Domain movements of HAP2 in the cap–filament
complex formation and growth process of the
Saori Maki-Yonekura*†, Koji Yonekura*†‡, and Keiichi Namba*†‡§¶
*Protonic NanoMachine Project, Exploratory Research for Advanced Technology, Japan Science and Technology Agency, 3-4 Hikaridai, Seika, Kyoto
619-0237, Japan;†Dynamic NanoMachine Project, International Cooperative Research Project, Japan Science and Technology Agency, 3-4 Hikaridai, Seika,
Kyoto 619-0237, Japan;‡Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan; and§Advanced
Technology Research Laboratories, Matsushita Electric Industrial Company, Limited, 3-4 Hikaridai, Seika, Kyoto 619-0237, Japan
Edited by Donald L. D. Caspar, Florida State University, Tallahassee, FL, and approved October 16, 2003 (received for review July 23, 2002)
The cap at the growing end of the bacterial flagellum is essential
for its growth, remaining stably attached while permitting the
insertion of flagellin transported from the cytoplasm through the
narrow central channel. We analyzed the structure of the isolated
cap in its frozen hydrated state by electron cryomicroscopy. The 3D
density map now shows detailed features of domains and their
connections, giving reliable volumes and masses, making assign-
ment of the domains to the amino acid sequence possible. A model
of the cap–filament complex built with an atomic model of the
filament allows a quantitative analysis of the cap domain move-
ments on cap binding and rotation that promotes the efficient self
assembly of flagellin during the filament growth process.
coli and Salmonella, the motor structure called the flagellar basal
body crosses both cytoplasmic and outer membrane and con-
tinues to the extracellular structure called the hook and the
filament. Assembly of the flagellum begins from basal body
construction, and a protein export apparatus homologous to the
type III protein secretion system (4) is attached on its cytoplas-
mic face (5). The export apparatus selectively transfers flagellar
axial proteins into the long narrow central channel of the
flagellum by the energy of ATP hydrolysis by FliI, a subunit of
the export apparatus (6, 7). These flagellar axial proteins travel
through the channel to the distal end, where assembly occurs (8,
9). A protein, called hook-associated protein 2 (HAP2, also
called FliD), forms a cap structure at the distal end of the
flagellum (10), which is essential for filament growth. The cap
remains stably attached while permitting flagellin insertion and
binding between the filament end and itself, thereby preventing
flagellin monomers from simply diffusing away. Isolated HAP2
forms a bipolar pair of pentamers in solution, but the native
capping function is performed by a pentamer (11–14).
The flagellar filament is a tubular structure constructed with
a single protein, flagellin. The filament is made of 11 protofila-
ments, whose lateral binding is axially staggered by about half a
subunit, producing five indentations at the ends of the filament
with a double indentation in one of these five, wider and deeper
a symmetry mismatch, between the 5-fold circular symmetry of
the cap and ?5.5-fold helical symmetry of the filament. Vonder-
viszt et al. (13) proposed a model of the capping interaction, and
Yonekura et al. (14) visualized the actual capping interactions by
electron microscopy and 3D image reconstruction, although at
a limited resolution. Based on the cap structure, with a pentag-
onal plate and five leg domains and the cap–filament structure
showing just one binding site open for flagellin, we proposed an
active role of the cap for the in vivo filament growth, namely a
rotary cap mechanism that promotes efficient flagellin assembly
(14). However, the boundary between the cap and the filament
end could not be well resolved.
acteria swim by rotating flagellar filaments (1–3), each of
To visualize the cap–filament interaction more clearly, we
have analyzed the structure of the cap at higher resolution by
frozen hydrated cap decamer. This map has more reliable
volume recovery, allowing docking of this map onto an atomic
model of the filament (K. Imada, K. Hasegawa, S.M.-Y., K.Y.,
F. A. Samatey, I. Yamashita, and K.N., unpublished results). The
interactions between distal flagellin subunits and the five leg
domains of the cap can now be analyzed more quantitatively,
including their conformational changes. Implications for the
flexibility in the domain arrangements of flagellar axial proteins
will be discussed below.
Materials and Methods
Sample Preparation. HAP2 was overproduced in E. coli cells,
strain BL21 (DE3), carrying pLysS and pKOT134 plasmids (15),
and was prepared as described (12). Chemical crosslinking was
used to stabilize the bipolar pair of caps. The crosslinking was
carried out by using a zero-length crosslinker 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (EDC), as
described (11, 12), with some improvements. Briefly, 1.0 mg?ml
HAP2 was incubated with 40 mM EDC and 40 mM sulfo
N-hydroxysuccinimide in 20 mM sodium phosphate buffer (pH
7.0) at room temperature. After 2 h, the reaction was terminated
by adding 500 mM Tris?HCl (pH 8.0) and 20 mM ?-mercapto-
ethanol to a final concentration of 167 and 6.7 mM, respectively.
The crosslinked products were dialyzed against 20 mM sodium
phosphate buffer (pH 7.0) and 200 mM NaCl.
Electron Cryomicroscopy. The crosslinked products of HAP2 were
suspended in the dialysis buffer containing 0.9% N-octyl-?-D-
glucoside at a protein concentration of 0.5 mg?ml just before the
preparation of frozen hydrated specimens. A holey carbon film
on a copper grid (QUANTIFOIL R1.2?1.3 Quantifoil Micro
Tools, Jena, Germany) was pretreated by glow discharge with
N-amylamine, and the sample solution was applied to the grid.
The grid was blotted and plunged into liquid ethane. Then, the
grids were examined by using an electron microscope, JEM-
3000SFF (JEOL), with a field emission gun operated at 100 kV
and at a specimen temperature of 4 K. Images were recorded on
SO-163 film (Eastman Kodak) at a nominal magnification of
?60,000. The electron dose was set to 13 ? 18 e?Å2.
Image Analysis. Electron micrographs were scanned and digitized
with a LeafScan 45 linear charge-coupled device densitometer
This paper was submitted directly (Track II) to the PNAS office.
Abbreviation: FSC, Fourier shell correlation.
¶To whom correspondence should be addressed. E-mail: email@example.com.
© 2003 by The National Academy of Sciences of the USA
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reduced by a factor of 2. The magnification was calibrated with
the layer line spacing measured in the computed Fourier trans-
form of the images of the straight flagellar filaments from the
SJW1660 strain of Salmonella recorded on the same electron
Overall procedure of image analysis and 3D image recon-
struction are described in Fig. 1. First, the phase reversal caused
by the contrast transfer function was corrected. Images recorded
at defocus levels of 15,000–20,000 Å were used for analysis.
Because HAP2 decamer images showed a tendency to bend at
the connection between the two pentamers (14), we treated each
pentamer separately. The end-on view images (Fig. 2A) were not
used, because it was not possible to exclude images of the
decamer. Only side-view images of the decamer (Fig. 2B) were
selected in electron cryomicrographs to make sure that only
pentamer images as halves of the HAP2 decamer were used for
image alignment. In total, 422 images of the decamers were
picked up by visual inspection. To treat each pentamer sepa-
rately, three points were manually marked on each decamer
image to indicate the center and both ends. Then, 844 pentamer
images were aligned roughly based on these marked points.
When we first tried conventional methods such as EMAN (16)
and SPIDER (17), which carry out 2D alignment by crosscorre-
lation, they showed a tendency to cause misalignment of images.
therefore used our own 1D projection method described below.
However, we later found out that the misalignment was due to
off-center placement of pentamer images in individual images of
the decamer being placed at the center. When we cut off
pentamer images and placed them at the center of the image box,
conventional alignment methods produced density maps similar
to that presented here (reconstruction by EMAN in Fig. 6, which
is published as supporting information on the PNAS web site).
Although there are significant differences between the two
reconstructions, particularly in the thin kinked rod subdomain
regions, the Fourier shell correlation (FSC) curve between the
two falls off to 0.5 at 26-Å resolution, which is comparable to
those of individual reconstructions. Therefore, the differences
indicate weak signal levels in those regions, probably because of
some structural flexibility. We present in this report the density
map obtained by the 1D method, which shows slightly better-
First, a running average along an ?5-fold axis was calculated for
in-plane rotational alignment. Then, two 1D density profiles
obtained by projection in two orthogonal directions were used
for translational alignment. We call the profile obtained by
projection along the 5-fold axis of the cap structure ‘‘projection
x’’ (x axis profile) and the profile by projection perpendicular to
the 5-fold axis, ‘‘projection z’’ (z axis profile). From the 1D
crosscorrelation between the images, the amount of x shift (shift
of the axis position) was roughly determined and further refined
as below. The amount of z shift (shift along the axis) was
obtained from the position of maximum slope in projection z,
which corresponds to the outer edge of the cap plate (14, 18).
After the alignment of the images, an averaged image was
obtained. By using the average as the new reference image, this
2D alignment procedure was iterated 10 times. In each step,
images that showed relatively large translation or angle of
rotation were excluded from the average to produce the refer-
ence for the next step. After the 10th cycle, the number of images
included in the average was 664.
The azimuthal orientation of the particles in individual images
was determined by projection x. The 3D density distribution of
the negatively stained HAP2 pentamer (14) was first projected
to produce 2D images at every 1° step of the azimuthal rotation
about the 5-fold axis, and then projection x was obtained from
each image and used as the initial reference. From 1D cross-
correlation between projection x of the reference and that of
each image, the azimuthal orientation was determined. The
Summary of the image analysis and 3D image reconstruction
complex and determination of its azimuthal orientation. (A) End-on views
and thin plates at both ends. (Bar ? 100 Å.) (C) Projection x profile for the
examples are shown. Thick lines represent those obtained from individual
images, and thin lines represent those from the reference image. The deter-
density profile correspond to the leg domains on two sides of the particle and
the trough to the central cavity. The interval of tick marks is 34.5 Å in the
were chosen to demonstrate how this profile changes sensitively by a small
change of angle.
Electron cryomicrographs of the frozen hydrated HAP2 decamer
Maki-Yonekura et al. PNAS ?
December 23, 2003 ?
vol. 100 ?
no. 26 ?
position of the 5-fold axis (x shift) was also refined in finer steps
at the same time. Projection x of the reference (thin solid lines
in Fig. 2C) changes its profile significantly depending on the
(thick solid lines in Fig. 2C), although still noisy, showed
structural features significant enough to give good crosscorre-
lation peaks, allowing us to determine the azimuthal orientation
reliably and accurately. Then, a 3D density distribution was
calculated by the back-projection method with a low-pass filter.
We used the same low-pass filter that we used for the recon-
struction of the cap–filament complex (18) to recover correct
volume. We used this reconstruction as the new reference after
four times. In each step, images with a crosscorrelation coeffi-
cient ?0.1 were excluded. The number of HAP2 molecules
included in the final image reconstruction was 531 ? 5 ? 2,655.
26 Å by using the 0.5 criterion in the FSC between two
reconstructions calculated from randomly chosen halves of the
data set. We also calculated the FSC for a density map recon-
structed without the symmetry enforcement (Fig. 7, which is
FSC curves between this density map and those rotated about
the 5-fold axis by 72°, 144°, 216°, and 288° all crossed an FSC of
0.5 at a resolution of 29 Å (Fig. 3). (See variance map in Fig. 8,
which is published as supporting information on the PNAS web
Model Building. An atomic model of the R type straight filament
was constructed (K. Imada, K. Hasegawa, S.M.–Y., K.
Yonekura, F. A. Samatey, I. Yamashita, and K.N., unpublished
results) with the atomic model of the flagellar protofilament
made of F41, a core fragment of flagellin (19), by using the
helical symmetry of the filament (20) and the two heavy atom-
binding positions as the fiducial marks. Details will be described
elsewhere. The density map of the cap obtained in the present
study was incorporated onto the atomic model of the filament by
using the graphics program O (21). The five leg domains of the
cap were cut off the plate domain by using XDISPMSK (22) and
rearranged without disconnection from the plate to fit into the
five indentations formed at the distal end of the filament.
Images of Frozen Hydrated HAP2 Decamer. The image contrast of
the frozen hydrated HAP2 decamer obtained at an accelerating
voltage of 300 kV was very poor because of its relatively small
size, with a molecular mass of ?250 kDa and a diameter slightly
larger than 100 Å. We therefore operated the same microscope
at an accelerating voltage of 100 kV to gain a higher contrast.
Because the amplitude contrast is significantly higher at lower
operation voltage (23), the image contrast was boosted up, and
the molecular shape became much clearer. A representative set
of images in end-on and side view is shown in Fig. 2 A and B,
Pentagonal particle images, called star-cap (15), correspond-
but the side views were rarely seen in the sample prepared in a
solution containing 200 mM NaCl and 20 mM sodium phosphate
buffer (pH 7.0). This observation indicated that the outer
surface of the cap plate is relatively hydrophobic and prefers to
be exposed to the air at the air–water interface. By adding a
detergent, n-octyl-?-D-glucoside, to the sample solution to 0.9%,
its critical micelle concentration, many side-view images ap-
peared as shown in Fig. 2B. The bipolar structure is clearly
visible in its elongated shape accentuated by the thin-plate
feature at both ends. The decamer particles had a tendency to
to those observed in negatively stained samples (11), indicating
that the bending flexibility is an intrinsic property and not an
artifact by negative staining.
3D Density Map of the Cap. Image analysis and 3D image recon-
struction were carried out as described in Materials and Methods.
The 3D density map of the HAP2 cap is shown in Fig. 4A. The
side view of the whole decamer is also shown in Fig. 4B, where
the density of the bottom half does not show up in many parts
due to misalignment resulting from the flexible linker between
the two pentamers, where the nature of disorder appears to be
twisting around the 5-fold axis rather than bending. The basic
architecture, consisting of a pentagonal plate that looks like a
flower with five petals and five leg-like domains underneath the
cap plate, is the same as the one in our previous map obtained
from images of negatively stained particles (14). In the new map,
FSC profile between two reconstructions calculated from two halves of the
entire data set with 5-fold symmetry enforcement. Colored lines represent
rotation for the reconstruction calculated without symmetry enforcement.
lines indicate the statistically expected error level in the FSC at 5?5 ? and 5 ?,
with and without 5-fold symmetry taken into account, respectively.
Oblique view of the pentamer (A) and side view of the decamer (B) , in a solid
surface representation with 100% volume recovery are shown. The 5-fold
symmetry is enforced. (Bar ? 50 Å.) (C) Averaged end-on view. (D) Projection
of the 3D reconstruction along the 5-fold axis. (E) Averaged side-view. (F)
Pojection of the 3D reconstruction perpendicular to the 5-fold axis.
Reconstructed 3D image of the HAP2 pentamer and decamer.
www.pnas.org?cgi?doi?10.1073?pnas.2534343100 Maki-Yonekura et al.
however, each domain shows much more detailed features
because of the significantly higher resolution. The leg domain is
divided into three parts: a large subdomain attached to the plate
through a narrow connection; a thin kinked rod subdomain that
extends toward the linker portion adjoining the two caps; and a
linker plate subdomain that adjoins the two sets of five leg
domains of the two caps. The narrow connection between the
in the previous map, strongly indicating that this connection is
flexible enough to allow the adjustment of leg domain orienta-
tion to fit into the indentations of the distal end of the filament
over the symmetry mismatch. The thin kinked rod subdomains
also appear to have flexibility, which is probably responsible for
the bending flexibility around the connection between the two
pentamers. The volume of these kinked rod subdomains is likely
to be underrepresented artificially by the contour level chosen to
represent appropriate volumes of the cap plate and large leg
subdomains, which occupy most of the volume of the cap dimer.
The flexible nature of this portion would be responsible for
variable conformations in individual decamer images, smearing
out the density distribution of the bottom half of the decamer
after averaging in the image reconstruction.
The cap plate is 30 Å thick, and its outer diameter is 145 Å,
just as observed for the cap plate in the cap–filament complex
(14). The axial length of the cap is 125 Å, as measured from the
outer surface of the cap plate to the center of the cap dimmer
(Fig. 4B). The large leg subdomain is ?55 Å long and 35 and 30
Å wide in its radial and azimuthal dimensions, respectively. The
diameter of the empty inner space between the five legs under-
neath the cap plate is ?40 Å at its narrowest position, which is
plate in the cap–filament complex (14). This agreement indicates
that, when the leg domains fit into the indentations at the end
of the filament, radial movements are not necessary, and azi-
muthal movements are sufficient to allow the binding over the
To check the validity of the method developed and used for
this study, the reconstructed 3D volume was projected along and
perpendicular to the 5-fold axis (Fig. 4 D and F). Those
projections were compared with the 2D averages of the end-on
and side-view images (Fig. 4 C and E), respectively. The close
similarity between them proves the validity of the method.
We estimated the molecular mass of each domain from the
rod, and linker plate subdomains occupy ?63%, 21%, 0.3%, and
15%, respectively, and the corresponding molecular masses are
?32, 11, 0.2, and 7.6 kDa, respectively. The value for the kinked
rod domain is underrepresented, as mentioned above. Possible
assignment of each domain on the amino acid sequence will be
Model of the Cap–Filament Complex. We docked the 3D density
map of the cap onto an atomic model of the R type straight
filament. The atomic model of the filament was constructed by
using the atomic model of the flagellar protofilament made of
F41, a core fragment of flagellin (19), as briefly described in
Materials and Methods (K. Imada, K. Hasegawa, S.M.-Y., K.
Yonekura, F. A. Samatey, I. Yamashita, and K.N., unpublished
results). The atomic model was nicely fitted into the density map
at 7-Å resolution obtained by electron cryomicroscopy (S.M.-Y.,
K. Yonekura, and K.N., unpublished results), further confirming
the correctness of the model. Although the filament model does
not contain domain D0 of flagellin, which forms the inner core
of the filament, the large leg subdomains appear to interact with
domains D1, which form the outer core of the filament, and
therefore important interactions can be inferred in this model to
identify the structural changes of the cap necessary for binding
and the rotary mechanism for filament growth.
First, the cap plate was placed on the filament end according
to the density map of the cap–filament complex (14), which
clearly shows the relative position and azimuthal orientation of
the cap plate to the helical array of the outer domains of flagellin
in the filament. The filament model shows a complicated surface
at its distal end but clearly demonstrates the shape, size, and
position of the five indentations. The five leg domains of the cap
orientation and position. We assumed flexibility in the connec-
tion between the cap plate and leg domain for the leg domain
movement. We also assumed flexibility in the connection be-
tween the large leg and the thin rod subdomains. The domains
that form the linker plate were removed, because they are
the indentations so that all of the leg domains have interactions
as uniform as possible with domains D1 of flagellin (Fig. 5). To
produce the inverted-L-shaped opening at one site and relatively
small gaps in the other four sites, as observed (14), one of the leg
domains (green in Fig. 5 A–D) had to be moved and reoriented
to a much larger extent than the other four, whereas the
remaining four fitted nicely into the indentations with relatively
small conformational changes (Fig. 5B).
The leg domain colored green in Fig. 5 A–D interacts with
domain D1 of only one flagellin subunit, whereas the other four
leg domains interact with two or three flagellin subunits (Fig.
5D). The changes in the position and orientation of the leg
domains on binding of the cap to the filament end are shown by
superimposition of modified leg domains on that of the isolated
cap (Fig. 5B). The azimuthal displacement and the change in
orientation are listed in Table 2. The largest displacement is 26
Å (the change in orientation, 33°), but the other displacements
are relatively small, all within a range of 8–16 Å (6–18°).
Displacements in the radial direction were not necessary.
The changes in the position and orientation of the leg domains
for each step rotation of the cap on binding of a flagellin subunit
are also shown by superimposing neighboring pairs of leg
domains (Fig. 5C). The displacements and the change in orien-
tation are summarized in Table 2. The largest displacement is
?21 Å (27°), but the other displacements are again relatively
small, all within a range of 4–13 Å (2–15°).
Fig. 5 D and E show cylindrical sections at domain D1. When
a new flagellin molecule (blue in Fig. 5E) that is exported from
the central channel binds to the filament, one of the leg domains
(green) is reoriented, making a new binding site in the next
position (Fig. 5E).
By electron cryomicroscopy of frozen hydrated HAP2 cap
dimers and 3D image analysis, we recovered the shape and
volume of each domain of the cap reliably. From a geometrical
consideration of these data, together with the surface structure
of the atomic model of the filament at its distal end (K. Imada,
K. Hasegawa, S.M.-Y., K. Yonekura, F. A. Samatey, I. Ya-
Table 1. Estimated volume and corresponding molecular mass of
Fraction, %Molecular mass, kDa
Volumes and fractions were obtained from the reconstructed 3D map.
The molecular mass was calculated from the volume fraction and the
molecular mass of HAP2, 49.7 kDa. Note that the value for ‘‘Thin rod’’ is
Maki-Yonekura et al. PNAS ?
December 23, 2003 ?
vol. 100 ?
no. 26 ?
mashita, and K.N., unpublished results) and the structural
features observed in the 3D density map of the cap–filament
complex (14), we built a model of the cap–filament complex and
identified possible ways interacting between domains of HAP2
and flagellin. Conformational changes of the cap on binding to
the filament end and rotation to promote the self assembly of
flagellin were preliminarily analyzed (27), but in the present
study, we actually measured domain movements involved in
Possible Domain Movements. The leg domains of the cap were
of the filament, where these pivot motions required changes in
the domain orientation up to ?33° (Fig. 5B). The changes in the
leg domain orientation required for a step rotation of the cap
(Fig. 5C) were even smaller. The narrow linker density connect-
ing the leg domains and the cap plate strongly suggests sufficient
flexibility for all these changes in the domain orientation.
Domain-Sequence Assignment. There is evidence indicating that
the leg domains are made of both terminal chains of HAP2.
Approximately 40 NH2-terminal and 50 COOH-terminal resi-
dues of HAP2, which are unfolded in its monomeric or pen-
tameric state in solution, are folded up to stabilize the cap–
filament complex or the cap dimer structure (13). In the density
map of the cap dimer, these terminal 90 residues would corre-
spond to two distal leg domains: the linker plate subdomain
adjoining the two caps and the thin kinked rod subdomain
connecting to the large leg subdomain. From their volume, the
molecular mass of these two domains together was estimated to
be 8 kDa (Table 1), which roughly agrees with that of the
terminal 90 residues, ?10 kDa. Considering the possible under-
representation of the volume of the thin kinked rod portion, this
domain-sequence assignment seems reasonable. The molecular
mass of the cap plate domain was estimated to be 31.5 kDa
(Table 1), which is close to the apparent molecular mass (29
kDa) of a proteolytic fragment of HAP2 (13). This further
supports the domain-sequence assignment for the terminal
complex built in five different side views, showing each of the five leg conformations and the gaps between the plate and the filament end. A front portion of
the model is trimmed off to show the leg domains and gaps clearly. The cap plate is red, and the five leg domains are white, blue, green, brown, and purple.
For the filament portion, the flagellin F41 subunits are represented as a space-filling model in yellow. (B) Pairwise comparison of the leg domain orientation in
of leg domain orientation between neighboring ones in the cap–filament model to show changes in their orientation by one-step cap rotation. Number labels
in B and C correspond to those in different views in A, whereas label 0 indicates the leg domain in the HAP2 decamer. Arrows in B and C indicate the direction
correspond to those in C. Domain colors are consistent in B–E. The figure was made with MOLSCRIPT (24), RASTER3D (25), VOLCUBE in SITUS (26), and our own in-house
Table 2. Displacement of large leg subdomains on cap binding
1, Å 2, Å 3, Å4, Å5, Å
The displacements for the cap binding were measured at the distal (cell-
proximal) end of the large leg subdomain, as marked by solid circle in Fig. 5B.
obtained by comparing neighboring pairs, where data for leg domain 1, for
in domain orientation.
www.pnas.org?cgi?doi?10.1073?pnas.2534343100 Maki-Yonekura et al.
Folding of HAP2 and Implications for Export and Assembly. These Download full-text
results suggest that the peptide chain of HAP2 is folded in a
similar manner to flagellin, having linearly connected domains
through all of the domains to the other end, and comes back. The
peptide chain of flagellin starts from domain D0, which forms
the inner core of the filament; goes out to D1, D2, and D3, which
is the outermost domain; and comes back to D0 through D2 and
D1 (19, 28, 29). The domains are connected by two chains
flexibility in the domain arrangement on flagellin. The assembly
process would be quicker if the domains are more or less folded
during the export process through the central channel. It has
recently been shown in the complete atomic model of the
filament, however, that the diameter of the channel is smaller
than the smallest dimension of the domains, suggesting that even
individual domains are unfolded during transport (30). Even in
that case, a flexibility in the domain arrangement would be
important for each flagellin domain to properly position, orient,
and bind to the indentation at the distal end of the filament just
after the domains get folded in the small cavity between the cap
plate and the distal end of the filament.
The peptide chain of HAP2 starts from the linker plate
subdomain, because removal of unfolded terminal chains results
in the loss of cap binding ability and decamer formation (13).
The chain then goes through the kinked rod and the large leg
subdomain and goes into the cap plate. Then, it comes back in
the opposite direction. A feature distinct from flagellin is that
the central portion of the chain forms the cap plate located near
the filament axis, instead of the outermost domain. But the
domain linkers are probably made of two antiparallel chains as
features are essential not only for assembly of HAP2 but also for
its specific functions, namely, the stable binding over the sym-
metry mismatch and the stepwise cap rotation to promote the
efficient self assembly of flagellin. The unfolded terminal re-
gions of HAP2 in its monomeric state would form ?-helical
coiled coil on binding to the filament end, just as flagellin, but
would go through repeated binding (folding) and unbinding
(unfolding) for the cap rotation. This is another distinct feature
from flagellin, whose terminal chains stay folded once the
molecule is built into the filament structure.
New Animation for the Rotary Cap Mechanism for the Filament
and its leg domain movements promoting the efficient growth of
the flagellar filament (14) to visualize more realistic motion of
this molecular complex at work (see Movie 1, which is published
as supporting information on the PNAS web site; also see
We thank K. Imada for the HAP2 sample; K. Imada (in our laboratory),
K. Hasegawa (Japan Synchrotron Radiation Research Institute, Hyogo,
Japan), F. A. Samatey (in our laboratory) and I. Yamashita (Matsushita
Electric Industrial Company, Kyoto) for making available the unpub-
lished atomic coordinates of a straight flagellar filament; and F. Oosawa
for support and encouragement.
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