned from Vibrio alginolyticus and shown to complement three mot mutations, motX94, motX115, and motX119, as well as a V. parahaemolyticus motX mutant. The motX94 mutant contains a frameshift at Val86 of MotX, while the motX115 and motX119 mutations comprise substitutions of Ala146 to Val and Gln 194 to amber, respectively. When MotX was overexpressed in Vibrio cells, the amount of MotY detected in the membrane fraction increased, and vice versa, suggesting that MotX and MotY mutually stabilize each other by interacting at the membrane level. When a plasmid containing the motX gene was introduced into motY mutants NMB117 (motY117) and VIO542 (motY542), the mutations were suppressed. In contrast, motY could not cause the recovery of any swarm-defective motX mutants studied. Considering the above evidence, we propose that MotX is more directly involved than MotY in the mechanical functioning of the Na(+)-type flagellar motor, and that MotY may stabilize MotX to support its interaction with other Mot proteins.     

Characterization of the periplasmic region of PomB, a Na+-driven flagellar stator protein in Vibrio alginolyticus

The stator proteins PomA and PomB form a complex that couples Na⁺ influx to torque generation in the polar flagellar motor of Vibrio alginolyticus. This stator complex is anchored to an appropriate place around the rotor through a putative peptidoglycan-binding (PGB) domain in the periplasmic region of PomB (PomB_C). To investigate the function of PomB_C, a series of N-terminally-truncated and in-frame mutants with deletions between the transmembrane (TM) segment and the PGB domain of PomB was constructed. A PomB_C fragment consisting of residues 135 to 315 (PomB_C5) formed a stable homodimer and significantly inhibited the motility of wild-type cells when overexpressed in the periplasm. A fragment with an in-frame deletion (PomB_ΔL) of up to 80 residues retained function, and its overexpression with PomA impaired cell growth. This inhibitory effect was suppressed by a mutation at the functionally critical Asp (D24N) in the TM segment of PomB, suggesting that a high level of Na⁺ influx through the mutant stator causes the growth impairment. The overproduction of functional PomA/PomB_ΔL stators also reduced the motile fractions of the cells. That effect could be slightly relieved by a mutation (L168P) in the putative N-terminal α-helix that connects to the PGB domain without affecting the growth inhibition, suggesting that a conformational change of the region including the PGB domain affects stator assembly. Our results reveal common features of the periplasmic region of PomB/MotB and demonstrate that a flexible linker that contains a “plug” segment is important for the control of Na⁺ influx through the stator complex as well as for stator assembly.     

Putative channel components for the fast-rotating sodium-driven flagellar motor of a marine bacterium.

The polar flagellum of Vibrio alginolyticus rotates remarkably fast (up to 1,700 revolutions per second) by using a motor driven by sodium ions. Two genes, motX and motY, for the sodium-driven flagellar motor have been identified in marine bacteria, Vibrio parahaemolyticus and V. alginolyticus. They have no similarity to the genes for proton-driven motors, motA and motB, whose products constitute a proton channel. MotX was proposed to be a component of a sodium channel. Here we identified additional sodium motor genes, pomA and pomB, in V. alginolyticus. Unexpectedly, PomA and PomB have similarities to MotA and MotB, respectively, especially in the predicted transmembrane regions. These results suggest that PomA and PomB may be sodium-conducting channel components of the sodium-driven motor and that the motor part consists of the products of at least four genes, pomA, pomB, motX, and motY. Furthermore, swimming speed was controlled by the expression level of the pomA gene, suggesting that newly synthesized PomA proteins, which are components of a force-generating unit, were successively integrated into the defective motor complexes. These findings imply that Na+-driven flagellar motors may have similar structure and function as proton-driven motors, but with some interesting differences as well, and it is possible to compare and study the coupling mechanisms of the sodium and proton ion flux for the force generation.     

Electron cryomicroscopic visualization of PomA/B stator units of the sodium-driven flagellar motor in liposomes

A motor protein complex of the bacterial flagellum, PomA/B from Vibrio alginolyticus, was reconstituted into liposomes and visualized by electron cryomicroscopy. PomA/B is a sodium channel, composed of two membrane proteins, PomA and PomB, and converts ion flux to the rotation of the flagellar motor. Escherichia coli and Salmonella have a homolog called MotA/B, which utilizes proton instead of sodium ion. PomB and MotB have a peptidoglycan-binding motif in their C-terminal region, and therefore PomA/B and MotA/B are regarded as the stator. Energy filtering electron cryomicroscopy enhanced the image contrast of the proteins reconstituted into liposomes and showed that two extramembrane domains with clearly different sizes stick out of the lipid bilayers on opposite sides. Image analysis combined with gold labeling and deletion of the peptidoglycan-binding motif revealed that the longer one, approximately 70 A long, is likely to correspond to the periplasmic domain, and the other, about half size, to the cytoplasmic domain.