Structure of Flagellar MotorProteins in Complex Allows forInsights into Motor Structureand SwitchingReceived for publication, May 7, 2012, and in revised form, June 12, 2012Published, JBC Papers in Press, August 15, 2012, DOI 10.1074/jbc.C112.378380

Armand S. Vartanian‡1, Aviv Paz§1, Emily A. Fortgang‡,Jeff Abramson§, and Frederick W. Dahlquist‡2

From the ‡Department of Chemistry and Biochemistry, University ofCalifornia, Santa Barbara, California 93106 and the §Department ofPhysiology, David Geffen School of Medicine, UCLA,Los Angeles, California 90095

Background:The flagellar motor generates bidirectionalrotation using the proteins FliG, FliM, and CheY andmembrane-bound stator complexes.Results: The structures of portions of FliG and FliM incomplex allow for insights into switching and structure.Conclusion: FliGMC and FliMM in a 1:1 complex mayform the C-ring of the motor.Significance: Understanding motor structure is key tounderstanding its mechanism.

The flagellar motor is one type of propulsion device of motilebacteria. The cytoplasmic ring (C-ring) of the motor interactswith the stator to generate torque in clockwise and counter-clockwise directions. The C-ring is composed of three proteins,FliM, FliN, and FliG. Together they form the “switch complex”and regulate switching and torque generation. Here we reportthe crystal structure of the middle domain of FliM in complexwith the middle and C-terminal domains of FliG that shows theinteraction surface and orientations of the proteins. In the com-plex, FliG assumes a compact conformation inwhich themiddleand C-terminal domains (FliGMC) collapse and stack togethersimilarly to the recently published structure of a mutant ofFliGMC with a clockwise rotational bias. This intramolecularstacking of the domains is distinct from the intermolecularstacking seen in other structures of FliG. We fit the complexstructure into the three-dimensional reconstructions of themotor and propose that the cytoplasmic ring is assembled from34 FliG and FliM molecules in a 1:1 fashion.

The flagellar motor is a rotary nano-turbine that drivesmotile bacteria. The motor generates torque through the freeenergy of an electrochemical gradient of extracellular cationsflowing through the stator in the membrane to the cytoplasmicrotor components (1, 2). The stator proteins MotA and MotB

form a membrane channel (MotAB) that generates torque atthe interaction site between MotAB and the rotor protein FliG(3, 4). The rotation direction results from modulation of theFliG/MotAB interaction that is regulated by the “switch com-plex” composed of FliM, FliG, and FliN.Multiple copies of eachprotein component form the cytoplasmic ring (C-ring)3 of themotor (5–8). The structure of the C-ring has been visualized bycryo-EMtomography, andmodels of how themotor proteins fitinto theC-ring have been published by several laboratories (24).X-ray crystallography has supplied the structure of parts ofevery protein that comprise theC-ring, yet an absolute arrange-ment has not emerged. The structure of the middle domain ofFliM (FliMM) has been solved alone (Protein Data Bank (PDB)2HP7) and in complex (PDB 3SOH) with the middle domain ofFliG (FliGM). In each structure, alone or in complex, FliMM hasassumed identical conformations, suggesting that the structureof FliM in the C-ring has been observed in the crystalstructures.The three domains of FliG, N-terminal, middle, and C-ter-

minal, have also been crystallized, but in each case, the domainsassume different orientations with respect to one another. Thestructures show the domains of FliG in two conformationseither held apart by extended linker helices as seen in the 1LKVand 3HJL structures or collapsed atop each other as seen in the3AJC structure. An interesting observation is that in all thesestructures, the middle and C-terminal domains of FliG form apseudo-armadillo repeat motif (ARM) stack. The apparentARM stack can form both intermolecularly (PDB 1LKV and3HJL) or intramolecularly (PDB 3AJC), but the orientation andinteractions of the two domains in all these structures aresimilar.Here we report the 1.9 Å resolution crystal structure of the

middle domain of FliM (FliMM) in complex with the middle(FliGM) and C-terminal (FliGC) domains of FliG (FliGMC) fromThermotogamaritima (see Fig. 1A). FliGMC assumes a compactconformation similar to the recently published FliGMC (PDB3AJC) structure from the Namba laboratory (9). Our structurealso contains an apparent intramolecular ARM stack as well asthe same interface between FliGM and FliMM as seen in the3SOH structure. Our structure was able to be modeled intothe three-dimensional cryo-tomography reconstructions of themotor provided byDeRosier laboratory, allowing for a proposalof the three-dimensional arrangement of themotor structure tobe formed.

EXPERIMENTAL PROCEDURES

FliG (residues 115–327) and FliM (residues 46–230) werecloned into the pJY5 vector. Proteins were expressed usingBL-21 Rosetta cells. The cell pellets were suspended in 25 mM

Tris, pH 7.5, 400 mM NaCl, 10 mM imidazole, 1 mM EDTA andlysedwith a French pressure cell. The lysatewas heat shocked at80 °C for 15min followed by centrifugation at 13 rpm in a Beck-The atomic coordinates and structure factors (code 4FHR) have been deposited in

the Protein Data Bank, Research Collaboratory for Structural Bioinformatics,Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

1 Both authors contributed equally to this work.2 To whom correspondence should be addressed. Tel.: 805-893-5326; E-mail:

Dahlquist@chem.ucsb.edu.

3 The abbreviations used are: C-ring, cytoplasmic ring; ARM, armadillo repeatmotif; r.m.s., root mean square.

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man JA-10 rotor. The clarified lysate was passed through anickel affinity column and eluted with 200 mM imidazole. Theeluant was cleaved with tobacco etch virus protease in 50 mM

Tris, pH 8.0. Following cleavage, the proteins were passed for asecond time through a nickel affinity column. The flow-through was collected and exchanged into 10 mM Tris, pH 7.9,10 mM NaCl. The proteins were mixed in a 1:1 ratio of 75 �M

each and crystallized in 50 mM calcium acetate, 0.1 M sodiumcacodylate, pH 6.0, and 25% v/v 2-methyl-2,4-pentanediol at20 °C in a hanging drop plate. The crystals were frozen in liquidnitrogen after being dipped into cryo-buffer containing 0.1 M

Tris, pH 8.8, 8% PEG 8000, 35% glycerol. The data sets werecollected at beamline 5.0.2 at the Advanced Light Source inBerkeley, CA. Images were processed and scaled usingHKL2000 (10). Because the data were highly anisotropic, ellip-soidal truncation and anisotropic scaling were performed usingthe UCLA Molecular Biology Institute diffraction anisotropyserver (11). The structure was solved usingT. maritima FliGMC(PDB 1LKV) and T. maritima FliM (PDB 2HP7) coordinates asa template for molecular replacement, refined using PHENIX(12). The structure was manually built in COOT (13).

RESULTS AND DISCUSSION

The FliGMC-FliMM complex (Fig. 1A) was generated bymix-ing truncated forms of FliG (residues 115–327) and FliM (resi-dues 46–230) in an equimolar ratio. Optimized crystals dis-played diffraction with Bragg spacing of 1.9 Å. The structurewas solved by molecular replacement using the previouslysolved structures of FliG (PDB 1LKV) and FliM (PDB 2HP7) assearch models (7, 14). The complex was refined to a resolutionof 1.9 Å with an Rfree of 24.7% and an Rwork of 21.7%. Completerefinement statistics are shown inTable 1. The overall architec-ture of the complex is cylindrical with a vertical height of �100Å and a diameter of�25Å, with a contact surface area between

FliM and FliG of 703 Å2 (15, 16). Four of the C-terminal resi-dues of FliM and seven residues of a loop in FliG were notobserved in the electron density and were presumed disor-dered. The unstructured region of FliG does not contain con-served residues, suggesting that this is a variable region amongspecies. Furthermore, the missing residues precede the con-served Gly-Gly hinge located between the domains of FliG andmay be unstructured due to the inherent flexibility in thisregion of the protein (17).Upon binding to FliGMC, FliMMdoes not undergo any signif-

icant conformational changes, with an r.m.s deviation of 0.6 Åas compared with the FliMM 2.0 Å resolution structure (PDB2HP7) or the 3.5 Å resolution structure of FliMM bound toFliGM both previously solved by the Blair and Crane laborato-ries (7, 18). FliMM consists of three �-strands (�1–�3) andthree �-helices (�1–�3) that form a pseudo-symmetric �/�/�three-layered sandwich. The highly conservedGGXGsequencemotif, located in the loop between helices�3–�1�, is involved inFliG binding and confirmed by both the 3SOH and this struc-ture (6, 19). In the 2HP7 structure, the two glycine residues ofthe GGXG motif are resolved, but the following four residues(PGEN) are disordered (7). Upon binding to FliGM, the loop inFliMM surrounding the GGXGmotif becomes ordered and sta-bilized and is observable in both the 3SOH and this structure.The FliGM/FliMM interaction surface is positioned directlyabove the secondary binding site of CheY located on FliMM thatwas identified by NMR experiments (20). When the responseregulator CheY is phosphorylated (CheY-P), it is able to bind toFliM to signal the switch in rotation direction. The proximity ofthe CheY-P binding site to the FliGM/FliMM interface couldexplain how the switch signal is propagated from FliM throughFliG toward the MotAB complex.FliGMC in complex with FliMM is predominantly �-helical

consisting of 14 helices arranged in an extended structure (Fig.1A). A right-handed super-helix is generated from the stacking

FIGURE 1. The FliGMC-FliMM complex. A, the FliGMC-FliMM complex. B, ex-panded view of the ARM stacking region. A green dashed line is shown for theresidues that could not be modeled in the structure. C, expanded view of theFliGMC and FliMM binding interface. Hydrogen bonding residues and resultinghydrogen bonds are shown in magenta. The black residues are the buriedhydrophobic residues at the binding interface. D, the FliGMC structuresequence. Helices are underlined, and the disorder residues are marked with astrikethrough.

TABLE 1Data collection and refinement statistics (molecular replacement)

Crystal 1

Data collectionSpace group C121Cell dimensionsa, b, c (Å) 125.96, 45.72, 98.84�, �, � (°) 90.0, 100.85, 90.0

Resolution (Å) 50–1.93 (2.0–1.93)aRsym or Rmerge 0.076 (0.44)I/�I 17.3 (1.65)Completeness (%) 99.5 (99.8)Redundancy 5.3 (4.6)

RefinementResolution (Å) 50–1.93No. of reflections 38,740Rwork/Rfree 21.7/24.7No. of atoms 3249Protein 3140Ligand/ionWater 109

B-factorsProtein 32.27Ligand/ionWater 29.0

r.m.s deviationsBond lengths (Å) 0.007Bond angles (°) 1.0

a Highest resolution shell is shown in parentheses.

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of pseudo-armadillo repeats fromhelices 1–4 (ARMM) andhel-ices 6–8 (ARMC) (Fig. 1B), mimicking the stacking observed ineukaryotic ARM motifs (21, 22). The ARMM/ARMC stackingresults in the burial of 14 hydrophobic residues forming a con-tact area of 745 Å2 (15, 16). HelixMC lies below ARMM andinteracts with helices 1–2 of FliGMC as well as parts of FliMM.The FliMM loop between helices �1� and �3 and residueswithin the helices themselves interactwithARMMandHelixMCof FliG, forming a network of bonding interactions. Fig. 1Cshows the packing of hydrophobic residues and hydrogenbonding between the two proteins. The C-terminal domain(HelicesC1–6) is located above the intramolecular ARM stackwith the charged HelixC5 positioned on top of the domain, freeto interact with MotAB (3). The C-terminal domain of FliG isprincipally connected to the ARM stack through the loopbetween helices 8 and C1, suggesting that changes in the posi-tion of FliGC relative to the ARM stack may be a possible com-ponent of motor switching.In two of the four FliG structures (PDB 1LKV and 3HJL), the

domains are held separate by extended linker helices (Fig. 2, Cand D) (14, 23). Brown et al. (14) suggested that HelixMC isstabilized through crystal contacts and postulated that thisregion would be flexible in vivo if the crystal packing repre-sented nonphysiological interactions. Furthermore, Van Wayet al. (17) used bioinformatic analysis in conjunction withmutational experiments to show that the conserved Gly-Glypair, at the end of HelixMC, acts as a flexible hinge between thetwo domains of FliGMC. The 3AJC structure (Fig. 2B) showsHelixMC in a new position as compared with the 1LKV and3HJL structures. The newposition ofHelixMCwas likely a prod-uct of the crystal packing facilitated by domain mobilitythrough the Gly-Gly pair (9). In the FliGMC-FliMM complex,HelixMC is packed against FliMMbeneathARMMas opposed tobeing pointed away from the ARM stack as in the 3AJC struc-ture. The multiple positions of HelixMC observed in the struc-tures suggest a degree of freedom that is likely limited whenFliGM binds to FliMM, allowingHelixMC to pack against FliMM.In the 3AJC structure, the position of HelixMC contacts theneighboring molecule, and movement of HelixMC in responseto CheY-P has been suggested to play a role in switching (9, 24).HelixMC in our complex structure is not in contact with neigh-boring molecules as it is in the 1LKV and 3AJC structures. Thissuggests that it may play a role in switching but not through thecommunication of multiple FliG monomers.Themajority of theC-ringmodels argue that theARMstack-

ing observed in the FliG structures is a central structural feature

of the C-ring. Alignments of the individual ARM domains ofFliGMC-FliMM complex versus the ARM domains of 1LKV,3HJL, and 3AJC show r.m.s deviation values�1.0Å. Thuswhenthe ARM domains stack, the ARM domain structures do notchange significantly whether the domains come from the samemolecule or from adjacent molecules. The crystal packing ofFliG structures 1LKV and 3HJL show ARM stacking betweenARMC andARMMof symmetry-related copies of FliG (Fig. 3,CandD) (14, 23). This results in anARMC-ARMM�1 stack. In theFliGMC-FliMM complex, theARMstacking is observedwithin asingle FliG protomer (Fig. 3A). The intramolecular ARM stack-ing is also assumed in the 3AJC structure (Fig. 3B), although theresidues connecting the domains are not observed (9).We pro-pose that ARM stacking is driven by the requirement to burythe hydrophobic residues that comprise theARMdomains, andthe intramolecular stack is driven by the binding to FliM as seenin the complex. Determining whether the ARM stacking isintra- or intermolecular in the C-ring requires further experi-mentation, but in the co-crystal, the presence of FliM appar-ently drove the ARM domains to stack within a single FliGMCmonomer, suggesting that the in vivo interaction could beintramolecular.The intramolecular ARM stacking we observed in the crystal

may represent a preassembly conformation of FliG. In complexwith FliM, FliG in the intramolecular stacking configurationmay be a precursor building block of the C-ring, and as they areassembled into the motor, FliG may undergo a transformationto the intermolecular interaction. The in vivo stacking may berepresented by either the stacking we observed in the co-com-plex structure or the intermolecular stacking observed in theother FliG structures. If the intermolecular stacking proposalwere correct, the rearrangement from cytoplasmic intramolec-ular stacking to the motor assembled intermolecular stackingwould require the 745 Å2 of contact surface between the ARMdomains to be broken and reassembled during the transition.This transition would have to overcome the energetics ofexposing the 14 buried hydrophobic residues to reassemble theARM stacks in themotor in the exact way they were previously,but now in the intermolecular stacked arrangement. Furtherexperimentation on intact isolated motors will be necessary tounambiguously determine the arrangement of FliG inside theC-ring.FliG and FliM have been extensively studied by mutational

analysis and biophysical techniques to determine the interac-

FIGURE 2. Comparison of the FliG structures. Four of the published FliGstructures show that FliG can assume either a compact or an extended struc-ture. Overall the individual domains retain their fold with their orientationvarying in each structure. A, FliGMC-FliMM complex. B, FliGMC (3AJC) (9).C, FliGMC (1LKV) (14). D, FliG full-length (3HJL) (23).

FIGURE 3. Comparison of the ARM stacking observed in structures andcrystal packing. The (�1) designates that these domains are from a neigh-boring molecule in the crystal packing. For clarity, the domains from neigh-boring molecules are shown in ribbon form and pale colors. A, ARM stackingobserved in the FliGMC-FliMM complex structure. B, ARM stacking observed inthe 3AJC crystal (9). C, ARM stacking observed in the 1LKV crystal (14). D, ARMstacking observed in the 3HJL crystal (23).

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tion site or sites (17, 19, 20, 25, 26). FliGM contains a conservedEHPQmotif that whenmutated disrupts the binding to FliMM.These same residues were shown to be in contact in the FliGM-FliMM co-crystal (PDB 3SOH) and in the complex presentedhere. The interaction surface between FliMM and FliGM hasalso been analyzed by NMR and chemical cross-linking. InNMR experiments, the resonances assigned to the EHP(Q/R)motif broaden when labeled FliGM is titrated with FliMM, sug-gesting that the surfaces seen in the crystal interact in solutionas well (20). Cross-linking experiments were designed from the3SOH structure and showed that FliGM is able to cross-link toFliMM in vivo, confirming that these faces are in contact in theassembled motor.Blair and co-workers (7, 18) have established that mutations

in the conserved hydrophobic patch in ARMC also affect bind-ing to FliMandhave performed chemical cross-linking of intactcells to determine whether FliGC is in contact with FliMM invivo (6, 19). Their cross-linking results show that the hydropho-bic patch of FliGC binds to the GGXG surface on FliMM asFliGM. In previous work (20), we used NMR to monitor theinteractions of FliGC andFliMM.This showed chemical shifts inthe resonances corresponding to the HelicesC1–6 region ofFliGC and not in the ARMC region, suggesting that the cross-linking results may not be present in solution. Compositiongradient multiangle light scattering was also performed tomeasure the affinity of FliGC and FliMM, resulting in aKd of 580�M. (data not shown). This low affinity suggests that the inter-action would probably not survive EM extraction. A model forthe C-ring presented by Blair and co-workers (7, 18) imaginesthat a portion of the FliGmolecules in the C-ring interacts withtwo FliMM domains, one via the FliGM domain and the othervia the FliGC domain. The remaining FliG molecules interactwith FliMM via the FliGC domain only. DeRosier and co-work-ers (27) suggest that this unusual arrangement allows formatching the 26–34-fold stoichiometry difference inferredfrom averaging of the EM reconstructions of the flagellarmotor. The yields of the cross-links they observe between thesedomains are relatively low, suggesting that these interactionsmay reflect the behavior of a subpopulation of both FliM andFliG.The FliGMC-FliMM complex was modeled into the high-res-

olution maps of the motor derived from three-dimensionalelectron micrograph reconstructions of a clockwise lockedmotor from Salmonella typhimurium (27, 28). TheC-ring fromthe three-dimensional reconstructions consists of two parts, aninner lobe with 26-fold symmetry and an outer lobe with34-fold symmetry. The inner lobe of the C-ring disappears inthree-dimensional reconstructions of amotor deleted for FliGN(29). This suggests that FliGN is only present in the inner lobeand that the outer lobe contains FliGMC, FliMM, and FliN that ismirrored by our construction of the C-ring.The outer lobe is divided into three parts consisting of an

upper, middle, and lower section. The upper section faces themembrane and is bridged to themiddle section by a thin strip ofdensity. Below the middle section is a ring that is believed tocontain the FliN tetramer and the C terminus of FliM. TheFliGMC-FliMM complex structure was docked into the densityby the programVEDAwith a correlation coefficient of 66.6 and

an R-factor of 58.0. The alignment positioned the ridge ofcharged residues in FliGC toward the membrane in position tointeract with the MotAB membrane channel (Fig. 4A). FliGMaligns nicely into density between the upper and middle sec-tions, interacting with both FliMM and FliG. The orientation ofFliMM in the middle lobe places the FliMM-FliMM subunitinterfaces in close proximity, as identified previously by disul-fide cross-linking experiments (7). Once we were satisfied withthe position of our complex, the FliN tetramer was docked intothe lower section of density. An expanded view of the docking isshown in Fig. 4C. The orientation of FliN to FliMwas estimatedfrom cross-linking experimental data and then refined by theVEDA fitting macro. Our x-ray structure and EMmodel fittingplaces the FliGC domain, FliM, and FliN subunits similarly tothe model proposed by Lee et al. (15) without invoking thecrystal packing ARMC-ARMM�1 notion they proposed. Fur-thermore, we observed a partial melting of HelixMC, suggestingthat HelixNMmight be destabilized as well in the complex. Thishas major implications with regard to some of the rigid bodymovements proposed for these proteins during the flagellarmotions (9, 18, 23).

FIGURE 4. Modeling the FliGMC-FliMM complex crystal structure and FliNtetramer into three-dimensional reconstructions of the motor. Maps ofthe flagellar motor were provided by the DeRosier laboratory. VEDA was usedto position the structures into the density; a total of 34 copies of the FliGMC-FliMM complex were positioned in the density as well as 34 copies of the FliNtetramer (30). A, side view of the assembled C-ring. B, bottom up view of theC-ring. C, expanded view of a portion of the C-ring illustrating the fit of theproteins inside of the density.

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The symmetry of the C-ring observed in the three-dimen-sional reconstructions shows a shift from 34-fold to 26-foldwhen going from the outer to inner rings, respectively (Fig. 4B).The modeling of the C-ring presented here shows FliG andFliM in a 1:1 stoichiometry, which implies that themismatch inthe symmetry is managed in the connection from the outer toinner rings.We postulate that only 26 out of the 34 of the FliGNdomains are able to bind to the 26 FliF copies located in theinner ring. The eight unbound FliGN domains would mostlikely be located unbound inside the inner space of the C-ringand would not participate in torque transfer from the outer toinner rings. This would allow for the motor to reconcile thesymmetrymismatch in the simplestmethod possible. The sym-metrymismatchmay be a dynamic part ofmotor assembly. Thethree-dimensional reconstructions showed that the C-ring var-ies between 31-, 33-, and 34-fold symmetries, and the free FliGNdomains may explain the ability of the C-ring to assume differ-ent copy numbers by displacing FliG-FliM complexes whennecessary.

Acknowledgments—We thank Dr. David DeRosier for providing thethree-dimensional maps of the flagellar motor. We also thank Dr.Gael Goret for the review of the density fitting.

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REPORT: Insights into Motor Structure and Switching

OCTOBER 19, 2012 • VOLUME 287 • NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 35783

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DahlquistArmand S. Vartanian, Aviv Paz, Emily A. Fortgang, Jeff Abramson and Frederick W.

Structure and SwitchingStructure of Flagellar Motor Proteins in Complex Allows for Insights into Motor

doi: 10.1074/jbc.C112.378380 originally published online August 15, 20122012, 287:35779-35783.J. Biol. Chem. 

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