crystallization communications
Acta Crystallographica Section F

Structural Biology
and Crystallization
Communications

Crystallization and preliminary X-ray analysis ofMotY, a stator component of theVibrio alginolyticus polar flagellar motor

ISSN 1744-3091

Akari Shinohara,a‡ Mayuko
Sakuma,b‡ Toshiharu
Yakushi,a,b§ Seiji Kojima,bKeiichi Namba,c,d Michio
Hommaa,b and Katsumi
Imadac,d*aSoft Nano-Machine Project, CREST, JST,
Chikusa-ku, Nagoya 464-8602, Japan, bDivisionof Biological Science, Graduate School of
Science, Nagoya University, Chikusa-ku,
Nagoya 464-8602, Japan, cGraduate School ofFrontier Biosciences, Osaka University,
1-3 Yamadaoka, Suita, Osaka 565-0871, Japan,
and dDynamic NanoMachine Project, ICORP,
JST, 1-3 Yamadaoka, Suita, Osaka 565-0871,
Japan‡ These authors contributed equally to thiswork.
§ Present address: Department of Bioscienceand Biotechnology, Shinshu University,
8304 Minami-minowa, Kami-ina,Nagano 399-4598, Japan.

Correspondence e-mail:kimada@fbs.osaka-u.ac.jp

Received 6 September 2006
Accepted 22 December 2006

# 2007 International Union of Crystallography
All rights reserved

Acta Cryst. (2007). F63, 89–92

The polar flagellum of Vibrio alginolyticus is rotated by the sodium motor. The
stator unit of the sodium motor consists of four different proteins: PomA, PomB,MotX and MotY. MotX and MotY, which are unique components of the sodiummotor, form the T-ring structure attached to the LP ring in the periplasmic space.
MotY has a putative peptidoglycan-binding motif in its C-terminal region andMotX is suggested to interact with PomB. Thus, MotX and MotY are thought tobe required for incorporation and stabilization of the PomA/B complex. In thisstudy, mature MotY composed of 272 amino-acid residues and its SeMet
derivative were expressed with a C-terminal hexahistidine-tag sequence,
purified and crystallized. Native crystals were grown in the hexagonal spacegroup P6122/P6522, with unit-cell parameters a = b = 104.1, c = 132.6 Å. SeMetderivative crystals belonged to the same space group with the same unit-cellparameters as the native crystals. Anomalous difference Patterson maps of theSeMet derivative showed significant peaks in their Harker sections, indicatingthat the derivatives are suitable for structure determination.

1. Introduction
Many bacteria swim by rotating a locomotive organelle called theflagellum. Rotation of the flagellum is driven by the motor, which isembedded in the bacterial membranes at the base of the flagellar
filament. The bacterial flagellar motor is a molecular machine that
couples the influx of specific ions to mechanical rotation of theflagellar filament. While the flagella of Escherichia coli and Salmonella typhimurium are driven by proton flow, the polar flagella ofmarine bacteria, such as Vibrio alginolyticus, V. parahaemolyticus andV. cholera, utilize sodium ions for rotation (Yorimitsu & Homma,2001).
The motor is composed of two distinct complex structures, therotor and the stator, and the torque is generated between these twostructures. The stator is a selective membrane ion-channel complex
composed of several proteins. The stator unit of the sodium motor
consists of four different proteins: PomA, PomB, MotX and MotY.
PomA and PomB are cytoplasmic membrane proteins and orthologues of MotA and MotB (Asai et al., 1997), which form a
proton-channel complex in the proton motor. Sato and Homma
demonstrated that the purified PomA–PomB complex, which iscomposed of four PomA and two PomB molecules (Sato & Homma,2000b; Yorimitsu et al., 2004), has a sodium-conducting activity (Sato& Homma, 2000a). Thus, the PomA–PomB complex was identified as
the sodium ion channel of the sodium motor.
MotX and MotY are unique components of the sodium motor ofVibrio and are believed to form a complex associated with the outermembrane from the periplasmic space. MotX and MotY aresynthesized with a signal sequence and exported to the periplasmic
space through the Sec system (Okabe et al., 2002). Mature MotY
consists of 272 amino-acid residues with a molecular weight of
32 000 Da. In the C-terminal region, MotY contains a putative
peptidoglycan-binding motif, which is also found in MotB, PomB,
outer membrane protein A (OmpA), peptidoglycan-associated lipoprotein (Pal) and RmpM (Grizot & Buchanan, 2004; Yakushi et al.,2005).
doi:10.1107/S1744309106055850

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crystallization communications
The null mutants of MotX and MotY showed nonmotile phenotypes (Okunishi et al., 1996; Okabe et al., 2001; Yagasaki et al., 2006);thus both proteins are indispensable for torque generation, but theiractual roles are unclear. MotX has been found to affect themembrane localization of PomB, suggesting that MotX interacts withPomB (Okabe et al., 2005). Recently, a new ring structure called the Tring has been found on the periplasmic side of the P ring of the hookbasal body purified from V. alginolyticus and the components of the Tring have been identified as MotX and MotY (Terashima et al., 2006).
In addition, PomA and PomB do not localize to the cell pole in nullmutants of MotX and MotY, suggesting that MotX and MotY areinvolved in the incorporation of the PomA–PomB complex into themotor by forming the T-ring complex.
To elucidate the rotation and construction mechanism of thesodium motor, we crystallized MotY in order to determine itsstructure. Here, we report the expression, purification, crystallizationand preliminary X-ray analysis of mature MotY with a C-terminal
hexahistidine tag and its SeMet derivative.

2. Materials and methods
2.1. Protein expression and purification

The gene encoding MotY tagged with a C-terminal hexahistidine
was transferred from pKJ503 (Okabe et al., 2002) into pJY19 (Yorimitsu et al., 2003), a derivative of plasmid pTTQ18 (Ampr; GEHealthcare Biosciences), using SalI–XbaI restriction sites andtransformed into E. coli strain BL21 (Novagen). Cells were grown at
303 K in LB medium [1% polypeptone (Wako), 0.5% yeast extract,
0.5% NaCl] containing 50 mg ampicillin per litre until the culture

density reached an OD660 of 1.2. Expression of MotY was inducedwith isopropyl -d-thiogalactoside (IPTG) at a final concentration of0.5 mM and culture was continued for 2 h. Cells were harvested bycentrifugation at 10 000g for 10 min and the pellet was suspended inbuffer (20 mM Tris–HCl pH 7.5, 100 mM NaCl, 25% sucrose, 0.5 mMPMSF). The cells were converted to spheroplasts by adding a solution
containing lysozyme and DNaseI (bovine, Sigma), followed by
gradual addition of a twofold volume of EDTA solution (1.5 mM
EDTA, 100 mM NaCl pH 8.0; Osborn et al., 1972; Osborn & Munson,1974). The spheroplast suspension was incubated for 20 min on ice
and centrifuged at 10 000g for 10 min in order to separate the periplasmic fraction from the spheroplast cells. NaCl was then added tothe supernatant to a final concentration of 500 mM and centrifuged at
100 000g for 60 min. The supernatant containing the mature MotY
protein was loaded onto an Ni–NTA Superflow column (Qiagen).
After washing with five column volumes of buffer (10 mM Tris–HCl
pH 7.5, 500 mM NaCl, 0.5 mM PMSF, 10 mM imidazole), proteins
were eluted with a stepwise gradient of imidazole from 40 to 200 mM.
The eluate was loaded onto a Superdex 75 (10/300) gel-filtration
column (GE Healthcare Biosciences) equilibrated and eluted with
10 mM Tris–HCl pH 7.5, 500 mM NaCl. The fractions containing
MotY were concentrated to 10 mg ml 1 by ultrafiltration using
AmiconUltra (Millpore). The purity of MotY was assayed by SDS–
PAGE and MALDI–TOF mass spectrometry (Voyager DE/STR,Applied Biosystems).
2.2. Crystallization

Initial crystallization screening was performed by the sitting-drop
vapour-diffusion technique using screening-solution kits (CrystalScreens I and II, Hampton Research; Wizard I and II, EmeraldBioSystems) at 293 K. Within a week, small hexagonal columnshaped crystals and needle-shaped crystals appeared using several
conditions containing PEG or NaH2PO4/K2HPO4 as precipitant. We
optimized the conditions by varying the precipitant concentration,pH, salts and additives using the sitting-drop method. The best
crystals were obtained from drops prepared by mixing 1 ml protein
solution with 1 ml reservoir solution containing 1.12 M NaH2PO4/
K2HPO4, 5% PEG 1000 and 0.1 M acetate pH 4.5 equilibrated
against 500 ml reservoir solution. The optimized hexagonal columnshaped crystals appeared at 293 K within one week and grew totypical dimensions of 0.5  0.1  0.1 mm (Fig. 1a).
2.3. Preparation and crystallization of SeMet-labelled MotY

Figure 1
Hexagonal column-shaped crystals of (a) MotY and (b) SeMet-labelled MotY. The
scale bar is 0.1 mm in length.

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Shinohara et al.  MotY

Mature MotY with a hexahistidine tag contains five methionine
residues in its 278-amino-acid sequence; therefore, we prepared andcrystallized SeMet-labelled MotY for phasing. SeMet MotY wasexpressed in B834(DE3)pLysS, which is a met autotroph (Leahy etal., 1992; Wood, 1966), carrying pJY19-motY-his6 using M9 medium
containing 0.2 g MgSO4, 4 g glucose, 40 mg of all l-amino acids
except Met, 40 mg seleno-l-methionine, 25 mg pyridoxine hydrochloride, 1.25 mg thiamine hydrochloride and 50 mg ampicillin perlitre. Cells were cultured at 303 K until the density of the medium
reached an OD660 of 0.4–0.5 and expression of MotY was inducedwith IPTG at a final concentration of 0.5 mM; culture was continuedfor 4 h. The labelled protein was then purified using the same
protocol as described for nonlabelled MotY. Crystals of SeMet MotY
were obtained under the same conditions as the native MotY crystals,
but only diffracted to 5 Å resolution. Therefore, we tested the SeMet
protein using other conditions under which small crystals of native
MotY had grown in the initial screening. Finally, crystals suitable for
X-ray experiments were obtained at 293 K using a reservoir solution
Acta Cryst. (2007). F63, 89–92

crystallization communications
Table 1
Summary of the data statistics.
Values in parentheses are for the highest resolution shell.
SeMet derivative

Space group
Unit-cell parameters
Wavelength (Å)
Resolution range (Å)
Observations
Unique reflections
Completeness (%)
Anomalous completeness (%)
Redundancy
Anomalous redundancy
I/(I)
Rmerge (%)
Ranom (%)

NativePeakInflection

Remote 1

Remote 2

P6122/P6522
a = b = 104.1, c = 132.6
1.176553.53–2.85 (3.00–2.85)40463 (5999)
10448 (1490)
99.6 (100)—
6.5 (6.9)—
11.4 (2.7)
8.7 (38.4)—

a = b = 104.7, c = 132.3
0.9791366.08–2.9 (3.06–2.9)
65286 (9668)
10022 (1410)
99.9 (100)99.5 (99.8)
6.5 (6.9)
3.6 (3.6)
16.3 (5.0)
8.0 (35.1)
4.9 (15.4)

0.97940
66.08–3.0 (3.16–3.0)
59007 (8795)
9095 (1290)
99.9 (100)99.5 (99.5)
6.5 (6.8)
3.6 (3.6)
17.0 (5.3)
8.4 (32.4)
4.0 (14.1)

0.986
66.08–3.0 (3.16–3.0)
59073 (8787)
9087 (1291)
99.9 (100)99.6 (99.5)
6.5 (6.8)
3.6 (3.6)
15.4 (4.7)
8.5 (33.1)
3.8 (14.2)

0.964
66.08–3.0 (3.16–3.0)
59348 (8841)
9086 (1292)
99.9 (100)99.6 (99.6)
6.5 (6.8)
3.6 (3.6)
14.6 (3.8)
9.1 (44.4)
4.6 (19.5)

containing 24% polyethylene glycol monoethyl ether (PEG MME)
2000, 0.36 M ammonium sulfate and 0.1 M Tris–HCl pH 8.5. They
were also hexagonal column-shaped crystals and grew to maximum
dimensions of 0.3  0.06  0.06 mm (Fig. 1b). The drop size, reservoir
volume and protein concentration were the same as for the nativecrystals.
2.4. Data collection and processing

X-ray diffraction experiments were performed at SPring-8(Harima, Japan) beamline BL41XU. Both native and SeMet crystals
were soaked in a solution containing 90%(v/v) of the respective
optimized reservoir solution and 10%(v/v) MPD for a few seconds,
immediately transferred into liquid nitrogen for freezing andmounted in a cryo-gas flow. The diffraction data were recorded on anADSC Quantum 315 CCD detector (Area Detector SystemsCorporation) under a 100 K nitrogen-gas flow for the native crystals
and a 35 K helium-gas flow (Rigaku cryocooling device) for theSeMet crystals. The diffraction data were indexed, integrated andscaled using the programs MOSFLM (Leslie, 1992) and SCALA fromthe CCP4 program suite (Collaborative Computational Project,
Number 4, 1994). The statistics of data collection are summarized inTable 1.

3. Results and discussion
MotY tends to form insoluble aggregates under low ionic strengthconditions (Okabe et al., 2005); hence, we kept the concentration ofNaCl at 500 mM throughout the purification. Typical protein solutions used for crystallization also contained 500 mM NaCl with
10 mM Tris–HCl pH 7.5 and 10 mg ml 1 MotY.
Initially, we were not able to obtain a sufficient amount of SeMet
MotY for crystallization. After spheroplast formation, most MotY
was precipitated with the cells. Soluble MotY was only recoveredfrom the periplasmic fraction at a very low level (Fig. 2a). We
analysed the molecular weight of MotY from the spheroplast fraction
by MALDI–TOF mass spectrometry and found that it had no signal
sequence (the observed molecular weight was 32 kDa and thecalculated monoisotopic weights of the His-tagged SeMet MotY withand without signal sequence were 34 400 and 32 100 Da, respectively). This observation means that the expressed protein shouldhave been translocated to the periplasmic space through the Secsystem. However, the protein was not in the periplasmic fraction(Fig. 2a, lane 4) and remained in the spheroplast fraction (Fig. 2a,lane 3). We then checked the spheroplast formation with a microActa Cryst. (2007). F63, 89–92

scope and found that the cell spheroplasts were not well produced.
While BL21 cells were used for expression of native MotY,
B834(DE3)pLysS cells were used for expression of SeMet MotY. We
thought that the T7 lysozyme expressed by the pLysS plasmid in thecells might have disturbed the formation of spheroplasts, as it isknown that control of lysozyme concentration is one of the key
factors in spheroplast formation. The T7 lysozyme is encoded on thepLysS plasmid, which carries the chloramphenicol-resistance gene.
Therefore, we cultured the cells without chloramphenicol in order todrop the pLysS plasmid and converted them into spheroplasts. In thisway, we recovered soluble SeMet MotY from the periplasmic fraction
and obtained a sufficient amount of the protein for crystallization(Fig. 2b).
MotY crystals diffracted to 2.9 Å resolution. The native crystals
belong to the hexagonal space group P6122/P6522, with unit-cellparameters a = b = 104.1, c = 132.6 Å. The Matthews coefficient (VM;Matthews, 1968) indicates the presence of a single MotY molecule in
the asymmetric unit, with a solvent content of 62%. The self-rotation
function map calculated using the program POLARRFN (Collaborative Computational Project, Number 4, 1994) showed no significant peak corresponding to local symmetry.
Although the crystallization conditions for the SeMet derivative
are quite different from those for native MotY, the unit-cell parameters of the derivative crystals, a = b = 104.7, c = 132.3 Å, werealmost the same as those of the native crystals (Table 1). The derivative crystals diffracted to 3.0 Å resolution, but were sensitive toFigure 2SDS–PAGE analysis of SeMet-labelled MotY expressed (a) with and (b) withoutchloramphenicol. MotY was detected by immunoblotting using an anti-MotYantibody. Lane 1, before induction; lane 2, after induction; lane 3, spheroplast
fraction; lane 4, periplasmic fraction.

Shinohara et al.  MotY91

crystallization communications

Figure 3(a) Bijovoet and (b) dispersive difference Patterson maps (w = 0.5 Harker section) calculated using the data from the SeMet derivative at 3.5 Å resolution. The contour lines
are drawn from 2.5 to 6.5, with an increment of 0.5.

X-ray irradiation. Therefore, we used a helium cryocooling device inorder to reduce the radiation damage. Statistics of the diffraction data
at four different wavelengths are summarized in Table 1. Bijvoet anddispersive difference Patterson maps of the SeMet derivative showedcommon clear peaks on the Harker sections (Fig. 3), suggesting theusefulness of these data for phasing by the MAD method.
We acknowledge H. Matsunami, M. Shimada, R. Iwase and T.
Tajima for assistance in data collection and N. Shimizu, M. Kawamoto
and K. Hasegawa at SPring-8 for technical help in the use of beamlines BL38B1 and BL41XU. This work was supported in part by
Grants-in-Aid for Scientific Research to KI (16310088 and 18074006)
and KN (16087207) and the ‘National Project on Protein Structuraland Functional Analyses’ (to KI and MH) from the Ministry ofEducation, Science and Culture of Japan and the Soft Nano-Machine
Project of Japan Science and Technology Agency (to TY and MH).

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Acta Cryst. (2007). F63, 89–92