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

Swarming and swimming of Pro 173 and Pro 222 mutants.In a previous mutational study of MotA, we found that the conserved proline residues Pro 173 and Pro 222 are important for motility (40). These residues are located at the cytoplasmic ends of membrane segments 3 and 4 in all MotA sequences reported thus far. Several mutations of Pro 173 and Pro 222 were isolated in the previous study, and their phenotypes were characterized by measurements of swarming rates in soft agar and assays of dominance of plasmid-borne alleles over wild-type motA on the chromosome. Here, we have isolated additional replacements of Pro 173 and Pro 222 and have undertaken more detailed measurements of their effects on motor function.

Additional replacements of residues Pro 173 and Pro 222 were isolated by targeted mutagenesis with oligonucleotides randomized at the corresponding codons. The resulting collection (old and new mutants together) included 19 different replacements of Pro 173 and 16 replacements of Pro 222. Swarming rates and dominance of the new mutations were measured as described previously (35) and are provided in Table 2. Some properties of the mutations isolated previously are also given in the legend to the table to facilitate discussion of all the mutants together. Examples of swarming phenotypes are shown in Fig. 2. As observed previously with a smaller collection of mutants (40), all replacements of Pro 222 prevented swarming. Most replacements of Pro 173 also prevented swarming, but seven (P173C, P173L, P173M, P173N, P173S, P173T, and P173V) allowed cells to swarm slowly, at rates from 10 to 50% of that of the wild type (the fastest swarmer was P173T, isolated in the previous study). Also as noted previously, many of the mutations, especially the Pro 222 replacements, were strongly dominant.

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Fig. 2.

Examples of swarming phenotypes of Pro 173 and Pro 222 mutants. The motA-defective strain MS5037 was transformed with plasmids (derivatives of pRF4) expressing the mutant MotA proteins indicated. The plate contained tryptone broth and 0.28% agar. It was inoculated with 1 μl of a saturated culture of each strain and photographed after 8 h at 32°C.

Next, swimming of the Pro 173 mutant strains in liquid medium was examined under the microscope. Mutants that did not swarm in soft agar also did not swim in liquid medium. The mutants that swarmed also swam, as expected, but five of them (P173C, P173L, P173M, P173N, and P173V) swam so unsteadily that accurate speed measurements were impossible. Two mutants (P173S and P173T) swam steadily enough to allow measurements of speed. At 32°C in a pH 7 buffer, cells of the P173S and P173T mutants swam at 10.6 ± 0.7 and 13.5 ± 0.5 μm/s, respectively, compared to 38 ± 1 μm/s for the wild-type control (mean ± standard error of the mean; n ≥ 50).

Protein stability.To determine whether the mutations affected the ability of the proteins to accumulate in cells, we used immunoblots to estimate the levels of mutant MotA proteins in cell membranes. An example is shown in Fig. 3. All of the Pro 222 mutant proteins were found at approximately the same level as that of the wild-type protein, except the P222F protein, which was not detected in cell membranes from either mid-log or saturated cultures. Most of the Pro 173 mutant proteins were also found at approximately the same level as that of the wild-type protein. Some (P173D, P173N, P173W, and P173Y) were present at relatively low levels in saturated overnight cultures but at nearly normal levels in mid-log cultures. The P173Q protein was not detected in either mid-log or saturated cultures but must nevertheless be present at some level, because it supports torque generation (at 50% of normal; Fig. 5). Thus, in the majority of cases the defects in motor function are not caused by failure of the proteins to accumulate.

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Fig. 3.

Immunoblot showing cellular levels of representative mutant MotA proteins. Cell membranes were prepared from mid-log cultures and electrophoresed and immunoblotted as described in Materials and Methods. w.t., wild type.

Load and isotope effects.In free-swimming cells the load on the flagellar motors is relatively light, because the flagellar filaments are thin and several filaments work together in a bundle (21). In this circumstance, the rotation rate of the motors is limited mainly by the rates of internal processes and not by the external load. MotA is not required for flagellar assembly, and MotA mutations are not expected to affect the shape or propulsive efficiency of the flagellar bundle. The slow swimming of the mutants thus suggests that a process occurring in the motor is made slower by the replacements of Pro 173. To learn more about the rate limitations in the mutants, we examined the effects of load and solvent isotope, by measuring swimming speeds in media containing various amounts of Ficoll and in media made with D₂O.

Both the wild type and the mutants swam more slowly in D₂O than in H₂O. In D₂O, wild-type cells swam at 82% ± 3% of their speed in H₂O (mean ± standard error of the mean for 100 cells in each medium), a value similar to the isotope effect reported previously (4). The P173S and P173T mutants were slowed to 80% ± 4% and 77% ± 3% of their speeds in H₂O, respectively (mean ± standard error of the mean; n = 79 to 100). The isotope effects in the mutants are thus similar to those in the wild type.

Effects of load on swimming speed are summarized in Fig.4 for the P173S and P173T mutants and a wild-type control. Two independent experiments for each mutant and three independent experiments for the wild type are shown. Both the wild type and the mutants swam more slowly as the viscous load was increased, but the load dependence was steeper in the mutants, especially P173S.

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Fig. 4.

Effects of viscous load on the swimming speeds of wild-type cells and the MotA mutants P173S and P173T. Swimming speeds and medium viscosity were measured at 32°C in media that contained Ficoll (average molecular weight, 400,000) at concentrations ranging from 0 to 16%, as described in Materials and Methods. The reciprocals of swimming speeds are plotted against the relative viscous load, which was obtained as the product of viscosity and swimming speed. Results are shown for three independent experiments with the wild type and two independent experiments with each mutant. Values are the means ± standard errors of the mean for at least 50 cells. Practically all of the uncertainty in the estimates of load comes from the measurements of swimming speed, because measurements of viscosity are much more precise. Errors along the two axes are therefore expected to be correlated, as indicated. Best-fit straight lines are drawn to guide the eye.

Motor torque in Pro 173 and Pro 222 mutants.When cells are tethered to coverslips by individual flagellar filaments, the motors turn the cell bodies at speeds between 1 and 20 Hz. The viscous load resisting rotation is large in this case and determines the rotation rate. When the medium viscosity is increased by a given factor, rotation rate decreases by the same factor, so that torque remains constant (17, 24). Because the torque of a motor turning a tethered cell is just balanced by the viscous drag opposing rotation, motor torque can be estimated from the rotation rate, cell size and radius of gyration, and medium viscosity. Cells of each Pro 173 and Pro 222 mutant were tethered to coverslips, and rotation speed, cell size, and radius of gyration were measured for all of the rotating cells in several microscope fields. Torques were then computed by using equations for the viscous drag on rotating cylinders, as described previously (4, 35). The results are shown in Fig.5 for all of the mutants that produced measurable torque.

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Fig. 5.

Motor torques in tethered cells of Pro 173 mutants. Flagellar filaments were sheared and tethered to coverslips on a microscope stage thermostated at 32°C, and cell rotation rate, size, and radius of gyration were measured. Torques were computed as described in Materials and Methods. Values are the means ± standard errors of the mean for the sample sizes indicated.

All mutations of residue Pro 222 completely eliminated motor rotation in tethered-cell experiments. This observation is consistent with the failure of these mutants to swarm and additionally shows that Pro 222 mutants do not produce any torque, even at low speeds. (Swarming requires not only that motors produce torque but that they rotate fast enough to form a flagellar bundle, so swarming measurements cannot rule out slow motor rotation. Swarming also requires chemotaxis, so such measurements cannot rule out defects in the response of the flagellar motors to chemotactic signals.)

Nine of the Pro 173 mutants did not produce torque. Ten produced measurable torque, with values ranging from about 40 to 80% of wild type. These included the seven mutants that swarmed and swam and three others (P173A, P173I, and P173Q) that did not swarm and that appeared immotile under the microscope. Evidently, the motors of these mutants can produce appreciable torque when rotating slowly but cannot turn fast enough to sustain swimming.

Function of the mutant proteins in proton conduction.Overexpression of MotA, together with a MotB fusion protein that contains the membrane segment and residue Asp 32, reduces the growth rate of cells by a factor of about 2 (38). This growth impairment has been shown to correlate with an increased permeability of the cytoplasmic membrane to protons (4, 33), so it can be used to assay proton conduction by the MotA-MotB complexes separately from other functions they may have in motor rotation. To determine whether mutations of Pro 173 and Pro 222 affect proton conduction as assayed by growth impairments, the mutations were transferred to plasmid pLW3 (38), which expresses the motA andmotB fusion genes from the trp promoter. The resulting plasmids were transformed into wild-type cells, and growth impairments were measured as described previously (4). Examples of growth curves and a summary of growth rates for all of the Pro 173 and Pro 222 mutants are shown in Fig.6.

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Fig. 6.

Proton conductance, as assayed by growth impairments, of MotA-MotB channels with mutations in Pro 173 and Pro 222. Wild-type cells were transformed with derivatives of plasmid pLW3 (38), which overexpresses MotA or its mutant variants and also a MotB-Rop fusion protein, both from the inducible trppromoter. (Left) Representative growth curves. Open symbols, uninduced cultures; filled symbols, cultures induced at t = 0 with indoleacrylic acid (final concentration, 100 μg/ml). Circles and triangles represent results of two independent experiments, which are fitted to solid and dashed lines, respectively. (Right) Summary of growth rates of the Pro 173 and Pro 222 mutants, with and without induction by indoleacrylic acid (mean ± standard deviation; n = 3).

All 16 mutations of Pro 222 eliminated or significantly reduced the growth impairment caused by overexpression of MotA and the MotB fusion protein. The replacements of Pro 222 thus not only prevent motor rotation but also reduce proton flow through the MotA-MotB complexes.

Most mutations of Pro 173 also reduced growth impairment. This was true even for most of the mutations that allowed motor function. The mutant proteins P173C, P173L, P173M, P173N, P173S, and P173T support torque generation and swarming (Fig. 4 and Table 2) but did not conduct protons well enough to give strong growth impairment in the overexpression experiment. Four mutants showed strong growth impairment. One of these (P173V) also swarmed and produced substantial torque when tethered. The other three (P173G, P173D, and P173E) were immotile and produced no torque in tethered cell experiments and thus appear to uncouple proton flow from motor rotation.

Residue Asp 32 of MotB was previously shown to be essential for proton conduction in the growth impairment assay. Growth impairment was eliminated or reduced by several different substitutions of Asp 32, including the mutation D32N (42). If Pro 173 has a role in regulating proton flow, as we suggest in the discussion below, then its side chain might be located in the channel near Asp 32. An aspartic acid residue introduced at position 173 in MotA might then augment or substitute for proton-conducting function(s) normally provided by Asp 32 of MotB. To test this, we measured the growth rates of cells overexpressing the P173D mutant MotA protein together with the D32N mutant MotB fusion protein. Whereas growth impairment was small in the MotB D32N mutant, larger growth impairment was observed in the P173D D32N double mutant (Fig. 7). Thus, aspartic acid at position 173 of MotA can increase proton flow through channels lacking Asp 32 of MotB. We next tested whether any MotB protein remained necessary for proton conduction when MotA contained the P173D replacement. A mutation in MotB that terminates translation after three residues was introduced into the overexpression plasmid, and growth rates were measured. When MotA was wild type and MotB was truncated after three residues, no growth impairment was observed. By contrast, when MotA harbored the P173D mutation, significant growth impairment was observed, even when MotB was truncated (Fig. 7).

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Fig. 7.

Proton conduction, as assayed by growth impairments, by MotA-MotB channels with mutations in both proteins. The mutations present in MotA and MotB are indicated. Q4Ter signifies a stop codon in place of codon 4 of motB (allele 33 in reference6). Black bars, uninduced; grey bars, induced with indoleacrylic acid. Values are the means ± standard deviations (n = 5 to 9). w.t., wild type.

Dominance of Pro 222 mutations.The strong dominance of certain Pro 222 mutations suggests that some Pro 222 mutant proteins are capable not only of substituting for wild-type protein in the motors but also of impeding (i.e., jamming) motor rotation (40). A clue to the function of Pro 222 would be provided if mutations of this residue were found to jam the motor more than other mutations of MotA. Mutations isolated by Garza et al. provide a means for testing this hypothesis (12, 13). Garza et al. found that certain mutations of the stator protein MotB are suppressible by certain mutations in the rotor protein FliG. Their results suggested that the MotB mutations cause the stator to become misaligned relative to the rotor and that the FliG mutations cause compensating movements in the rotor that restore a functional alignment. Detailed physiological studies suggested that the FliG mutations increase the clearance between the rotor and the stator (13). To determine whether the dominance of Pro 222 mutations can be modulated by changes in rotor-stator clearance, we measured the dominance of several Pro 222 mutations in strains containing the FliG mutations that are believed to increase clearance. These experiments used afliG null strain transformed with one plasmid encoding the mutant FliG proteins and another plasmid encoding the dominant nonfunctional MotA proteins (or wild-type MotA as a control). The strains thus contained the wild-type motA gene on the chromosome, allowing dominance of the plasmid-borne motAalleles to be assessed. The results for the strongly dominant MotA mutations P222R and P222M and controls with wild-type MotA are shown in Fig. 8.

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Fig. 8.

Dominance of Pro 222 mutations of MotA and modulation of this dominance by mutations in fliG. The plates were inoculated with 1 μl of saturated cultures of the indicated strains and incubated at 32°C. Strains that contained only wild-type MotA were incubated for 6 h (top) or 7.5 h (bottom); others were incubated for 7.5 to 8 h. Three isolates of each strain are shown. w.t., wild type.

The dominance of the motA mutations depended upon whichfliG allele was present. When FliG was wild type and the P222R or P222M mutant MotA proteins were expressed from plasmids, swarming was slow and the swarms consisted mainly of trails of satellite microcolonies. Trails occur when only a few of the cells in a population are motile, and the motile cells usually produce daughter cells that are immotile. When FliG contained the mutation E192K, swarming was faster and the swarms were more uniform in density, indicating that more of the cells were motile (Fig. 8). The E192K mutation in FliG also increased swarming rate somewhat in the control expressing wild-type MotA, as noted previously by Garza et al. (13). The enhancement was smaller than that observed with the dominant MotA mutations, however, and the overall appearance of the swarms was not affected. The FliG mutation G194S had the opposite effect, increasing the dominance of the P222M and P222R mutations so that swarming was eliminated completely. It also slowed swarming when MotA was wild type, but by only a small factor (Fig. 8). Other MotA mutations, which were dominant to various degrees, gave qualitatively similar results (data not shown).

Role of proline 173 of MotA.Mutations of Pro 173 gave complex phenotypes, in many cases impairing but not eliminating motor function. Replacements that supported some motor function tended to be uncharged residues of medium size. Motor performance was diminished under both low load and high load in Pro 173 mutants. In some cases (P173A, P173I, and P173Q), motor speed at low load was decreased so much that cells did not swarm at all and were immotile in liquid medium, even though they produced significant torque when tethered. Thus, mutations of Pro 173 can slow some step(s) in the operation of the motor, in some cases severely. In the two mutants that swam steadily enough to allow measurements of speed (P173S and P173T), the speed was more sensitive to viscous load than wild type (Fig. 3) but no more sensitive to solvent isotope (D₂O versus H₂O). Given the limited precision of the swimming speed measurements, we cannot rule out the possibility that Pro 173 mutations affect a proton transfer step(s). Our results do suggest, however, that proton transfer is not the main step affected. Instead, the increased sensitivity of the mutants to viscous load suggests that Pro 173 functions in a step that involves rotor movement. An alternative possibility is that it functions in a step that immediately precedes or follows rotor movement, with the first of the two steps being a rapid pre-equilibrium. In either case, the results imply a link between Pro 173 and events at the rotor-stator interface.

The Pro 173 mutants that retained partial function showed subnormal torque in tethered cell experiments. This contrasts with most MotA mutants characterized previously, which swim more slowly than the wild type but produce normal torque when tethered (4). A similar decrease in torque was observed in the MotB mutant D32E, which was the only replacement of MotB Asp 32 that retained any function, producing about half of wild-type torque when tethered. Several studies of flagellar motor physiology suggest that motor torque in tethered cell experiments is determined by the efficiency of coupling between proton flow and motor rotation and that this coupling efficiency is normally very high. Specifically, at the high loads (and consequently low speeds) of tethered cells, torque is proportional to the protonmotive force (24), is nearly independent of temperature and solvent isotope (17), and remains constant as the viscosity of the medium is increased (24). These observations suggest that each turn of the motor is coupled to the movement of a fixed number of protons and that the rotation rate of tethered cells is determined by thermodynamic rather than kinetic factors. (If the motor is tightly coupled, then the motor torque × 2π will equal the energy available from the proton gradient to drive each revolution.) Thus, the diminished torque of the Pro 173 mutants (and the MotB mutant D32E) is most likely caused by a decreased efficiency of coupling proton flow to motor rotation. An alternative possibility is that the mutant MotA proteins are not incorporated efficiently into the motors, so that each motor contains fewer torque generators than normal (a wild-type motor has about eight [3]). This alternative is less likely, because most of the Pro 173 mutations that reduced motor torque were moderately or strongly dominant (Table 2). Another possibility is that the mutations introduce rate limitations so severe that they limit motor torque even at the low rotation speeds of tethered cells. This alternative is ruled out by the observation that several of the Pro mutants with reduced torque were able to swarm and swim, implying that their motors can rotate much faster than the maximum speeds of tethered cells.

The growth impairment results also point to a coupling role for Pro 173. Certain mutations of Pro 173 prevented motor rotation yet still permitted proton flow as assayed by growth impairments. Like the diminished torque at high load, this property is unlike most MotA mutations characterized previously, which prevented both motor rotation and growth impairment. Exceptions were mutations of certain charged residues of MotA that interact with FliG and that are believed to function in rotor-stator interactions but not in proton conduction (20, 41). Since proton flow in the growth impairment assay involves just MotA and the MotB fusion protein, it is not expected to depend on interactions with FliG. The Pro 173 replacements that prevented motor rotation but allowed proton flow were acidic (Asp and Glu) or introduced the smallest possible side chain (Gly). Growth impairments were much weaker when MotA contained the amide replacements P173N or P173Q or the second-smallest replacement, P173A. Proton flow through the MotA-MotB channels is thus very sensitive to the side chain at position 173, suggesting that this residue might be located in the channel.

The growth impairments observed in MotA MotB double mutants also suggest that Pro 173 of MotA is inside the channel and is probably near Asp 32 of MotB. Although Asp 32 of MotB is normally required for proton flow through the channels (as assayed by growth impairments), this requirement is obviated when an aspartic acid residue is present at position 173 of MotA. Even terminating MotB after three residues did not eliminate the growth impairment when MotA had aspartic acid at position 173. These results suggest that the side chain of residue 173 is located inside the channel and that MotB does not have a large role in forming the membrane-embedded parts of the channel but serves mainly to provide the critical aspartic acid residue. This is consistent with the arrangement of membrane segments of MotA and MotB suggested by tryptophan-scanning mutagenesis studies (29, 30).

Overall, our results cast Pro 173 in a central role, on the one hand influencing the movement of the rotor and on the other hand regulating proton flow across the membrane.

Role of proline 222 of MotA.All replacements of Pro 222 abolished motor rotation in both the swarming and tethered cell experiments and also eliminated or reduced proton flow as assayed by growth impairments. Even residues with side chains comparable in size to proline did not support any motor function, suggesting that the special conformational constraints imposed by a proline residue are essential at this position. We propose that Pro 222 regulates the conformation and/or conformational changes of the MotA-MotB complex. The diminished growth impairments observed in the Pro 222 mutants also implicate this residue in proton conduction. This finding and the location of Pro 222 near the cytoplasmic surface of the membrane suggest that it is in the general vicinity of Asp 32. We cannot conclude that the side chain of Pro 222 is very near Asp 32 or is necessarily inside the channel, however.

The Pro 222 mutations are most notable for their dominance. This dominance is stronger than that of other MotA mutations that have been studied (4, 40). It is comparable to what is seen with mutations in Asp 32 of MotB, which also abolish or nearly abolish motility when the mutant protein is expressed in wild-type cells (42). We have suggested that protonation and deprotonation of Asp 32 might drive conformational changes in the stator that regulate the movement of the rotor (42). (Such a conformation-based model for coupling contrasts with models in which protons influence rotor movement more directly by binding to the rotor at some step.) If Pro 222 has a role in these hypothesized conformational changes, then mutations of Pro 222 might jam the movement of the rotor, which could account for their strong dominance.

In support of the proposal that Pro 222 mutations affect the rotor-stator interface, we found that the dominance of Pro 222 mutations was modulated by fliG mutations that are believed to increase the clearance between rotor and stator. Pro 222 mutations (P222M and P222R) that were strongly dominant in the presence of wild-type FliG were less dominant when FliG harbored the mutation K192E. This result is expected if the K192E mutation increases the clearance between rotor and stator, and if rotor-stator jamming is a factor contributing to the dominance of the Pro 222 mutations. The FliG mutation G194S had the opposite effect, however, increasing the dominance of the Pro 222 mutations. In the studies of Garza et al. (12, 13), the G194S mutation suppressed more motBmutations than did the K192E mutation, and its presence in otherwise wild-type motors caused a larger reduction in torque. Those results suggest that the G194S mutation causes a larger increase in rotor-stator clearance. The increased dominance of the Pro 222 mutations in the presence of the FliG mutation G194S might therefore be caused by a decrease in the torque generated by the wild-type MotA molecules present rather than by increased jamming by the mutant MotA proteins.

Additional insight into the putative jamming might be obtained by measuring the tethered torque of cells expressing the Pro 222 mutant proteins and by measuring the resistance of mutant motors to rotation by external torques. Berry and Berg have used optical tweezers to study torque-speed relationships in wild-type motors over a range of motor speed (1). They found that motor torque was constant over a range of speeds, including negative speeds (i.e., with rotation driven backwards by the external torque). Ishihara et al. used the flow of viscous medium past stationary cells to apply external torque to the flagella of several nonmotile mutants of Salmonella typhimurium (16a). They found that the mutant flagella could be rotated by an external torque, implying that the mutations studied (including two in motA) did not introduce large barriers to rotation. The flagella did not exhibit rotational Brownian movement in the absence of an external torque, however, suggesting that there were some small barriers to rotation.

Sequence context of the key proline residues of MotA.An alignment of selected MotA sequences (Fig.9) shows that no other residues are conserved in the segments around Pro 173 and Pro 222. Evidently, no other side chains in these segments of MotA are critical for function. A feature that is conserved is the spacing between the proline residues and some invariant glycine residues in the membrane segments, suggesting that the positions of Pro 173 and Pro 222 relative to the membrane-embedded parts of the channel are an important feature that has been conserved through evolution. The conformation of MotA in the segments around these proline residues is likely to be important for function. Secondary structure predictions suggest that the segments flanking both Pro 173 and Pro 222 are α-helices (Fig. 9). The Pro residues would introduce kinks in these helices and, as described below, might mediate conformational changes coupled to the protonation and deprotonation of Asp 32.

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Fig. 9.

Alignment of selected MotA sequences in the segments around Pro 173 and Pro 222. M3 and M4 indicate parts of membrane segments 3 and 4. Coils below the sequences indicate segments predicted to be α-helical by a neural-net algorithm (27).

Lessons gleaned here about the roles of Pro 173 and Pro 222 in E. coli are likely to be relevant to the flagellar motors of many species. MotA homologs are known in several species besides those shown in Fig. 9 (including Aquifex aeolicus, Bordetella pertussis, Campylobacter jejuni, Clostridium acetobutylicum, Helicobacter pylori, Pseudomonas aeruginosa, Shewanella putrefasciens, Thermotoga maritima, Vibrio alginolyticus, Vibrio cholerae, and Yersinia pestis), and in all cases proline residues are found at positions corresponding to 173 and 222.

A model for motor rotation.Figure10illustrates a proposed mechanism of torque generation in the flagellar motor. The model shown is only one of several possibilities, but it is offered as a concrete example of how proton movements might be coupled to rotor movements at the putative Asp 32-Pro 173-Pro 222 site. The model has two main features. First, we suggest that when the rotor and the stator are appropriately aligned, interactions across the rotor-stator interface induce a conformational change in the stator that allows a proton to enter from the periplasm and bind to Asp 32. This channel-gating step involves mainly Pro 173. (Such a role for Pro 173 in gating proton flow, as opposed to a role in forming the proton pathway, would not imply an increased deuterium isotope effect for Pro 173 mutations.) The triggering site that controls the timing of this gating step might consist of the charged residues of MotA and FliG that have been shown to interact at the rotor-stator interface (41). Electrostatic interactions at the rotor-stator interface could also serve to drive some movement of the rotor, and this function is also indicated in the figure. Second, we suggest that the binding of the proton to Asp 32 causes a further conformational change in the stator, which alters the rotor-stator interface to drive and/or restrict the movement of the rotor. In the model drawn, most movement of the rotor is driven actively either by electrostatic interaction or by direct contact with the stator. Alternatively, in a rectified Brownian movement model, rotor movement would be driven mainly by thermal energy, with conformational changes in the stator serving to provide restraints that prevent reverse movements. We suggest that Pro 222 functions in this rotor-driving (or latching) conformational change. To complete the cycle, the rotor moves further forward, allowing the proton to exit to the cytoplasm and restoring the stator to its initial state.

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Fig. 10.

Proposed mechanism for coupling proton flow to motor rotation at the Asp 32-Pro 173-Pro 222 site. (A) Mechanism of motor rotation. The diagram shows a single stator complex and a small section of the rotor. A series of projections on the edge of the rotor, drawn as white triangles, interact with points projecting from the stator so that relative movement of rotor and stator is restricted. Other sites on the rotor and stator, colored gray and termed trigger sites, interact so that when the rotor and stator are properly aligned, a conformation change is triggered in the stator. The trigger sites might be formed from the charged residues of MotA and FliG that have been shown to interact at the rotor-stator interface (41). Other elements pictured are the functionally important residues Asp 32 of MotB and Pro 173 and Pro 222 of MotA. In an initial state (panel i), Asp 32 is unprotonated, and the trigger sites on the rotor and stator are close to each other but not yet aligned. A small movement of the rotor, which might be accelerated by electrostatic interactions between the rotor and stator, brings the trigger sites into alignment so that they can interact to promote a conformational change in the stator (panel ii). This conformational change, which is suggested to involve Pro 173, opens a gate in a gate in a proton channel to the periplasm. A proton enters and binds to Asp 32 (the filled oval signifies the protonated site), triggering a further conformational change in the stator (panel iii). This conformational change affects contacts at the rotor-stator interface to drive movement of the rotor, toward the right for the geometry pictured. Next, the proton is released from Asp 32 to the cytoplasm, and the stator returns to its starting conformation. The rotor is driven farther to the right at this step because the stator engages the next projecting site on the rotor (panel iv). The net result is transfer of one proton across the membrane and movement of the rotor to the right by one rotor subunit. (B) A mechanism for switching the direction of rotation. Switching could occur by a coordinated movement of the projecting sites on the rotor relative to the trigger sites. The events in the stator would be the same as those pictured in part A, but for the geometry shown at the bottom, the rotor would move to the left.