Insights into the stator assembly of the Vibrio
flagellar motor from the crystal structure of MotY
Seiji Kojima*, Akari Shinohara†, Hiroyuki Terashima*, Toshiharu Yakushi*†‡, Mayuko Sakuma*, Michio Homma*†§,Keiichi Namba¶储, and Katsumi Imada§¶储*Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya 464-8602, Japan; †Soft Nano-Machine Project, Core
Research for Evolutional Science and Technology, Japan Science and Technology Agency, Chikusa-Ku, Nagoya 464-8602, Japan; ¶Graduate School of Frontier
Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan; and 储Dynamic NanoMachine Project, International Cooperative Research
Project, Japan Science and Technology Agency, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
Edited by Donald L. D. Caspar, Florida State University, Tallahassee, FL, and approved March 18, 2008 (received for review January 11, 2008)

Rotation of the sodium-driven polar flagellum of Vibrio alginolyticus requires four motor proteins: PomA, PomB, MotX, and MotY.
PomA and PomB form a sodium-ion channel in the cytoplasmic
membrane that functions as a stator complex to couple sodium-ion
flux with torque generation. MotX and MotY are components of
the T-ring, which is located beneath the P-ring of the polar flagellar
basal body and is involved in incorporation of the PomA/PomB
complex into the motor. Here, we describe the determination of
the crystal structure of MotY at 2.9 Å resolution. The structure
shows two distinct domains: an N-terminal domain (MotY-N) anda C-terminal domain (MotY-C). MotY-N has a unique structure.
MotY-C contains a putative peptidoglycan-binding motif that isremarkably similar to those of peptidoglycan-binding proteins,
such as Pal and RmpM, but this region is disordered in MotY.
Motility assay of cells producing either of the MotY-N and MotY-C
fragments and subsequent biochemical analyses indicate that
MotY-N is essential for association of the stator units around therotor, whereas MotY-C stabilizes the association by binding to the
peptidoglycan layer. Based on these observations, we propose amodel for the mechanism of stator assembly around the rotor.
bacterial flagellum 兩 peptidoglycan binding 兩 sodium driven motor 兩 T-ringT

he bacterial flagellum is a rotating organelle that drives cell
movement in response to environmental stimuli (1). The
flagellum is driven by a reversible rotary motor embedded in thecell membrane at the base of the filament. The motor is poweredby the transmembrane electrochemical gradient of a specific ion.
Whereas the flagella of Escherichia coli and Salmonella enterica
serovar Typhimurium use proton flux, the polar flagellum ofVibrio alginolyticus uses a flow of sodium ions (2, 3).
The motor consists of two parts, the rotor and the stator, andtorque is generated between them. The stator is a transmembrane
ion-channel composed of several different proteins. The stator
units are produced and assembled around the rotor after completion of the hook–basal body structure. Even a single stator unit candrive rotation (4, 5). Although the stator unit of the Escherichia coliand Salmonella motors consists of two proteins, MotA and MotB,
which form a proton-channel, the sodium-ion motor of V. alginolyticus requires four proteins, PomA, PomB, MotX, and MotY, toform a functional stator (6–8).
PomA and PomB are orthologs of MotA and MotB (6) andform a complex in the cytoplasmic membrane with the stoichiometry PomA4PomB2 (3, 9, 10). In their C-terminal periplasmic
region, PomB and MotB both have a putative peptidoglycanbinding motif that is well conserved among outer-membraneproteins, such as outer membrane protein A (OmpA) from E.
coli (11), peptidoglycan-associated lipoprotein (Pal) from E. coli(12), and reduction modifiable protein M (RmpM) from Neisseria meningitidis (13). Thus, PomB and MotB are thought toanchor the stator unit to the peptidoglycan layer (14, 15).
MotX and MotY are unique to the sodium-ion motors ofVibrio species. Null mutants of motX and motY exhibit nonmotile
7696 –7701 兩 PNAS 兩 June 3, 2008 兩 vol. 105 兩 no. 22

phenotypes (7, 8, 16), indicating that both proteins are essential
for torque generation, but their actual roles remain unclear.
MotX and MotY are translated with an N-terminal signal
sequence, which is cleaved during their export to the periplasmic
space, probably through the Vibrio equivalent of the E. coli Sec
system (17). Mature MotY is a 32-kDa polypeptide with 272
amino acid residues. MotX and MotY form a complex in the
periplasm, and MotX rapidly degrades in the absence of MotY,
suggesting that complex formation stabilizes MotX (16, 18).
MotX affects membrane localization of PomB, suggesting that
MotX interacts with PomB (18).
The C-terminal region of MotY contains a putative peptidoglycan-binding motif similar to those of PomB and MotB,
suggesting that MotY also binds to peptidoglycan (7). Recently,
we discovered a ring-like structure (the T-ring) composed of
MotX and MotY and localized beneath the P-ring of V. alginolyticus (19). In the absence of MotX or MotY, PomA and PomB
do not localize to the flagellated cell pole, demonstrating thatthese two proteins are involved in the incorporation and/or
stabilization of the PomA/PomB complex within the motor (19).
To understand the role of MotY in the assembly and functionof the sodium-ion motor, we purified and crystallized (20) MotY
from V. alginolyticus and determined its structure at 2.85 Å
resolution. This structure and subsequent biochemical analyses
clearly reveal distinct roles for the N- and C-terminal domains of
MotY and provide insights into the process of stator complex
assembly around the rotor.
Results
Overall Structure of MotY. The asymmetric unit in the crystal

contained a single MotY molecule. The atomic model of MotY
was constructed from Val-1 to Gln-271 (Fig. 1). (This numbering
does not include the 21 N-terminal residues that comprise thesignal sequence). Residues Thr-203 through Lys-209, Lys-244
through Arg-261, Val-272, and the C-terminal hexahistidine tag
were invisible in the electron density map, probably because ofdisorder, and are therefore missing in the model. Two distinct
domains are present: an N-terminal domain (Val-1 throughAuthor contributions: S.K. and A.S. contributed equally to this work; M.H., K.N., and K.I.
designed research; S.K., A.S., H.T., T.Y., M.S., and K.I. performed research; S.K., A.S., H.T.,
and K.I. analyzed data; and S.K., A.S., and K.I. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates have been deposited in Protein Data Bank,
www.pdb.org (PDB ID code 2ZF8).
‡Present address: Faculty of Agriculture, Tamaguchi University, 1677-1 Yoshida, Yamaguchi

753-8575, Japan.
§Towhom correspondence may be addressed. E-mail: g44416a@cc.nagoya-u.ac.jp or
kimada@fbs.osaka-u.ac.jp.

This article contains supporting information online at www.pnas.org/cgi/content/full/
0800308105/DCSupplemental.
© 2008 by The National Academy of Sciences of the USA

www.pnas.org兾cgi兾doi兾10.1073兾pnas.0800308105

a mixed ␤-sheet; ␤11, ␤12, and ␤13 are parallel, and ␤14 isanti-parallel to them. The residues connecting ␤12 and ␣4, and␤13 and ␤14, are invisible. The fold of MotY-C shows remarkable similarity to other OmpA/MotB-like proteins, such as thecore domain of Pal (Fig. 2B) and the OmpA-like domain of
RmpM (Fig. 2C) (13). MotY-C can be superimposed on RmpM
[PDB ID code 1R1M (13)], E. coli Pal (PDB ID code 1OAP),
and Haemophilus influenzae Pal [PDB ID code 2AIZ (21)], withrmsds for C␣ atoms of 2.2 Å, 2.6 Å, and 1.9 Å, respectively. The
corresponding sequence identities are 26%, 27%, and 21% (Fig.
2D). The major structural difference is that the residues connecting ␤13 and ␤14 are disordered in MotY, whereas they form
helical structures in Pal and RmpM. The N terminus of helix ␣4
in MotY is also one turn shorter than in the corresponding
helices of Pal and RmpM.

Fig. 1. Overall structure of MotY. (A) Stereoview of the C␣ ribbon drawingof MotY. (B) The N-terminal and C-terminal domains are shown in blue andyellow, respectively. The disulfide bond between Cys-25 and Cys-147 and theArg-262 residue are displayed in ball-and-stick format.

Tyr-154; MotY-N), and a C-terminal domain (Ser-155 throughVal-272; MotY-C), with the approximate dimensions of 30 ⫻40 ⫻ 50 Å and 25 ⫻ 30 ⫻ 40 Å, respectively. The amino acidsequence and elements of secondary structure are shown insupporting information (SI) Fig. S1.
Structure of MotY-N. MotY-N is composed of two ␤-sheets,
sheet-A and sheet-B, and two ␣-helices, ␣1 and ␣2. The two␤-sheets form a distorted ␤-sandwich with a deep cleft, and thetwo ␣-helices cover the opening of the cleft. Each sheet contains
five ␤-strands: ␤1, ␤6, ␤7, ␤9, and ␤10 in sheet-A; and ␤2, ␤3,
␤4, ␤5, and ␤8 in sheet-B (Fig. 1 A). The strands are anti-parallel
except for ␤1, which lies parallel to ␤10 in sheet-A. Sheet-B
twists and extends toward MotY-C. Helix ␣1, which connects
strands ␤8 and ␤9, is located just above the cleft and covers thehydrophobic core formed by the two ␤-sheets. Helix ␣2, which
is followed immediately by MotY-C, lies on sheet-B along strand
␤3. The C-terminal end of helix ␣2 is anchored to sheet-B
through a disulfide bridge between Cys-25 in ␤3 and Cys-147 in␣2 (Fig. 1B). The two Cys residues are conserved in MotY fromvarious bacteria with polar flagella (Fig. S1). This disulfide
seems to be important for stabilizing MotY-N. MotY proteins
containing the C25S, C147S, and C25S/C147S residues substitutions were susceptible to proteolysis and resulted in cells with
reduced or lost motility (16).
Structure of MotY-C and Its Similarity to Other OmpA/MotB-Like
Domains. MotY-C forms an ␣/␤ sandwich composed of ␣3 and ␣4and ␤11, ␤12, ␤13, and ␤14 (Fig. 1 A and 2 A). The strands form
Kojima et al.

structure of H. influenzae Pal bound to the peptidoglycan
precursor UDP-NAM-L-Ala-␥-D-Glu-L-Lys-D-Ala-D-Ala revealed that the peptidoglycan-binding pocket is formed by theloop between ␤1 and ␣2, the loop between ␤2 and ␣3, and ␣4(21). Although the overall structure of the C-terminal domain of
MotY is very similar to Pal (Fig. 2 Aand Band Fig. 3), two of thethree chains forming the binding pocket are disordered in the
MotY crystal structure. The residues between ␤12 and ␣4 andbetween ␤13 and ␤14 in MotY, which correspond to the ␤2-␣3loop and helix ␣4 in Pal, are invisible in the electron density map.
The C-terminal regions of ␤12 and ␤13 are slightly twisted andseparate from ␣3; thus, the peptidoglycan-binding site is wide
open in the MotY structure.
The conformation of the ␤11-␣3 loop of MotY differs fromthe corresponding ␤1–␣2 loop of Pal (Fig. 3), and the loops
contain different numbers of residues (Fig. 2D). The residues inthis loop are not conserved among the OmpA/MotB-like proteins, although Phe-36 and Asp-37 in Pal interact directly withthe peptidoglycan precursor (Fig. 2D and 3B). The loop structureof RmpM deviates from the loops in both MotY and Pal. Thisloop may interact with peptidoglycan in different ways in these
proteins. However, structure at the bottom of the peptidoglycanbinding site is well conserved. Leu-215 and Arg-219 in ␣4 of
MotY are in the same relative positions as Leu-82 and Arg-86 in
Pal; these residues interact directly with the peptidoglycan
precursor (21). The orientation of Arg-219 in MotY is supported
by His-165, just as Phe-34 supports Arg-86 in Pal.
MotY-N Is Essential for Motility. To elucidate the roles of the twodomains of MotY, we analyzed the motility of ⌬motY (GRF2)
cells expressing either MotY-N or MotY-C fused to a signal
peptide at its N terminus. Expression of full-length MotY
complemented the motility defect of GRF2 cells, whereas expression of MotY-N did not restore swarming in semisolid agar
(Fig. 4A). However, ⬇10% of the cells producing MotY-N swam
to some extent when viewed by dark-field microscopy, indicatingthat the stator complex was installed into the motor even without
MotY-C, albeit inefficiently. No motility was observed
when MotY-C was expressed alone in GRF2 cells (Fig. 4A).
Neither MotY-N nor MotY-C exhibited negative dominancewhen expressed from a multicopy plasmid in wild-type cells (datanot shown).

MotY-N Is Responsible for Interaction with both MotX and theBasal-Body. MotY interacts directly with MotX (18) and preventsdegradation of MotX in the cell (16). Recently, we found that
MotY, together with MotX, forms the T-ring structure beneaththe P-ring (19). The ⌬motX strain had a smaller T-ring than thewild-type, and no T-ring was obser ved in ⌬motY and⌬motX⌬motY strains, suggesting that MotY interacts with thebasal body. GRF2 cells expressing MotY-N and MotY-C were
PNAS 兩 June 3, 2008 兩 vol. 105 兩 no. 22 兩 7697

BIOCHEMISTRY

Disordering of the Putative Peptidoglycan-Binding Site. The NMR

Fig. 2. Comparison of OmpA/MotB-like domains. C␣ ribbon representation of OmpA/MotB-like domain structures. (A) The C-terminal domain of MotY
(MotY-C). (B) Pal of E. coli (1OAP). (C) The C-terminal domain of RmpM from N. meningitidis (1R1M). (D) Structure-based sequence alignment of the
OmpA/MotB-like domains. The aligned sequences are: MotY, the C-terminal domain of V. alginolyticus MotY; Pal㛭Ec, E. coli Pal; Pal㛭Hi, H. influenzae Pal; RmpM,
N. meningitidis RmpM; OmpA㛭Ec, E. coli OmpA; PomB㛭Va, V. alginolyticus PomB; and MotB㛭Ec, E. coli MotB. Residues highlighted in yellow are conserved in allseven proteins. Regions of secondary structure of MotY, Pal㛭Ec, Pal㛭Hi, and RmpM are indicated below the corresponding sequences as follows: Red line, ␣-helix;
green line, ␤-strand. The secondary structure elements for MotY are labeled above the sequence. The residues contributing to the binding of the peptidoglycan
precursor in Pal㛭Hi are indicated by blue boxes. The Arg-262 residue of MotY, which is important for motility and is conserved in OmpA/MotB-like proteins, ismarked with an asterisk. Disordered residues in the MotY crystal structure are shown in cyan. The pink bars above the sequences represent the regions used toalign the structures.

grown to exponential phase, harvested, disrupted by sonication,and subjected to ultracentrifugation to isolate the insoluble
membrane fraction and the soluble fraction. Both domains weredetected in the soluble fraction (Fig. 4 Band C). MotX was also
detected when intact MotY or MotY-N was coexpressed but notwhen MotY-C was coexpressed. These observations suggest that
MotY-N is responsible for stabilizing the MotX structure, presumably through direct interaction between MotX and MotY-N.
To investigate the association of each domain with the basal
body, we isolated basal bodies from TH2 cells (a ⌬motY strain

Fig. 3. The peptidoglycan-binding regions of MotY (A) and Pal㛭Hi complexed with the peptidoglycan precursor (B). The residues contributing tobinding of the peptidoglycan precursor and the corresponding residues in
MotY (shown by the blue boxes in Fig. 2D) are indicated by blue balls. The
polypeptide chains are drawn as C␣ ribbon diagrams, with color coding goingfrom green to red from the N terminus to the C terminus. The peptidoglycan
precursor is shown in a ball-and-stick representation.
7698 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0800308105

derived from the multiflagellated KK148 mutant) (19) expressing either MotY-N or MotY-C and analyzed the preparation byimmunoblotting (Fig. 4D). Although both proteins were found in
the whole-cell fraction (Fig. 4D, lanes 5 and 7), only MotY-N was
detected in the basal body fraction (Fig. 4D, lanes 6 and 8). MotX
was detected in both the whole-cell and basal body fractions
when MotY-N was coexpressed (Fig. 4D, lanes 5 and 6), confirming that MotY-N prevents degradation of MotX. The P-ringprotein FlgI was detected in all fractions, indicating that the basal
body was properly isolated. These results suggest that MotY-N
binds both to the flagellar basal body and MotX.
Discussion
The crystal structure of MotY revealed that the protein consistsof two distinct domains: the N-terminal MotY-N and the Cterminal MotY-C. MotY-C, which contains a putative peptidoglycan-binding sequence motif, showed remarkable structuralsimilarities to the peptidoglycan-binding domains of Pal (21) and
RmpM (13), but the MotY-N structure was unique.
Several experiments indicated that MotY-N is essential forformation of a functional stator complex, whereas MotY-C is not(Fig. 4). A ⌬motY strain expressing MotY-N had a weak but
significant ability to swim, and significant amounts of MotY-N
and MotX were detected in a purified basal body fraction,although the T-ring structure of this strain was difficult tovisualize by electron microscopy (data not shown). These results
suggest that MotY-N connects the basal body to MotX and that
the PomA/PomB complex associates with MotX to form thefunctional stator complex around the rotor. MotY-N alone does
not associate strongly with the basal body, but the partial T-ring
structure made of the MotY-N/MotX complex must be sufficient
to allow at least a few PomA/PomB stator complexes to beincorporated into the motor.
Kojima et al.

The similarity between MotY-C and other peptidoglycanbinding proteins suggests that MotY interacts with the peptidoglycan layer much like those proteins. In the crystal structure,
however, the putative peptidoglycan-binding site of MotY-C was
more widely open than in the structures determined for Pal and
RmpM (Fig. 2), and most of the loops forming the peptidoglycan-binding pocket were disordered (Fig. 3). The disordered
loops appear to be intrinsic to monomeric MotY and not an
artifact of the crystallization conditions, because the native
crystal obtained at pH 4.5, for which the model was refined, andthe Se-Met derivative crystal obtained at pH 8.5 both show thedisordered conformation (data not shown). A disordered peptidoglycan-binding pocket may be an important property for freeMotY, because, after secretion into the periplasm, MotX andMotY presumably must diffuse around until they collide with thebasal body and form the T-ring structure. Therefore, MotY
should not bind tightly to the peptidoglycan layer before it
encounters the basal body. Interaction with the basal body may
induce folding of the disordered chains into a functional peptidoglycan-binding pocket.
A motY mutant containing the R262Q residue substitution
exhibits greatly decreased swarming motility in semisolid agar
(16). Arg-262 is near the N terminus of the ␤14 strand in MotY-C(Fig. 1B), and this residue is conserved in various proteins of the
OmpA/MotB family (Fig. 2D). Although Arg-262 is near theputative peptidoglycan-binding site, its side chain extends awayfrom the binding pocket. Thus, it is unclear why the R262Q
substitution affects motility so strongly. One possible explanation is that Arg-262 may interact with a peptide chain of anotherpeptidoglycan strand cross-linked to the peptidoglycan chain
bound to the binding site. Another possibility is that Arg-262 isinvolved in association of MotY with the basal body to induce
proper folding of the peptidoglycan-binding pocket.
MotX and MotY are essential for torque generation by thesodium ion-driven polar flagellar motor of V. alginolyticus. Basedon the results presented here, we propose a model for stator
Kojima et al.

assembly (Fig. 5). MotY may form a complex with MotX anddiffuse around the periplasm with little or no affinity for the
peptidoglycan layer (Fig. 5A). When the complex associates withthe basal body via MotY-N, conformational changes are induced
that greatly increase the affinity of MotY-C for peptidoglycan.
The PomA/PomB complex also forms before association with

Fig. 5. Model of stator assembly in the sodium ion-driven polar flagellar
motor of Vibrio. MotY is shown as a space-filling model colored blue for
MotY-N and yellow for MotY-C. MotX, PomA, and PomB are shown in orange,
dark green, and light green, respectively. (A) MotX and MotY form a complex
and diffuse around in the periplasm without binding to the peptidoglycan
layer. (B) Interaction of the MotX/MotY complex with the flagellar basal body
triggers a conformational change in MotY-C that increases its affinity for the
peptidoglycan layer, and the complex associates tightly with the basal body
via MotY-N to form the T-ring. The PomA/PomB stator complex diffuses
through the membrane. (C) The PomA/PomB complexes assemble around therotor through association with MotX in the T-ring and binding to the peptidoglycan layer via the C-terminal domain of PomB.
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BIOCHEMISTRY

Fig. 4. Characterization of MotY-N and MotY-C. (A) Motility assays with GRF2 cells expressing MotY variants. Cells containing pSU41 (vector), pAS101 (MotY),
pAS102 (MotY-N), and pAS103 (MotY-C) were inoculated into semisolid VPG agar and incubated at 30°C for 5 h. The fraction of cells swimming in liquid medium
for each of the strains is shown at Right. (B and C) MotY-N prevents MotX degradation. Supernatants (Upper for MotY detection) and pellets (Lower for MotX
detection) of lysates after ultracentrifugation were analyzed by SDS/PAGE followed by immunoblotting. Samples are for GRF2 (⌬motY) cells containingthe pSU41
vector (lane 1 and 4), pAS101 (full-length MotY, lane 2 and 5), pAS102 (MotY-N, lane 3), and pAS103 (MotY-C, lane 6). Antibodies used for detection of proteins
are shown on the left. Because we could not detect MotY-N with the affinity-purified MotY antibody, we used an MotY antiserum obtained from another rabbit
for detection of MotY-N (␣-MotY1). For detection of MotY and MotY-C, the affinity-purified antibody (␣-MotY2) was used. Full-length MotY can be detectedby both ␣-MotY1 and ␣-MotY2. (D) Association of MotY-N and MotY-C with the flagellar basal body. Samples of whole cells (W) and the basal bodies (B) of KK148
cells (lanes 1 and 2), TH2 cells expressing full-length MotY (lanes 3 and 4), TH2 cells expressing MotY-N (lanes 5 and 6), and TH2 cells expressing MotY-C (lanes
7 and 8) were analyzed by SDS/PAGE. The presence of the flagellar P-ring protein FlgI, MotX, and MotY (or its domains) were visualized by immunoblotting.
Antibodies used for detection of proteins are shown on the left. As noted in C, MotY fragments were detected by ␣-MotY1 (for MotY-N) and ␣-MotY2 (forMotY-C). A number of extraneous bands appeared in the MotY blots are unidentified proteins that cross-reacted with the MotY antibodies.

the motor and diffuses around in the cytoplasmic membrane(Fig. 5B). When the PomA/PomB complex encounters the basal
body, association of the periplasmic C-terminal domain of PomBwith MotX positions PomB to bind to the peptidoglycan layer inthe proper orientation to interact productively with the rotor(Fig. 5C).
The proton-driven motors of E. coli and Salmonella do notneed MotX or MotY to function, and a ⌬pomAB⌬motX⌬motY
strain of Vibrio cholerae can swim by using proton motive force
if it contains a plasmid that encodes the E. coli MotA/MotB
complex (22). Furthermore, a stator complex composed of
PomA and PotB, which is a chimeric protein containing the
N-terminal transmembrane domain of PomB and the C-terminal
periplasmic domain of E. coli MotB, is functional in V. alginolyticus without MotX or MotY (23). The PomA/PotB complex,
which forms a sodium-ion channel, also allows the E. coli
flagellar motor to rotate by using a sodium-ion current (23).
These observations indicate that the periplasmic region of MotB
can carry out the combined functions of the periplasmic domainof PomB, MotX, and MotY. Pseudomonas aeruginosa, which also
has a polar flagellum, has a MotY homolog but no MotX (24).
Comparative studies of the Vibrio, P. aeruginosa, E. coli, andSalmonella flagellar motors should elucidate the variety ofmechanisms used to assemble stator complexes around theflagellar motors of ␥ Proteobacteria.
Materials and Methods
Preparation and Crystallization of MotY. Details of expression, purification, andcrystallization of MotY with a hexahistidine tag are described in ref. 20.
Briefly, hexagonal P6522 crystals of MotY with unit cell dimensions a ⫽ b ⫽104.1 Å and c ⫽ 133.1 Å were grown from a solution containing 1.12 M
NaH2PO4/K2HPO4, 5% PEG 1000, and 0.1 M acetate (pH 4.5) by the sitting drop
vapor-diffusion method. Se-Met labeled MotY was prepared and crystallized
in a solution containing 24% polyethylene glycol monoethyl ether 2000, 0.36
M ammonium sulfate, and 0.1 M Tris䡠HCl (pH 8.5). Although the crystallization
conditions for the Se-Met derivative were different from those for native
MotY, the unit cell dimensions (a ⫽ b ⫽ 104.7 Å and c ⫽ 132.3 Å) were almost
the same as those of the native crystals.

of the residues were located in the most favorable and allowed regions,
respectively. Structural refinement statistics are summarized in Table S1.
Expression and Fractionation of MotY Fragments in Vibrio Cells. The plasmid
pSU41 (Krm, vector), pAS101 (encoding full-length motY in pSU41), pAS102(encoding MotY-N, residues 1–154 of MotY in pSU41), and pAS103 (encoding
MotY-C, residues 154 –272 in pSU41), were introduced into the ⌬motY strain
GRF2 (16) by electroporation (30). MotY fragments were constructed with thenative signal sequence fused at their N termini so that they are secreted to theperiplasmic space. Transformants were grown exponentially at 30°C in vibrio
peptone/tryptone glycerol (VPG) medium (1% bactotryptone, 0.4% K2HPO4,
3% NaCl, and 0.5% glycerol) containing kanamycin 100 ␮g/ml. The cells were
harvested and washed once with buffer [20 mM Tris䡠HCl (pH 8.0), 500 mM
NaCl], then resuspended in the same buffer. The cells were disrupted bysonication (Branson Ultrasonics), unbroken cells were removed by centrifugation (12,000 ⫻ g for 5 min), and the solution was ultracentrifuged(148,781 ⫻ g for 30 min). The fractionated samples were precipitated bytrichloroacetic acid if necessary, then analyzed by SDS/PAGE followed byimmunoblotting. Because we could not detect MotY-N with the affinitypurified MotY antibody, we used an MotY antiserum obtained from anotherrabbit for detection of MotY-N. For detection of MotY and MotY-C, theaffinity-purified antibody was used.
Isolation of the Flagellar Basal-Body. Full-length MotY, MotY-N, and MotY-C
were expressed in the ⌬motY strain TH2 (19) from plasmids pAS101, pAS102,
and pAS103, respectively. The strain TH2 was derived from multipolarflagellated mutant KK148 (31) so that we can obtain high yields of flagellar
basal bodies from multiple polar flagella. The cells were grown in the VPg
medium exponentially, and isolation of the basal bodies from each strain was
carried out as described in ref. 19. Isolated basal body samples were analyzed
by SDS/PAGE followed by immunoblotting. For detection of the P-ring protein
FlgI, we used anti-Salmonella FlgI antibody that cross-reacts with FlgI of V.
alginolyticus.
Motility Assay. GRF2 cells were transformed with the respective plasmids, andfresh colonies were inoculated onto the VPG soft agar plates (1% bactotryptone, 0.4% K2HPO4, 3% NaCl, 0.5% glycerol, and 0.25% bact-agar) containing
kanamycin 100 ␮g/ml and incubated at 30°C for 5 h. To measure the fractionof swimming cells, cells of each construct were grown exponentially in the VPg
medium at 30°C, harvested and suspended in TMN300 medium [50 mM
Tris䡠HCl (pH 7.5), 5 mM MgCl2, 5 mM glucose, and 300 mM NaCl]. Then, a small
amount of the cell suspension was diluted 10-fold into the fresh TMN300
medium, motility of the cells was observed at room temperature under adark-field microscope, and images were recorded on videotapes. The fractionof swimming cells was determined by counting the ratio of motile to total cells
among the total cells in one video image.

Data Collection and Structure Determination. X-ray diffraction data werecollected at the synchrotron beamline BL41XU of SPring-8 (Harima, Japan).
The statistics of the data are described in ref. 20. The crystals were frozen inliquid nitrogen. Because the Se-Met derivative crystals were highly sensitive to
X-ray, we used a helium cryocooling device to reduce the radiation damage.
The data were processed with MOSFLM (25) and scaled with SCALA (26). Phase
calculation was performed by using SOLVE (27). The best electron-density map
was obtained from MAD phases from the Se-Met derivative data, followed bydensity modification with DM (26). The model was constructed with O (28) andwas refined against the native data at 2.85 Å, using CNS software (29). During
the refinement process, iterative manual modifications were performed byusing ‘‘omit map.’’ The refinement converged to an R factor of 29.0% and afree R factor of 31.2%. The Ramachandran plot showed that 71.8% and 25.5%

ACKNOWLEDGMENTS. We thank I. Kawagishi for invaluable discussions, S.
Nagashima for help with machine setup for modeling and refinement; S.
Tatematsu for technical support; N. Shimizu, M. Kawamoto, K. Hasegawa, andM. Yamamoto at SPring-8 for technical help in use of beamlines; F. Oosawa, S.
Asakura, and S. Hayashi for continuous support and encouragement during
this work; K. Kutsukake (Okayama University, Okayama, Japan) for providing
anti-Salmonella FlgI antibody. This work was supported in part by Ministry of
Education, Science and Culture of Japan Grants-in-Aid for Scientific Research
18074006 (to K.I.) and 16087207 (to K.N.), the National Project on Protein
Structural and Functional Analyses (K.I. and M.H.), and the Soft Nano-Machine
Project of the Japan Science and Technology Agency (A.S., T.Y., and M.H.).

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PNAS 兩 June 3, 2008 兩 vol. 105 兩 no. 22 兩 7701