plosPLoS BiolplosbiolPLoS Biology1544-91731545-7885Public Library of ScienceSan Francisco, USA10-PLBI-RA-9322R310.1371/journal.pbio.1000616Research ArticleBiophysicsBiophysics/Macromolecular Assemblies and MachinesMicrobiologyStructural Insight into the Rotational Switching Mechanism of the
Bacterial Flagellar MotorRotational Switching of the Flagellar
MotorMinaminoTohru12ImadaKatsumi13*KinoshitaMiki1NakamuraShuichi1¤MorimotoYusuke V.1NambaKeiichi1*Graduate School of Frontier Biosciences, Osaka
University, Osaka, JapanPRESTO, JST, Saitama, JapanDepartment of Macromolecular Science, Osaka
University, Osaka, JapanBrayDennisAcademic EditorCambridge University, United Kingdom* E-mail: kimada@fbs.osaka-u.ac.jp (KI); keiichi@fbs.osaka-u.ac.jp (KN)
The author(s) have made the following declarations about their contributions:
Conceived and designed the experiments: TM KI. Performed the experiments: TM
KI MK SN YVM. Analyzed the data: TM KI SN YVM. Contributed
reagents/materials/analysis tools: TM KI. Wrote the paper: TM KI KN.
Current address: Department of Applied Physics, Tohoku University, Sendai,
Japan
The authors have declared that no competing interests exist.
52011105201195e100061639201029320112011Minamino et alThis is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited.How Bacteria Shift Gears
Structural analysis of a clockwise-biased rotation mutant of the bacterial
flagellar rotor protein FliG provides a new model for the arrangement of FliG
subunits in the motor, and novel insights into rotation switching.
The bacterial flagellar motor can rotate either clockwise (CW) or
counterclockwise (CCW). Three flagellar proteins, FliG, FliM, and FliN, are
required for rapid switching between the CW and CCW directions. Switching is
achieved by a conformational change in FliG induced by the binding of a
chemotaxis signaling protein, phospho-CheY, to FliM and FliN. FliG consists of
three domains, FliGN, FliGM, and FliGC, and
forms a ring on the cytoplasmic face of the MS ring of the flagellar basal body.
Crystal structures have been reported for the FliGMC domains of
Thermotoga maritima, which consist of the FliGM
and FliGC domains and a helix E that connects these two domains, and
full-length FliG of Aquifex aeolicus. However, the basis for
the switching mechanism is based only on previously obtained genetic data and is
hence rather indirect. We characterized a CW-biased mutant
(fliG(ΔPAA)) of Salmonella enterica by
direct observation of rotation of a single motor at high temporal and spatial
resolution. We also determined the crystal structure of the FliGMC
domains of an equivalent deletion mutant variant of T. maritima
(fliG(ΔPEV)). The FliG(ΔPAA) motor produced torque
at wild-type levels under a wide range of external load conditions. The
wild-type motors rotated exclusively in the CCW direction under our experimental
conditions, whereas the mutant motors rotated only in the CW direction. This
result suggests that wild-type FliG is more stable in the CCW state than in the
CW state, whereas FliG(ΔPAA) is more stable in the CW state than in the CCW
state. The structure of the TM-FliGMC(ΔPEV) revealed that
extremely CW-biased rotation was caused by a conformational change in helix E.
Although the arrangement of FliGC relative to FliGM in a
single molecule was different among the three crystals, a conserved
FliGM-FliGC unit was observed in all three of them. We
suggest that the conserved FliGM-FliGC unit is the basic
functional element in the rotor ring and that the PAA deletion induces a
conformational change in a hinge-loop between FliGM and helix E to
achieve the CW state of the FliG ring. We also propose a novel model for the
arrangement of FliG subunits within the motor. The model is in agreement with
the previous mutational and cross-linking experiments and explains the
cooperative switching mechanism of the flagellar motor.
Author Summary
The bacterial flagellum is a rotating organelle that governs cell motility. At
the base of each flagellum is a motor powered by the electrochemical potential
difference of specific ions across the cytoplasmic membrane. In response to
environmental stimuli, rotation of the motor switches between counterclockwise
and clockwise, with a corresponding effect on the swimming direction of the
cell. Switching is triggered by the binding of the signaling protein
phospho-CheY to FliM and FliN, and achieved by conformational changes in the
rotor protein FliG. The actual switching mechanism, however, remains unclear. In
this study, we characterized a fliG mutant of
Salmonella that shows an extreme clockwise-biased rotation,
and determined the structure of a fragment of FliG (FliGMC) of the
equivalent mutant variant of Thermotoga maritima.
FliGMC is composed of two domains and covers the regions
essential for torque generation and FliM binding. We showed that the mutant
structure has a conformational change in the helix connecting the two domains,
leading to a domain orientation distinct from that of the wild-type FliG. On the
basis of this structure, we propose a new model for the arrangement of FliG
subunits in the rotor that is consistent with the previous mutational studies
and explains how cooperative switching occurs in the motor.
This work was supported in part by Grants-in-Aid for Scientific Research to K.I.
(18074006) and K.N. (16087207 and 21227006). This research was supported in part
by the National Science Foundation through TeraGrid resources provided by the
National Center for supercomputing Applications under grant number
TG-MCB060069N. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.Introduction
Bacteria such as Escherichia coli and Salmonella
enterica swim by rotating multiple flagella, which arise randomly over
the cell surface. Each flagellum is a huge protein complex made up of about 30
different proteins and can be divided into three distinct parts: the basal body, the
hook, and the filament. The basal body is embedded in the cell envelope and acts as
a reversible motor powered by a proton motive force across the cytoplasmic membrane.
The hook and the filament extend outwards in the cell exterior. The filament is a
helical propeller that propels the cell body. The hook connects the basal body with
the filament and functions as a universal joint to transmit torque produced by the
motor to the filament. The flagellar motor can exist in either a counterclockwise
(CCW) or clockwise (CW) rotational state. CCW rotation causes the cell to swim
smoothly in what is termed a run, whereas brief CW rotation of one or more flagella
causes a tumble. The direction of motor rotation is controlled by environmental
signals that are processed by a sensory signal transduction pathway to generate
chemotaxis behavior [1]–[3].
Five flagellar proteins, MotA, MotB, FliG, FliM, and FliN, are involved in torque
generation. Two integral membrane proteins, MotA and MotB, form the stator, which
converts an inwardly directed flux of H+ ions through a
proton-conducting channel into the mechanical work required for motor rotation. The
FliG, FliM, and FliN proteins form the C ring on the cytoplasmic side of the MS
ring, which is assembled from 26 subunits of a single protein, FliF, and this
complex acts as the rotor of the flagellar motor [1]–[3]. An electrostatic interaction
between the cytoplasmic loop of MotA and FliG is thought to be involved in torque
generation [4],[5] and in stator assembly around the rotor [6]. The
protonation-deprotonation cycle of a highly conserved aspartic acid residue in MotB
is coupled to the movement of the MotA cytoplasmic loop to generate torque [7]–[9].
Because FliG, FliM, and FliN are also responsible for switching the direction of
motor rotation, their assembly is called the switch complex [10]. Binding of a chemotactic
signaling protein CheY-phosphate (CheY-P) to FliM and FliN is presumed to induce
conformational changes in FliG that result in a conformational rearrangement of the
rotor-stator interface, allowing the motor to spin in the CW direction [11],[12]. The switching
probability is also affected by motor torque, suggesting that the switch complex
senses the stator-rotor interaction as well as the concentration of CheY-P [13],[14]. Recently,
turnover of FliM and heterogeneity in the number of FliM subunits within functioning
motors have been reported [15],[16]. The turnover rate is increased by the presence of
CheY-P, implying that turnover of FliM may be directly involved in the switching
process [15].
FliG forms a ring on the cytoplasmic face of the MS ring with 26-fold rotational
symmetry [17],[18]. FliG consists of three domains, FliGN,
FliGM, and FliGC. FliGN is responsible for
association with the cytoplasmic face of the MS ring [17],[19], and FliGM and
FliGC are required for an interaction with FliM [20]. The FliGM domains of
adjacent subunits are fairly close to each other in the FliG ring [21]. The crystal
structure of FliGMC of Thermotaoga martima
(Tm-FliGMC) shows that FliGM and FliGC are
connected by an extended α-helical linker (helix E) [22]. The linker contains two
well-conserved Gly residues and hence might be flexible [22]. This finding is supported by
genetic analyses of FliG and a computer-generated prediction of its secondary
structure [23],[24]. Critical charged residues, which are responsible for an
interaction with MotA [4]–[6], are clustered together along a prominent ridge on
FliGC[25]. It has been
shown that the elementary process of torque generation by the stator-rotor
interaction is symmetric in CCW and CW rotation [26], although the torque-speed
curves are distinct between them [27].
A recent report on the full-length FliG structure of Aquifex
aeolicus has shown two distinct conformational differences between the
full-length FliG and FliGMC structures [28]. The helix E linker is held in a
closed conformation by packing tightly against an α-helix (helix n), which
connects FliGN to FliGM in a way similar as helix E connects
FliGM and FliGC in the full-length FliG structure. Helix E
is dissociated from FliGM in the Tm-FliGMC structure,
resulting in its being in an open conformation. The conformation of FliGC
is also different in these two structures. Combined with the previous genetic data,
it has been proposed that the closed conformation represents FliG during CCW
rotation and that switching to CW rotation may be accompanied by the dissociation of
helix E from FliGM to form an open conformation.
The S. enterica FliG(ΔPAA) mutant protein has three-amino-acid
deletion at positions 169 to 171. Motors containing this protein are extremely CW
biased [29]. The
mutant motors remain in CW rotation even in the presence of a cheY
deletion, indicating that the motor is locked in the CW state [29]. Therefore, it is likely
that binding of CheY-P to FliM may introduce a conformational change in FliG similar
to the one introduced by the in-frame PAA deletion. To elucidate the switching
mechanism, we crystallized a fragment of a T. maritima FliG mutant
variant, FliGMC(ΔPEV), which contains a deletion equivalent to
S. enterica FliGMC(ΔPAA), and determined its
structure at 2.3 Å resolution. Based on the structural difference among
full-length A. aeolicus FliG, wild-type Tm-FliGMC, and
its deletion variant, we suggest that a reorientation of helix E relative to
FliGM is important for switching and propose a new model for the
arrangement of FliG subunits in the motor.
ResultsCharacterization of S. enterica fliG(ΔPAA)
Mutant
The motors of the fliG(ΔPAA) mutant rotated only CW (Figure
S1A), whereas wild-type motors rotated exclusively CCW under our
experimental conditions. The motors of the deletion mutant produced normal
torque under a wide range of external-load conditions, indicating that the
deletion does not affect the torque generation step (Figure
S1B). Introduction of a cheA-Z deletion, which causes
wild-type motors to spin exclusively CCW [30], into the
fliG(ΔPAA) mutant did not change the CW-locked
behavior. These results are in good agreement with a previous report [29].
Switching between the CW and CCW states is highly cooperative [31]–[34]. The switching
mechanism can be explained by a conformational spread model, in which a
switching event is mediated by conformational changes in a ring of subunits that
spread from subunit to subunit via nearest-neighbor interactions [34],[35]. Therefore we
investigated rotation of a single motor composed of wild-type and mutant FliG
subunits at different ratios. FliG(ΔPAA) inhibited expansion of wild-type
colonies in semi-solid agar (Figure
1A), even when its expression level was ca. 5-fold lower than the
level of wild-type FliG expressed from the chromosome (Figure 1B). Bead assays revealed that the
decrease in colony expansion results from an increase in both switching
frequency and prolonged pausing (Figure 1C). In addition, a low level expression of FliG(ΔPAA)
partially increased the colony expansion of the ΔcheA-Z
smooth-swimming mutant, presumably because switching now occurred (Figure 1D, upper and middle
panels). These results suggest that even a small fraction of FliG(ΔPAA) in a
motor can affect the CW-CCW switching.
10.1371/journal.pbio.1000616.g001
Dominant-negative effect of FliG(ΔPAA) on motility of wild-type
cells.
(A) Motility of SJW1103 cells (wild-type) transformed with pET19b
(indicated as Low-V), pTrc99A (indicated as High-V), pGMK4000
(pET19b/His-FliG(ΔPAA), indicated as Low-FliG(ΔPAA)), and
pGMM4500 (pTrc99A/His-FliG(ΔPAA), indicated as High-FliG(ΔPAA))
in semi-solid agar plates. (B) Expression levels of FliG and
His-FliG(ΔPAA). Immunoblotting, using polyclonal anti-FliG antibody,
of whole cell proteins. Lane 1, MKM1/pET19b (indicated as
ΔfliG/V); lane 2, SJW1103/pET19b (indicated as
WT/V); lane 3, SJW1103/pGMK4000 (indicated as
WT/Low-His-FliG(ΔPAA)). Arrows indicate positions of FliG and
His-FliG(ΔPAA). (C) Measurement of CCW and CW rotation of the
flagellar motor by bead assays. We used SJW46
(fliC(Δ204–292)) as a host because it
produces flagellar motors with the sticky flagellar filaments, which are
easily labeled with polystyrene beads. CCW, counterclockwise rotation;
CW, clockwise rotation. Upper panel: SJW46 carrying pET19b. Middle
panel: SJW46 carrying pGMK4000. Bottom panel: SJW46 carrying pGMM4500.
(D) Effect of FliG(ΔPAA) on motility of a
ΔcheA-Z mutant. Upper panel: Motility of
SJW3076 (ΔcheA-Z) transformed with pET19b, pGMK3000
(pET19b/His-FliG), or pGMK4000 in semi-solid agar. Middle panel:
measurement of CCW and CW rotation of the flagellar motor of
MM3076iC/pGMK4000. Bottom panel: measurement of CCW and CW rotation of
the flagellar motor of MM3076iC/pGMM4500.
The CW-CCW transition, which is very fast in wild-type motors, became
significantly longer in mixed motors (Figure 1), suggesting that, as proposed
previously [24], the motor can exist in multiple states. A much
higher expression of FliG(ΔPAA) completely inhibited wild-type motility
(Figure 1D) and did not
increase the colony size of the ΔcheA-Z mutant in
semi-solid agar plates because of the extreme CW-biased rotation of its flagella
(Figure 1C and D, lower
panel), in agreement with data showing that a higher expression level of
wild-type FliG is required for complementation of the
fliG(ΔPAA) mutant (Figure S2). Therefore, we conclude that
wild-type FliG is more stable in the CCW state than in the CW state, whereas
FliG(ΔPAA) is more stable in the CW state than in the CCW state.
Limited Proteolysis of FliG and FliG(ΔPAA)
To identify structural differences between the CW and CCW states of FliG, we
carried out limited trypsin proteolysis of the wild-type and mutant FliG
proteins and analyzed the products by matrix-assisted laser desorption
ionization time-of-flight (MALDI-TOF) mass spectrometry and N-terminal
amino-acid sequencing (Figure
2). Both the wild-type and mutant FliG proteins were cleaved between
helix E and FliGC, producing the T1 and T2a fragments. This indicates
that there is a flexible region between them. The T1 fragment derived from
FliG(ΔPAA) was less stable than the T1 fragment from wild-type FliG,
suggesting that the deletion causes a conformational change in FliGM
and helix E. In contrast, the T2a fragment was more stable in FliG(ΔPAA)
than in the wild-type. The T2a fragment derived from the wild-type FliG protein
was detected by MALDI-TOF but not on SDS-PAGE gels, indicating that the
wild-type T2a fragment is rapidly converted into the T2 fragment. These results
suggest that the deletion also influences the conformation in the region between
helix E and FliGC.
10.1371/journal.pbio.1000616.g002
Conformation of FliG in solution.
(A) Protease sensitivity of His-FliG (left panel) and His-FliG(ΔPAA)
(right panel). Arrowheads indicate intact molecule and proteolytic
products on SDS-PAGE gels with labels corresponding to those in the
diagram shown in (B). (B) Proteolytic fragments identified by MALDI-TOF
mass spectroscopy and N-terminal amino acid sequencing.
Structural Comparison of Tm-FliGMC and
Tm-FliGMC(ΔPEV)
We tried crystallizing both wild-type FliG and FliG(ΔPAA) from S.
enterica but did not succeed in obtaining crystals. It has been
reported that the crystal structure of a fragment (residues 104–335) of
T. martima FliG (Tm-FliGMC) consists of
FliGM, FliGC, and helix E connecting the two domains
([22]; PDB
ID, 1lkv). FliGC can be further divided into two sub-domains
(FliGCN and FliGCC). Therefore, we introduced the
deletion (ΔPEV), equivalent to ΔPAA, into Tm-FliGMC
(Tm-FliGMC(ΔPEV)) and determined its structure at 2.3 Å
resolution by X-ray crystallography (Figure 3).
10.1371/journal.pbio.1000616.g003
Comparison of the structures of Tm-FliGMC(ΔPEV),
Tm-FliGMC, and Aa-FliG.
Cα ribbon representation of (A) Tm-FliGMC(ΔPEV), (B)
Tm-FliGMC (PDB code 1lkv), and (C) Aa-FliG (PDB code
3hjl), color coded from purple to red going from the N- to the
C-terminus. The FliGM-FliGC unit with helix E is
surrounded by broken line in the upper panels. The white and black
arrowheads in the upper panels represent view directions of the middle
and the lower panels, respectively. (A, upper panel) Two possible
connections between the M-domain and the C-domain (FliGCN and
FliGCC) in the Tm-FliGMC(ΔPEV) crystal are
shown. Because the residues between G186 and V195 are invisible in the
density map, G186 can be to either V195 or V195'. The two possible
C-domains are indicated by vivid and dull colors. (B, C, upper panel)
The orientation of the Tm-FliGMC and Aa-FliG molecule is
adjusted to that of Tm-FliGMC(ΔPEV) by the M-domain
(colored cyan). FliGCN and FliGCC of an adjacent
molecule related by crystallographic symmetry are shown by dull yellow
and dull pink, respectively. The middle panels show comparison of the
FliGM-FliGC unit structures. All the elements
of secondary structure are labeled in alphabetical order from the N- to
the C-terminus, except for “n,” which is not found in the
Tm-FliGMC structure. The lower panels are viewed from the
right of the middle panels.
FliGM, FliGCN, and FliGCC are composed of five
(n, A–D), three (F–H), and six (I–N) helices, respectively
(Figure 3). Since the
residues between G186 and V195 are invisible in the crystal, there are two
possible ways to connect FliGM with FliGCN: one is to
connect FliGM with its adjacent FliGCN (G186 to V195 in
Figure 3A upper panel
and Figure
S3A), and the other is with a distant FliGCN (G186 to
V195' in Figure 3A
upper panel and Figure S3A). The Cα distance between G186 and V195, and G186 and
V195' is 16.9 Å and 27.9 Å, respectively. Therefore, to connect
with the distant FliGCN, the invisible chain would have a fully
extended conformation. We thus conclude that the connection with the adjacent
FliGCN is more plausible.
Compared with the structure of wild-type Tm-FliGMC, FliG(ΔPEV)
showed a significant conformational change in the hinge between helix E and
FliGM, leading to a very different orientation of helix E
relative to FliGM (Figure 3A and B, and Figure 4A and C). As a result, some of the residues in
FliGM are exposed to solvent in the
Tm-FliGMC(ΔPEV) structure. This result is in good agreement with
the data obtained by limited proteolysis (Figure 2). Thus, the conformational
difference in the FliGM-helix E hinge between the wild-type and
mutant structures may represent the conformational switch between the CW and CCW
states of the motor.
10.1371/journal.pbio.1000616.g004
Structural comparison of the FliGM-FliGC
unit.
(A) Comparison of Tm-FliGMC(ΔPEV) and wild-type
Tm-FliGMC (PDB code 1lkv). A
FliGM-FliGC unit of wild-type
Tm-FliGMC, which is composed of FliGM of one
subunit and FliGC of the neighboring subunit related by
2-fold crystallographic symmetry, is superimposed onto
Tm-FliGMC(ΔPEV) using Cα atoms of V117-L165 and
G196-F236 for least-square fitting. FliGM with helix E and
FliGC of Tm-FliGMC(ΔPEV) are colored cyan
and blue, respectively. FliGM with helix E and
FliGC of wild-type Tm-FliGMC are yellow and
orange, respectively. (B) Comparison of Tm-FliGMC(ΔPEV)
with Aa-FliG (PDB code 3hjl). A FliGM-FliGC unit
of Aa-FliG, which is composed of FliGM of one molecule and
FliGC of the neighboring molecule related by 2-fold
crystallographic symmetry, is superimposed onto
Tm-FliGMC(ΔPEV) using Cα atoms of the same region
used in (A). Tm-FliGMC(ΔPEV) is shown in the same color
as in (A), and FliGM and FliGC of
Aa-FliGMC are shown in green and red, respectively. (C)
Comparison of the orientation of helix E. The
FliGM-FliGCN units of wild-type
Tm-FliGMC and wild-type Aa-FliGMC are
superimposed on Tm-FliGMC(ΔPEV). The models are shown in
the same colors used in (A) and (B).
The C-terminal half of helix E is disordered and protrudes into the solvent
channel in the Tm-FliGMC(ΔPEV) crystal (Figure
S3A). In contrast, helix E in the wild-type crystal is stabilized by
forming an anti-parallel four-helix bundle structure with the E helices of three
adjacent subunits related by crystallographic symmetry (Figure S3B)
[22].
Therefore, the orientation of FliGC relative to FliGM is
different between the wild-type and the deletion variants (Figure 3A and B upper panel). Because the
disordered region of helix E is far from the PEV deletion, we conclude that
helix E has a highly flexible nature, which may be responsible for the switching
mechanism, as suggested before [23],[24].
Tm-FliGMC(ΔPEV) also showed a conformational difference in the
H–I loop, resulting in a rigid body movement of FliGCC relative
to FliGCN (Figure 3A and
B middle and lower panels, and Figure 4A). This movement is consistent with
the limited proteolysis data because, in the Tm-FliGMC(ΔPEV)
structure, FliGCC almost covers D199, which is the residue
corresponding to R198 in S. enterica FliG. It is, however,
unclear how the deletion affects the conformation of the H–I loop, because
neither direct contact between FliGCC and helix E nor significant
structural difference in FliGCN is observed.
Comparison of the Structure of Tm-FliGMC(ΔPEV) with A.
aeolicus FliG
The crystal structure of full-length A. aeolicus FliG (Aa-FliG)
showed that the conformation of helix E and the orientation of FliGCN
relative to FliGCC are quite distinct from those of wild-type
Tm-FliGMC[28]. We compared
the Aa-FliG structure with the Tm-FliGMC(ΔPEV) structure and
found that the conformation of helix E and the relative conformation of
FliGCC to FliGCN are also different in those two
structures (Figure 3A and C,
and Figure 4B and C). The
conformational differences are greater than those between Tm- FliGMC
and Tm-FliGMC(ΔPEV). The conformation of helix E in Aa-FliG seems
to be stabilized by interactions of helix E with FliGM and helix n in
the crystal (Figure S3C). As mentioned earlier, the conformation of helix E and
the orientation of FliGCC to FliGCN are also different
between the wild-type and mutant Tm-FliGMC structures. Therefore,
these conformational differences among the three structures strongly suggest
that both helix E and the linker connecting FliGCN to
FliGCC are highly flexible.
Interaction between FliGM and FliGCN
The interaction between FliGM and FliGCN, which share the
armadillo repeat motif [36] that is often responsible for protein-protein
interaction, is very tight in the Tm-FliGMC(ΔPEV) crystal, in
agreement with a previous report [28]. FliGM and FliGCN can be
identified as a single domain, although it is unclear whether the two domains
belong to the same molecule or not because the residues between Gly-186 and
Val-195 are invisible in the crystal (Figures 3A and S3A). The
interaction surface between FliGM and FliGCN is formed by
the C-terminal portion of αB, αC, and αD of FliGM, and
αF, αG, and the N-terminal portion of αH of FliGCN,
respectively (Figure 5A and
B). The interface is highly hydrophobic. Ala-143, Ala-144, Leu-147,
Leu-156, Leu-159, Ile-162, and Ala163 of FliGM, and Ile-204, Met-205,
Leu-208, Ile-216, Leu-220, Leu-227, and Ile-231 of FliGCN are mainly
involved in the tight domain interaction. Leu-159 is located at the center of
the hydrophobic interface (Figure
5C). Around the hydrophobic core, hydrophilic interactions between
Arg-167 and Glu-230, and Gln-155 and Thr-212, also contribute to the domain
interaction (Figure 5C).
These interactions are also conserved in the wild-type Tm-FliGMC and
Aa-FliG crystals, in which FliGM interacts with FliGCN of
an adjacent molecule related by crystallographic symmetry (Figures 3 and S3B). The
FliGM-FliGCN unit in the wild-type
Tm-FliGMC structure can be superimposed onto that in
Tm-FliGMC(ΔPEV) with root mean square deviation of 0.46
Å for corresponding Cα atoms (Figure 4A and C), and that in Aa-FliG with
0.79 Å (Figure 4B and
C). These observations support the idea that the
FliGM-FliGCN unit is a functionally relevant structure
[28]. This is
in good agreement with the previous mutational study showing that most of the
known point mutations that affect FliM-binding [37] are located either on the
bottom surface of the FliGM-FliGCN unit or on the
interaction surface between FliGM and FliGCN (Figure 6A and C).
10.1371/journal.pbio.1000616.g005
Domain interface between FliGM and
FliGC.
The two domains are colored cyan and magenta, respectively. (A) Structure
of Tm-FliGMC(ΔPEV). The secondary structure elements are
labeled as in Figure
3. (B) Structure of Tm-FliGMC(ΔPEV) viewed
from the direction of arrow in (A). (C) Stereo view of the domain
interface between FliGM and FliGCN. The boxed area
in (B) is shown. Side chains of the residues contributing strongly to
the interaction are shown in a ball-and-stick representation, with
carbon, nitrogen, and oxygen atoms indicated by yellow, blue, and red
balls, respectively. Bonds are shown with colors of the domains to which
they belong.
10.1371/journal.pbio.1000616.g006
A plausible model for arrangement of FliG subunits in the
rotor.
(A) A primary sequence alignment of FliGMC from T.
maritima (TmFliG), Salmonella Typhimurium
(StFliG), and Escherichia coli (EcFliG). The regions
involved in the structure models of Tm-FliGMC and
Tm-FliGMC(ΔPEV) are shown in black bars above and
below the Tm-FliG sequence, respectively. The α-helical regions are
indicated by thick bars labeled with the same codes used in Figure 3. The region
of the three-amino-acid deletion is shown by the magenta bar. The
charged residues essential for the motor function are highlighted in
cyan. The EHPQR motif is highlighted in green, and the other residues
thought to be related to FliM-binding are shaded highlighted in yellow
[21],[37]. In vivo
cross-linking experiments using various Cys-substitution mutants of
FliGM have shown that residues indicated by blue arrows
are located near the residues indicated by red ones. The
Cys-substitution sites that did not show any cross-linked products are
indicated by green arrows [22]. Blue and red boxes
indicate point mutations that bias the motor rotation to CCW and CW,
respectively [38]. The residues within magenta boxes can give
rise to CCW or CW-biased mutants, depending on the substitutions. The
numbers under the boxes represent the number of the cluster to which the
indicated residues belong. (B–D) Mapping of various mutation sites
identified in previous studies on the model of
Tm-FliGMC(ΔPEV). A stereo pair of the
Tm-FliGMC(ΔPEV) subunits, color coded from blue to
red going from the N- to the C-terminus, is shown in each panel.
(B–C) Stereo diagram of the subunit arrangement model. (B) The
charged residues essential for motor function are shown in stick
representation colored in cyan. Residues at which substitutions affect
the direction of motor rotation are indicated by balls: blue, CCW motor
bias; red, CW motor bias; magenta, CCW or CW motor bias, depending on
the substitution. The clusters of residues targeted by mutations are
surrounded by ellipsoids and labeled (1, 2, and 3). (C) Residues
involved in FliM binding are indicated by balls: yellow, residues at
which substitutions decrease FliM binding; green, the EHPQR motif. (D)
Residues substituted with Cys for in vivo cross-linking experiments are
shown by balls. Residues indicated in blue cross-linked to residues
indicated in red. Residues that produced no cross-linking products are
colored in green.
Discussion
The default direction of the wild-type flagellar motor of Salmonella
enterica is CCW, and the binding of CheY-P to FliM and FliN increases
the probability of CW rotation. CheY-P binding induces conformational changes in
FliM and FliN that are presumably transmitted to FliG, which directly interacts with
MotA to produce torque [1],[2]. Mutations located in and around helix E FliG, which
connects the FliGM and FliGC domains, generate a diversity of
phenotype, including motors that are strongly CW biased, infrequent switchers, rapid
switchers, and transiently or permanently paused, suggesting that helix E is
directly involved in the switching of the flagellar motor [24]. However, it remains unclear
how helix E affects the switch.
To investigate the switching mechanism, we characterized an extreme CW-biased
S. enterica mutant in which an in-frame deletion of three
residues, Pro-169, Ala-170, and Ala-171, in FliG caused an extreme CW-biased
rotation even in the absence of CheY. Motors containing the FliG(ΔPAA) protein
showed normal torque generation under a wide range of external-load conditions
(Figure 1 and Figure 1S). Thus, the
conformational change in FliG induced by ΔPAA is presumably similar to one
induced by CheY-P binding to FliM and FliN. Limited proteolysis revealed that
ΔPAA induces conformational changes in the hinge between FliGM and
helix E (Figure 2). This result
is in agreement with the crystal structure of Tm-FliGMC(ΔPEV), which
shows that the orientation of helix E relative to FliGM has changed
significantly compared to wild-type FliG (Figure 3).
FliG forms a ring on the cytoplasmic face of the MS ring [17],[18]. In vivo disulfide
cross-linking experiments using Cys-substituted FliG proteins have suggested that
helix A is close to the D–E loop of the adjacent FliG molecule in the FliG
ring [21]. Both a
conserved EHPQR motif in FliGM and a conserved surface-exposed
hydrophobic patch of FliGCN are important for the interactions with FliM
[21]. Because
the conserved charged residues on helix M in FliGCC are responsible for
its interaction with MotA [4],[5],[25], which is embedded in the cytoplasmic membrane, helix M
must lie on top of FliGCC[21],[28]. Considering those
facts in light of the crystal structure of Tm-FliGMC(ΔPEV) described
here, we propose a new model for arrangement of FliG subunits in the motor (Figures 6 and 7).
10.1371/journal.pbio.1000616.g007
Possible models for cooperative switching.
(A) The most plausible model. Two adjacent FliG molecules are colored yellow
and green. The conformational change of the hinge between FliGM
and helix E not only changes the orientation within its own subunit but also
influences the orientation of the neighboring subunit through the
interaction between helix E and FliGM of the neighbor. (B)
Another possible model. Helix E in one subunit is linked to
FliGCN in the adjacent subunit. Therefore, a single
functional unit consists of FliGM and helix E of one molecule and
FliGCN and FliGCC of the other molecule. Three
adjacent FliG molecules are colored yellow, green, and cyan.
FliGM of the cyan molecule, and FliGCN and
FliGCC of the yellow molecule are not shown. (C) The
cooperative switching model proposed by Lee et al. Three FliG molecules are
colored by yellow, green, and cyan. The FliGM-FliGC
units are surrounded by broken lines. The closed conformation (left panel,
helix E interacts with FliGCN) changes to the open conformation
(right panel, helix E dissociates from FliGCN), inducing the
rotation of the FliGM-FliGC unit and additional
rotation of FliGCN. The box in the FliGM indicates
helix A. The open circles represent the sites linked to the D–E loop
(colored red) by in vivo disulfide cross-linking. (D) Possible orientation
of the FliGM-FliGC unit in the rotor. The hydrophilic
surface and the hydrophobic core layers of the cytoplasmic membrane are
shown in orange and yellow, respectively.
In the proposed model, the conserved charged residues on helix M are located on the
top of the FliGM-FliGC unit and the EHPQR motif is present at
the bottom of the unit (Figure 6B and
C). The conserved hydrophobic patch, and most of the point mutation sites
involved in the interaction with FliM, is localized at the bottom of the
FliGMFliGCN units around the EHPQR motif or on the
interface between the FliGM and FliGCN. The D–E loop and
helix E interact with the FliGM domain in the neighboring subunit, in
agreement with data of in vivo cross-linking experiments, which show that residues
117 and 120 (118 and 121 in T. martima) on helix A of one subunit
lie close to residues 166 and170 (167 and 171 in T. martima) on the
D–E loop of the neighboring subunit [21]. In fact, these residues are
very close to each other in our model in positions in which disulfide-crosslinking
should occur. Moreover, the position of Cys residues that do not participate in
disulfide cross-linking are far from each other in the model (Figure 6D).
Our model can also explain the results of mutational studies of CW and CCW-biased
fliG mutants [37],[38]. The mutation sites are widely distributed from helix A
to the H–I loop. Most of them are localized in three regions in our model
(Figure 6A and B). In the
first region, the CCW-biased mutations, which are located on helix A, affect
residues close to residues targeted by CW-biased mutations, which are on a segment
between helix D and E of the adjacent subunit (Figure 6A and B, 1). Because these residues are distributed on the
interaction surface between the neighboring subunits, they presumably affect
cooperative changes in subunit conformation. A second cluster of residues targeted
by CW-biased mutations is located on the C-terminal half of helix B and the
E–F loop (Figure 6A and B,
2). These mutations may
change the orientation of the E–F loop and probably alter the orientation of
helix E, resulting in unusual switching behavior. The third cluster of residues
affected by mutations causing a CW switching bias is located near the loop between
helices H and I (Figure 6A and
B, 3). This region
determines the relative orientation of FliGCC to the
FliGM-FliGCN unit, and therefore the mutations may change the
orientation of FliGCC to cause anomalous switching behavior.
Helix E is directly involved in the switching mechanism, but how does the structure
of helix E affect the orientation of the FliGM-FliGC unit?
Since the D–E loop and helix E interact with FliGM in the
neighboring subunit, we propose that a hinge motion of helix E may directly change
the orientation of the neighboring FliGM domain (Figure 7A). This mechanism could explain the
cooperative switching of the motor. The conformational changes of FliM induced by
association or dissociation of CheY-P may trigger conformational changes in the
FliGM-FliGC unit that it contacts, leading to a large
change in the interaction between FliGCC and MotA. The conformational
change in one unit is probably accompanied by a conformational change in the loop
between FliGM and helix E. This change could influence the orientation of
the neighboring subunit through the interaction between helix E and FliGM
of the neighbor, thereby propagating the conformational change to the neighboring
subunit (Figure 7A).
If helix E actually contacts the more-distant FliGCN in the crystal
structure, an alternative interaction could be responsible for the cooperative
switching (Figure 7B). However,
the same general mechanism involving changes in the conformation of helix E would
still be responsible for the cooperative switching.
Recently, Lee et al. have proposed a model for FliG arrangement and switching based
on the structural differences in Aa-FliG and Tm-FliGMC[28]. In the crystal
structure of Aa-FliG, the hydrophobic patch in FliGM is covered by the
N-terminal hydrophobic residues of helix E (closed conformation), whereas the patch
is exposed in Tm-FliGMC (open conformation). Because mutations that may
disturb the hydrophobic interaction result in strong CW-bias in motor rotation [38], the
structures of Aa-FliG and Tm-FliGMC are proposed to be in the CCW and CW
states, respectively [28]. The hydrophobic patch is also exposed in the
Tm-FliGMC(ΔPEV) structure, although the conformation of helix E
is different from that of Tm-FliGMC. Since ΔPAA in S.
enterica FliG (ΔPEV in T. maritima) caused an
extreme CW-bias, it is possible that the dissociation of helix E from
FliGM leads to CW rotation. In our model, however, the hydrophobic
patch of the FliGM is covered by the hydrophobic residues in the
C-terminal half of helix E of the adjacent subunit. This arrangement raises the
possibility that the closed conformation of helix E found in the Aa-FliG structure
is an artifact of crystal packing.
Lee et al. assume that the FliGM-FliGC unit is present in the
rotor ring, and hence is in agreement with the results of most of mutational
studies. However, the arrangement of the subunits and the mechanism of switching are
different than in our model. In their model, dynamic motion of helix E and helix n
induces a large conformational change of the FliGM-FliGC unit,
including the rotation of FliGM-FliGCN unit and relative to
the FliGCC to the unit, leading to a change in the arrangement of the
charged residues on helix M (Figure
7C) [28].
Cooperative switching is explained by the strong interaction between
FliGCN of one subunit and FliGCC of the adjacent subunit.
However, helix A of one subunit and the D–E loop of the adjacent subunit are
always at a considerable distance in both the CW and CCW states. Hence, their model
cannot explain the in vivo disulfide cross-linking experiments (Figure 7C) [21]. Since our new model can
explain the cross-linking data, it appears to be more plausible than the model
proposed by Lee et al. [28].
Although our model is consistent with most of the previous experimental data, it
still contains ambiguity. The available density map of the basal body obtained by
electron cryo-microscopy is not high enough to allow fitting of the atomic model.
Thus, a higher-resolution rotor-ring structure will be required to build a more
precise model to explain the molecular mechanism of directional switching.
Materials and MethodsBacterial Strains, Plasmids, and Media
S. enterica strains and plasmids used in this study are listed
in Table 1. L-broth, soft
agar plates, and motility media were prepared as described [39],[40]. Ampicillin was added to
a final concentration of 100 µg/ml.
10.1371/journal.pbio.1000616.t001
Strains and plasmids used in this study.
Strains and Plasmids
Relevant Characteristics
Source or Reference
Salmonella
SJW1103
Wild type for motility and chemotaxis
[48]
SJW46
fliC(Δ204–292)
[49]
SJW2811
fliG(ΔPAA)
[10]
SJW3076
Δ(cheA–cheZ)
[30]
MKM1
ΔfliG
[19]
MM3076iC
Δ(cheA–cheZ),
fliC (Δ204–292)
[50]
MMG1001
ΔfliG
fliC(Δ204–292)
This study
Plasmids
pET19b
Expression vector
Novagen
pTrc99A
Expression vector
Pharmacia
pGKM3000
pET19b/His-FliG
[19]
pGKM4000
pET19b/His-FliG(ΔPAA)
This study
pGMM3500
pTrc99A/His-FliG
This study
pGMM4500
pTrc99A/His-FliG(ΔPAA)
This study
pGMM5000
pET22b/Tm-FliGMC(ΔPEV)
This study
Motility Assay
Fresh colonies were inoculated on soft tryptone agar plates and incubated at
30°C.
Bead Assay for Motor Rotation
Bead assays were carried out using polystyrene beads with diameters of 0.8, 1.0,
and 1.5 mm (Invitrogen), as described before [8]. Torque calculation was
carried out as described [8].
Preparation of Whole Cell Proteins and Immunoblotting
Cultures of S. enterica cells grown at 30°C were centrifuged
to obtain cell pellets. The cell pellets were resuspended in SDS-loading buffer,
normalized in cell density to give a constant amount of cells. Immunoblotting
with polyclonal anti-FliG antibody was carried out as described [41].
Purification of His-FliG and His-FliG(ΔPAA) and Limited
Proteolysis
His-FliG and His-FliG(ΔPAA) were purified by Ni-NTA affinity chromatography
as described before [39]. His-FliG and its mutant variant (0.5 mg/ml) were
incubated with trypsin (Roche Diagnostics) at a protein to protease ratio of
300∶1 (w/w) in 50 mM
K2HPO4-NaH2PO4 pH 7.4 at room
temperature. Aliquots were collected at 0, 5, 15, 30, 60, 90, and 120 min and
trichloroacetic acid was added to a final concentration of 10%. Molecular
mass of proteolytic cleavage products was analyzed by a mass spectrometer
(Voyager DE/PRO, Applied Biosystems) as described [42]. N-terminal amino acid
sequence was done as described before [42].
Purification, Crystallization, Data Collection, and Structure Determination
of Tm-FliGMC(ΔPEV)
Tm-FliGMC(ΔPEV) was purified as described previously [23]. Crystals of
Tm-FliGMC(ΔPEV) were grown at 4°C using the hanging-drop
vapor-diffusion method by mixing 1 µl of protein solution with 1 µl
of reservoir solution containing 0.1 M sodium phosphate-citrate buffer pH
4.2–4.4, 36%–50% PEG200, and 200 mM NaCl. Initially,
we tried to solve the structure by the molecular replacement method using
Tm-FliGMC structure (PDB ID: 1 lkv) as a search model. However,
no significant solution was obtained, even though individual domains were used
as search models. Therefore, we prepared heavy-atom derivative crystals and
determined the structure using the anomalous diffraction data from the
derivatives.
Derivative crystals were prepared by soaking in a reservoir solution containing
K2OsCl6 at 50% (v/v) saturation for one day.
Crystals of Tm-FliGMC(ΔPEV) and its Os derivatives were soaked in
a solution containing 90%(v/v) of the reservoir solution and
10%(v/v) 2-Methyl-2,4-pentanediol for a few seconds, then immediately
transferred into liquid nitrogen for freezing. All the X-ray diffraction data
were collected at 100 K under nitrogen gas flow at the synchrotron beamline
BL41XU of SPring-8 (Harima, Japan), with the approval of the Japan Synchrotron
Radiation Research Institute (JASRI) (Proposal No. 2007B2049). The data were
processed with MOSFLM [43] and scaled with SCALA [44]. Phase calculation
was performed with SOLVE [45] using the anomalous diffraction data from
Os-derivative crystals. The best electron-density map was obtained from MAD
phases followed by density modification with DM [44]. The model was
constructed with Coot [46] and was refined against the native crystal data to
2.3 Å using the program CNS [47]. About 5% of the
data were excluded from the data for the R-free calculation. During the
refinement process, iterative manual modifications were performed using
“omit map.” Data collection and refinement statistics are summarized
in Tables
S1 and S2, respectively.
Supporting Information
Effects of the in-frame deletion of residues PAA of S.
enterica FliG on the direction of flagellar motor rotation and
torque generation. (A) Measurement of CCW and CW rotation of the flagellar
motor. Rotation individual flagellar motors of SJW46 transformed with
pGMK3000 (pET19b/His-FliG, indicated as WT) (left) or pGMK3000
(pET19b/His-FliG(ΔPAA), indicated as FliG(ΔPAA)) (right) were
carried out by tracking the position of 1.0 µm bead attached to the
sticky flagellar filament. Measurements were made at ca. 23°C. CCW,
counterclockwise rotation; CW, clockwise rotation. (B) Measurements of the
rotational speeds of single flagellar motors labeled with 0.8 µm
(right), 1.0 µm (left), and 1.5 µm (middle) beads.
(0.06 MB TIF)
Motility assays for complementation of the motility of a
ΔfliG null mutant (left) and a
fliG(ΔPAA) mutant transformed with pET19b
(indicated as Low-V), pTrc99A (indicated as High-V), pGMK4000
(pET19b/His-FliG(ΔPAA), indicated as Low-FliG(ΔPAA)), and pGMM4500
(pTrc99A/His-His-FliG(ΔPAA), indicated as High-FliG(ΔPAA)) in
semi-solid agar. The plates were incubated at 30°C for the length of
time indicated.
(0.31 MB TIF)
Molecular packing in the crystal. (A) Stereo view of the molecular packing of
Tm-FliGMC(ΔPEV) in the P62
crystal, projected down the c axis. Molecules are indicated by Cα
backbone traces. A pair of FliG molecules related by two-fold
crystallographic symmetry is highlighted in cyan and yellow for
FliGM and FliGC, respectively. Other molecules are
shown in grey. G186 and V195 are indicated by blue and magenta balls,
respectively. G186 can be connected to V195 (solid line) or V195'
(dashed line). (B) Stereo view of four symmetry-related molecules of
Tm-FliGMC that form the inter-molecular four-helix bundle
structure in the P6422 crystal (PDB code: 1lkv).
FliGM and FliGCN of the subunit colored by cyan
form the FliGM-FliGCN units with FliGCN and
FliGM of the subunit colored by yellow, respectively, and
FliGM and FliGCN of the subunit colored by green
form the FliGM-FliGCN units with FliGCN and
FliGM of the subunit colored by orange, respectively. (C)
Stereo view of the molecular packing of Aa-FliG in the
P21 crystal (PDB code: 3hjl), projected
down the c axis. The molecules related by crystallographic
21 symmetry are colored by cyan and
yellow. The cyan molecule located in the centre of the panel is labeled, and
helix n and helix E of the center molecule are highlighted in orange.
(2.96 MB TIF)
Data collection statistics.
(0.04 MB PDF)
Refinement statistics.
(0.03 MB PDF)
We thank M. Kihara for her kind gift of pGMK3000 and pGMK4000, cloning
Tm-FliGMC(ΔPEV) into a pET19b vector, critical reading of the
manuscript, and helpful comments. N. Shimizu, M. Kawamoto, and K. Hasegawa at
SPring-8 provided technical help with the use of beam lines. This research was
supported in part by the National Science Foundation through TeraGrid resources
provided by the National Center for supercomputing Applications; we would like to
specifically thank Susan John for assistance with the allocation and technical help.
These experiments were originally designed by M. Kihara and the late R. M. Macnab,
who passed away suddenly on September 7, 2003. This manuscript is duly dedicated to
both M. Kihara and the late R. M. Macnab.
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