Locations of terminal segments of flagellin in the filament structure and their roles in...

J. Mol. Biol. (1997) 270, 222±237

Locations of Terminal Segments of Flagellin in theFilament Structure and Their Roles in Polymerizationand Polymorphism

Yuko Mimori-Kiyosue, Ferenc Vonderviszt and Keiichi Namba*

International Institute forAdvanced Research, MatsushitaElectric Industrial Co., Ltd.3±4 Hikaridai, Seika619-02, Japan

Present addresses: Y. Mimori-KiAxis Project, 17 Minamimachi, NaKyoto 600 Japan; F. Vonderviszt, DPhysics, University of VeszpreÂm, Vu.10. H8201, Hungary.

Abbreviations used: CTF, contraF40, F27, major proteolytic fragmethe numbers indicate approximateF(#N-#C), proteolytic fragments ofand #C indicate the sequence posit

0022±2836/97/270222±16 $25.00/0/m

Terminal regions of ¯agellin, about 180 NH2 and 100 COOH-terminalresidues, are well conserved and play important roles in polymerizationand polymorphism of bacterial ¯agellar ®laments. About 65 NH2 and 45COOH-terminal residues are disordered in the monomeric form, butbecome folded upon ®lament formation. Taking advantage of the factsthat relatively small segments can be cleaved off these disordered terminiby limited proteolysis, and isolated fragments still form straight®laments, locations of those terminal segments have been mapped out inthe ®lament structure by electron cryomicroscopy and helical imagereconstruction. The fragments studied are F(1-486), F(20-494), F(1-461),F(30-461) and F(30-452). Regardless of the size and terminal side oftruncation, the structures of the ®laments reconstituted from the trun-cated fragments all have identical subunit packing arrangements of theLt-type symmetry. Structural differences compared to the ®lament recon-stituted from intact ¯agellin are found only around the ®lament axis,namely in the inner-tube region, and no obvious changes are observed inthe outer-tube or the outer part of the ®lament. Truncation of only a fewterminal residues results in misfolding of the inner-tube domains andtheir aggregation around the ®lament axis; further truncation reduces thedensities of different parts of the aggregate. The ®lament reconstitutedfrom F(30-461) fragment shows complete disappearance of the densitycorresponding to the inner-tube. When a further nine residues areremoved, the spoke-like features left on the inner wall of the outer-tubebecome signi®cantly smaller. Based on the structures and radial mass dis-tributions of the ®laments obtained, the previous amino acid sequenceassignment to the morphological domains has been con®rmed andfurther re®ned. The roles of terminal segments in the assembly regu-lation, and those of the double-tubular structure in the polymorphicmechanism are discussed.

# 1997 Academic Press Limited

Keywords: bacterial ¯agellum; ®lament structure; electron cryomicroscopy;helical image analysis; domain organization
*Corresponding author
yosue, ERATO, Cellkadoji, Shimogyo,

epartment ofeszpreÂm, Egyetem

st transfer function;nts of ¯agellin, wheremolecular masses;

¯agellin, where #N

ions of fragments.

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Introduction

Bacteria swim by rotating the ¯agellar ®laments.During the straight swimming phase, the ¯agellar®laments form a bundle behind the cell body,where the ®laments are all in a left-handed super-coiled form, each acting as a propeller driven by arotary motor at the base of the ®lament (Berg &Anderson, 1973; Silverman & Simon, 1974; Larsenet al., 1974). Bacteria tumble once every fewseconds to change their swimming direction forchemotactic behavior. The tumbling is triggered by

# 1997 Academic Press Limited

Locations and Roles of Flagellin Termini 223

quick reversal of motor rotation, which produces atwisting torque and transforms the ®laments intoright-handed supercoils momentarily (Macnab &Ornston, 1977). This allows the bundle to fall apartsmoothly, and then the uncoordinated propellingforces change the orientation of the cell quickly.Thus, the dynamic properties of the ¯agellar®laments play an essential role in bacterialchemotaxis. The ¯agellar ®laments can also betransformed into various but distinct polymorphicforms including two straight forms, in response toamino acid replacements in ¯agellin (Leifson, 1960;Iino & Mitani, 1966; Martinez et al., 1968; Asakura& Iino, 1972; Iino et al., 1974; Hyman &Trachtenberg, 1991; Kanto et al., 1991) and tochemical changes in the environment (Kamiya &Asakura, 1976, 1977) as well as to mechanicalforces (Macnab & Ornston, 1977; Hotani, 1982).

The ¯agellar ®lament is composed of a singleprotein, ¯agellin. Flagellin of a wild-type strain ofSalmonella typhimurium SJW1103 consists of 494amino acid residues, and the molecular mass is51.5 kDa (Kanto et al., 1991). The ®lament is a tubu-lar structure composed of 11 proto®laments, nearlylongitudinal arrays of subunits (O'Brien & Bennett,1972), and is built by a self-assembly process(Asakura et al., 1964). The supercoiled forms of the®laments are thought to be constructed from amixture of two distinct subunit conformations of¯agellin arranged in a regular manner (Asakura,1970; Calladine, 1975, 1976, 1978). There are actu-ally two types of straight ¯agellar ®laments havingdistinct helical symmetries; the 11-start helix repre-senting the proto®lament is left-handed in one ofthe two and right-handed in the other, and there-fore they are called the L- and R-type, respectively(Kamiya et al., 1979). The straight ®laments isolatedfrom mutant strains SJW1660 and SJW1655, bothof which are point mutants of SJW1103 (Hyman &Trachtenberg, 1991; Kanto et al., 1991), have the he-lical symmetry of the L- and R-type, respectively,and these two types of ¯agellins can be copolymer-ized to form different types of supercoiled ®la-ments depending on the mixing ratio (Kamiya et al.,1980). Therefore, these two subunit conformationsor modes of packing are thought to represent thetwo that co-exist in the supercoiled ®laments (Ka-miya et al., 1979, 1980).

Structural studies of the ¯agellar ®laments havebeen carried out by electron microscopy(Shirakihara & Wakabayashi, 1979; Trachtenberg &DeRosier, 1987, 1988, 1991; Ruiz et al., 1993,Mimori et al., 1995; Morgan et al., 1995) and X-ray®ber diffraction (Namba et al., 1989, Yamashitaet al., 1995). Particularly with recent advances inelectron cryomicroscopy, such as the use of a cryo-stage cooled to liquid helium temperature to re-duce the radiation damage (Fujiyoshi et al., 1991)and a stable ®eld emission source that increasesimage quality through higher coherence (Tuggel &Swanson, 1985), or developments in helical imageanalysis to average high resolution Fourier data byproper CTF correction (Unwin, 1993, Mimori et al.,

1995; Morgan et al., 1995), the structures of the L-and R-type ®laments have been revealed at around10 AÊ resolution by Morgan et al. (1995) and Mimoriet al. (1995), respectively. The ®lament shows adensely packed core region out to a radius ofabout 60 AÊ , a central channel with a diameter ofabout 30 AÊ , and well-resolved outer parts that ex-tend out to a radius of 115 AÊ . There are two radialregions of high density in the core region, from 15to 30 AÊ (domain D0 as labeled in Figure 7) andfrom 35 to 60 AÊ (D1), where many rod-like featuresextend nearly axially and are packed continuouslyin both axial and lateral directions. These two re-gions form a pair of concentric double-tubularstructures. The two concentric tubes are called theinner and outer-tubes, and are connected by radialspoke-like densities. The outer part consists of twomajor domains: one of them is a vertical columndomain (D2); the other is a projection that extendsout from the lower part of the vertical column(D3). The L- and R-type ®laments show no signi®-cant differences in the overall structure of the sub-units, and the subunit orientation is only slightlychanged according to the different tilt of the proto-®laments. This indicates that the conformationalchange responsible for the polymorphism is smalland seems to occur in the core region of the ®la-ment (Mimori-Kiyosue et al., 1996).

Although ¯agellin is well folded in the ®lament(Aizawa et al., 1990), its terminal regions, residuesfrom 1 to 65 and from 451 to 494, have noordered tertiary structure in the monomeric state(Vonderviszt et al., 1989). Using highly speci®cproteolytic enzymes, terminal segments of varioussizes can be removed from these disordered terminalregions by limited proteolysis. The isolated frag-ments still form straight ®laments, but their stabilityrequires high salt concentrations and the poly-morphic ability is lost (Vonderviszt et al., 1991). Thisindicates that the major part of the disordered termi-ni is not essential for polymerization but importantfor structural stability of the ®laments and confor-mational changes involved in the polymorphism.In our previous report (Mimori-Kiyosue et al.,1996), the structures of straight ®laments reconsti-tuted from fragments missing 19 NH2-terminal re-sidues or eight COOH-terminal residues weredescribed. Regardless of the terminal side of trun-cation and conformational preferences of original¯agellins, the ®lament structures are all in thesame symmetry, which differs from both the L orR-type, and has been named Lt-type. Although theLt-type structure can only be produced by a recon-structive transformation either from the L- or R-type, the local subunit packing arrangements areidentical to the R-type and only slightly but dis-tinctly different from the L-type. Structural differ-ences are found only around the ®lament axis,where the inner-tube structure is destroyed andthe inner-tube domains are misfolded and aggre-gated. No obvious changes are observed in theouter-tube or the outer part of the ®lament.The results indicate that direct interaction of the

Figure 1. Electron micrographs of frozen hydrated¯agellar ®laments. Images are: a, intact ®lament ofSJW1655 (18,000 AÊ underfocus); b, F(20-494) ®lament(50,000 AÊ ); c, F(30-461) ®lament (33,000 AÊ ). Symbols atthe bottom indicate peak positions in the axially pro-jected density distributions with vertical lines and the®lament diameter with a horizontal bar. A pair of longlines represent the outer-tube peak positions in a, b andc; a pair of short thin lines in a are the inner-tube peakpositions; a short thicker line on the ®lament axis in brepresents the aggregated inner-tube domains. Horizon-tal bars represent 230 AÊ .

224 Locations and Roles of Flagellin Termini

termini, most likely in the inner-tube domainand between the subunits, is responsible forproper folding of the inner-tube domains, whichis essential for building the stable ®lamentstructure with polymorphic ability. This obser-vation also shows a strong two-state preferenceof the intersubunit interactions of ¯agellin even inthe absence of the inner-tube structure.

Here, we have analyzed the structures of straight®laments reconstituted from ¯agellin fragmentswith larger sizes of terminal truncation to map outthe locations of the terminal segments. The assign-ment of the terminal amino acid sequence to themorphological domains given in our previous re-ports has been re®ned, and possible roles of theterminal segments in the molecular mechanism ofassembly regulation and polymorphism are dis-cussed.

Results

Structural stability of the filamentsreconstituted from fragments

Puri®ed ¯agellin fragments were polymerized insolutions containing various concentrations of(NH4)2SO4 as described by Vonderviszt et al.(1991). Although the (NH4)2SO4 concentrationcould be slightly reduced once the ®laments arepolymerized, relatively high (NH4)2SO4 concen-trations, from 0.7 to 1.5 M depending on the size ofterminal truncation, were still required to keep the®laments in their stable forms. Therefore, frozenhydrated specimens had to be prepared in thosesolutions. The fragments we studied are F(1-486),F(20-494), F(1-461), F(30-461) and F(30-452), wherethe NH2 and COOH-terminal positions of the frag-ments are indicated in the parentheses. Even in thesolutions containing (NH4)2SO4 at concentrationshigh enough to stabilize the ®laments, their mech-anical stability was signi®cantly lower than thoseof the straight ®laments reconstituted from intact¯agellins. This made it more dif®cult to preparefrozen hydrated ®laments with no apparent bend-ing, and therefore many trials were necessary to®nd good ®lament images in the electron micro-graphs. The fragment F(59-494), which lost most ofthe disordered NH2-terminal region, could also bepolymerized into straight ®laments, and the ®la-ments were observed to be straight and stableunder the dark-®eld microscope, but their mechan-ical stability was too low even in the solution con-taining 1.5 M (NH4)2SO4 to produce images ofnicely straight ®laments when embedded in thevitreous ice ®lm. Therefore, helical image recon-struction of this ®lament was not possible, whileits symmetry was identi®ed to be the same as theothers.

Characteristics of the filament images

Filament images on the electron micrographsshowed basically three types of characteristic

features depending on the size of truncation(Figure 1). The clearest feature is in the image of aF(30-461) ®lament in Figure 1c, in which two paral-lel lines of high density are visible along the ®la-ment axis. These two lines correspond to the sideview of the outer-tube with a diameter of 90 AÊ inthe core of the ®lament. The two parallel lines arealso visible in the image of a F(20-494) ®lament inFigure 1b, although less clearly, and no clear linescan be observed in the image of the intact ®lamentin Figure 1a. In the axial projections of theseimages, the density distributions show differentnumbers of peaks: the intact ®lament shows fourpeaks, corresponding to a pair of inner- and outer-tube densities; the F(20-494) ®lament shows threepeaks, corresponding to a pair of the outer-tubedensity and a slightly lower peak at the ®lamentaxis; the F(30-461) ®lament shows only a pair ofthe outer-tube peaks (peak positions are indicatedat the bottom of Figure 1). These characteristic fea-tures correspond to different structures in the

Figure 2. Computed diffraction patterns of ®laments of intact and truncated ¯agellins. Diffraction patterns in a and bare of the intact straight ®laments representing the two conformational states of ¯agellin subunits: a, SJW1660; b,SJW1655. Diffraction patterns in c to f are of the ®laments of truncated ¯agellins of SJW1655: c, F(20-494); d, F(1-461);e, F(30-461); f, F(30-452). The numbers in the Figure denote the Fourier-Bessel orders of the layer-lines or the startnumbers of the helices to which these layer-lines correspond. In a and b, the axial spacing of the layer-line labeled 1is 26.4 AÊ and 25.6 AÊ , respectively, and in c to f the spacing of the layer-line labeled 3 is 26.5 AÊ . The layer-line labeled3 is not observed in e and f, because the ®rst CTF nodes are located in these regions due to the large amounts ofdefocus.


region inside the outer-tube, as described later inthe density maps deduced by three-dimensionalimage reconstruction. The images simply show theintact inner-tube structure in Figure 1a, misfoldedand aggregated mass of the inner-tube domainsin Figure 1b, and no mass of the inner-tubedomains in Figure 1c, all without any changes inthe outer-tube or the outer part of the ®lament.

Symmetry of the filaments reconstituted fromtruncated flagellins

Figure 2 shows Fourier transforms of all thestraight-type ®laments of truncated fragments weanalyzed, together with those of the L- and R-type®laments as references. The four transforms inFigure 2c to f all show almost identical layer-linespacings and very similar amplitude distributions.They are also indistinguishable from the Fouriertransforms of the ®laments reconstituted with F(1-486) fragments of SJW1103, SJW1660 and SJW1655¯agellins, all of which have the symmetry namedLt-type as reported in our previous work (Mimori-Kiyosue et al., 1996).

Each layer-line is labeled for its Fourier-Besselorder. In Figure 2a and b the order of the axialheights of a closely located pair of layer-lines la-beled ÿ5 and 6 indicates the tilt direction or helicalhandedness of the 11 proto®laments: when thelayer-line labeled ÿ5 is above that labeled 6, the 11

proto®laments are tilted to the left, and vice versa.Since the spacing between those two layer-lines isthe same as the axial height of the layer-line la-beled 11, which arises from the 11 proto®laments,a larger spacing between the two layer-lines corre-sponds to a larger tilt angle of the proto®laments.Although the Fourier-Bessel orders labeled inFigure 2c to f are slightly different from those inFigure 2a and b, the characteristic features indi-cated by relative layer-line positions still hold; theaxial heights of a closely positioned pair of layer-lines labeled ÿ4 and 7 indicate that the 11-strandedproto®laments are tilted to the left with a tilt anglemuch larger than that of the L-type symmetry.

The Fourier-Bessel orders for the Lt-type aredifferent from those for the L- and R-type, becausethe Lt-type structure can only be produced by a re-constructive transformation either from the L- orR-type structure, while the L- and R-type struc-tures are interconvertible to each other by a displa-cive transformation, as described by Mimori-Kiyosue et al. (1996). Here, the word reconstructivemeans that the transformation requires breakingand reforming of subunit bonding with some struc-tural deformation between the two processes,while displacive means only small mutual displa-cement between subunits, as have been used fordescribing the lattice transformation during thecontraction of bacteriophage sheath by Moody(1967).

Figure 3. Layer-line positions pre-dicted from the symmetry andamplitude distributions of averageddata. a, Predicted layer-line pos-itions for the ®laments of truncated¯agellins, which are determined bythe helical symmetry, 368 subunitsper 267 turns, with the pitch of6.46 AÊ for the 1-start helix (Mimori-Kiyosue et al., 1996) and the outerradius of 120 AÊ . Continuous linesrepresent the layer-line positionsused for data collection and ®lledcircles represent the start positionsof layer-lines. The numbers of (n, l),where n is the order of Fourier-Bes-sel component and l is the layer-line order, are indicated for the (3,65) and (2, 166) layer-lines. Overlapof higher order layer-lines does notoccur within the resolution of theFigure. b, Layer-line amplitude dis-tributions of the averaged data ofthe F(20-494) ®lament. The resol-ution is limited to 10 AÊ in both aand b.


Data averaging and evaluation

The procedure of the image analysis is fully de-scribed by Mimori et al. (1995). Structure factors onthe layer-lines were extracted from individualFourier transforms, corrected for CTF, and thenaveraged over many images after aligning thestructures for axial rotation and translation. Thestructural symmetry and repeat distance used inthe analysis were determined by X-ray diffraction(Mimori-Kiyosue et al., 1996). The symmetries ofthe ®laments determined by X-ray and electroncryomicroscopy were very close, indicating highreliability of the data obtained by electron cryomi-croscopy.

Structure factors were extracted from each Four-ier transform on the layer-lines shown in Figure 3aout to 1/7 AÊ ÿ1. In the Lt-type symmetry, overlapof higher Bessel orders does not occur within thisresolution, because the layer-line spacing is verysmall (1/1724 AÊ ÿ1). The layer-line amplitude distri-butions of the averaged data for the F(20-494) ®la-ment are shown in Figure 3b. Due to largeamounts of defocus and mechanical instability ofthe ®laments as compared to the case of intact ®la-ments, the amplitude decay toward higher resol-ution is more prominent.

The statistics of the data averaging are listed inTable 1, in comparison with that for the R-type ®la-ment from SJW1655, whose structure was deduced

at 9 AÊ resolution (Mimori et al., 1995). Althoughthe statistics show that the quality of the data inthe present study is almost as equally high as thatof the R-type ®lament, it is simply because the stat-istics were obtained using data in rather limited re-gions that showed small phase residuals betweenthe near- and far-side transforms. It was importantto limit the data in order to maximize the accuracyof the alignment. The up-down differences of thephase residuals, indicative of how clearly the struc-tural polarity is distinguished, tend to be smallerfor the Lt-type ®laments than for the R-type ®la-ment. This also indicates that the qualities of thedata are not as good as that of the R-type.

After averaging over many images, the structurefactors were evaluated in the same way as de-scribed by Mimori et al. (1995). When Fourier databeyond 1/14 AÊ ÿ1 or 1/12 AÊ ÿ1, depending on the ®-lament, were included in the calculation of the den-sity maps, the maps showed signi®cantly noisierfeatures at the contour level that covers the mol-ecular volume of the ®lament. The estimated®gures of merit (j�Fjj/�jFjj, where Fj is the struc-ture factor of the jth image) were also low in thoseregions (data not shown). Therefore, the resolutioncutoffs for the ®nal density-map calculationswere determined to be 14 AÊ for the F(1-461) andF(30-452) ®laments, and 12 AÊ for all the others.

Table 1. Statistics of alignment of the ®lament images in averaging

Flagellin fragment n R-factor Phase residual (�) U-D difference (�)

1655 ¯agellin 16 0.18 � 0.05 14 � 3 67 � 41103F(1-486) 16 0.18 � 0.06 16 � 4 45 � 81660F(1-486) 15 0.18 � 0.04 16 � 5 51 � 51655F(20-494) 25 0.18 � 0.05 11 � 3 48 � 41655F(1-461) 11 0.17 � 0.04 14 � 2 53 � 11655F(30-461) 15 0.16 � 0.03 13 � 3 54 � 51655F(30-452) 12 0.25 � 0.06 21 � 8 46 � 1

The number of averaged ®lament images are given in the column under n. R-factor and phase resi-dual are the averages of the values between individual images and their average. U-D difference isthe average difference between the above phase residuals for the correct and opposite orientationsof the ®lament. R-factor is de®ned as �jFj ÿ Favgj/�jFavgj, where Fj is the amplitude of individualimages and Favg is that of the averaged image. These values were derived during the alignment ofthe ®laments using selected (5 to 14) layer-lines, including major layer-lines, such as l: 7, 29, 36, 65having the Fourier-Bessel orders n: ÿ11, ÿ4, 7, 3, respectively. The cut-off level of amplitude wasset to 0.1 of the maximum. The values for 1655 ¯agellin are those given by Mimori et al. (1995).


Structure of the outer part of the filaments

The structures of the outer parts of the ®lamentsreconstituted from truncated fragments are allidentical to each other. We have already shown inour previous work that it is so for the F(1-486) ®la-ments from SJW1103, SJW1660 and SJW1655 ¯agel-lins and the F(20-494) ®lament from SJW1655¯agellin (Mimori-Kiyosue et al., 1996). We havenow con®rmed that all the Lt-type ®laments, in-cluding the F(1-461), F(30-461) and F(30-452) ®la-ments, have the same structure on the outersurface of the ®lament. Figure 4a and b show sideviews of the structures of the F(20-494) and F(30-461) ®laments, respectively, representing those ofthe Lt-type ®laments, in comparison with theR-type ®lament in Figure 4c.

Figure 4. Side view of the density maps of various ®lamen494); b, F(30-461); c, intact ¯agellin. Flagellin and fragmentsdata out to 12 AÊ resolution are included for calculating theare very similar, except for the obvious difference of the(R-type ®lament) are tilted to the right (c); those of the ®lamThe scale bar represents 100 AÊ .

There are no obvious changes in the outer shapeof the subunits between the Lt- and R-type ®la-ments; only the subunit orientations are slightlydifferent according to the different tilt angles of theproto®laments. The difference in the subunit orien-tation can be recognized in the directions of theouter projections, which extend out from the lowerpart of the vertical-column domain. The outer pro-jection points down slightly in the R-type ®lamentand points up a little in the Lt-type ®lament(Figure 4). This indicates that the local subunitpacking interactions are very similar to each other.Close comparison of the cylindrical-surface latticeshas actually con®rmed the local lattices of the Lt-and R-type to be identical, while those of the Lt-and L-type are also similar but distinct from eachother (Mimori-Kiyosue et al., 1996).

ts in solid surface representation. The ®lament of: a, F(20-are from SJW1655. A 300 AÊ long segment is shown. The

density maps. The surface appearances of these ®lamentssubunit lattice: the proto®laments of SJW1655 ®lamentents of truncated ¯agellin are tilted to the left (a and b).

Figure 5. Axial view of the density maps of various ®la-ments in solid surface representation. The ®laments of:a, ¯agellin; b, F(1-486); c, F(20-494); d, F(1-461); e, F(30-461); f, F(30-452). All the ®laments were prepared fromSJW1655, except for the F(1-486) ®lament, which wasmade from SJW1660. A 50 AÊ thick cross-section contain-ing 11 subunits in two turns of the 1-start helix isshown. Two maps are shown for each ®lament: ones inthe left column are at a contour level that represents ap-proximately 90% of the correct volume of the ®lament;the others in the right column at contour levels of 1.6,1.4, 1.4, 1.4, 1.6, and 1.7 times those of the left ones, fora, b, c, d, and e, respectively. The maps in the right col-umn show the progressive mass depletion and changes


Structure of the core region

Figure 5 shows end-on views of the ®lamentsreconstituted from intact ¯agellin and fragmentswith progressive truncation. Each ®lament struc-ture is shown at two different contour levels: theone on the left represents the ®lament surface thatcovers about 90% of the correct molecular volume,and the other on the right is contoured at higherlevels to show ®ner details of density distributionsin the core region.

In the core of the intact ®lament (Figure 5a),there is a pair of concentric double-tubular struc-tures composed of the inner-tube from 15 to 30 AÊ

and the outer-tube from 35 to 60 AÊ in radius,which are connected by radial spoke-like densitiesbetween them, and the central channel with a di-ameter of about 30 AÊ , as shown previously (Mi-mori et al., 1995; Morgan et al., 1995). Structuraldifferences in the Lt-type ®laments as comparedwith the intact ®lament are observed only in the re-gion inside the outer-tube. The F(1-486) ®lament(Figure 5b) lacks the central channel; by truncationof eight COOH-terminal residues the inner-tubedomains appear to be misfolded and aggregatedaround the axis, as already described by Mimori-Kiyosue et al. (1996). The F(20-494) and F(1-461)®laments (Figure 5c and d, respectively) showsimilar appearances to the F(1-486) ®lament at thelower contour level (the left column), while themass distributions inside the outer-tube are signi®-cantly different as shown at the higher contour(the right column). Within the aggregated massaround the axis, it appears that the segment com-posed of the 19 NH2-terminal residues is locatednear the axis, and the segment of the 33 COOH-terminal residues is distributed relatively off theaxis. This is also shown in the radial density distri-butions in Figure 6a, in which the density is highernear the axis (dotted line) when the COOH-term-inal segment is cleaved off, whereas the density

in the distribution of the inner-tube domain mass moreclearly than those in the left. Data out to 12 AÊ resolutionare included, except for the F(1-461) and F(30-452) ®la-ments (d and f, respectively), for which the data out to14 AÊ resolution are included. The intact ®lament (a) hasa central channel with a diameter of about 30 AÊ , whilethere is no central channel in the maps of the F(1-486),F(20-494) and F(1-461) ®laments (b, c and d, respect-ively), and aggregated inner-tube domains ®ll in theregion inside the outer-tube. The features that look likethe central channel in the maps on the right side of cand d merely indicate that the density there is relativelylower, but there are no hollow channels as shown in theleft maps. The mass inside the outer-tube is reduced aslarger terminal segments are removed, and ®nally alarge central hole with a diameter a little larger than60 AÊ appears in the maps of the F(30-461) and F(30-452)®laments (e and f, respectively), showing a very goodcorrelation between the sizes of removed segments andthe mass reduction in the maps, which is also demon-strated in Figure 6 and Table 2. The scale bar represents100 AÊ .

Figure 6. Radial density distribution pro®les of the ®la-ments of truncated ¯agellins in comparison with that ofthe intact ®lament. In both a and b, continuous linesrepresent the density distribution of the intact ®lamentof SJW1655, and broken lines represent that of the F(1-461) ®lament. The other lines in a are: dot-dashed line,F(1-486) ®lament; broken line, F(20-494) ®lament; and inb: dot-dashed line, F(30-461) ®lament; broken line, F(30-452) ®lament. All the pro®les show close similarity inthe region outside a radius of 35 AÊ , which correspondsto the outer-tube (D1) and the outer part (D2 and D3),while differences are found only inside this radius,where the mass reduction is well correlated with thesizes of terminal truncation as shown in Table 2.


peak is located relatively off the axis (broken line)when the NH2-terminal segment is removed. Incontrast, the F(30-461) and F(30-452) ®laments(Figure 5e and f, respectively) show a large holeproduced by complete loss of the inner-tube do-mains, while the outer-tube remains unchanged.This is clear also in Figure 6b, where the radialdensity peak at around 25 AÊ (continuous line),which corresponds to the inner-tube, is almostcompletely removed (broken and dot-dashedlines).

The three ®laments, F(1-486), F(20-494) and F(1-461) (Figure 5b, c and d, respectively), have thespoke-like connections between the outer-tube andthe aggregated mass inside it at the same positionrelative to the outer domain as in the intact ®la-ment (Figure 5a). Even when the mass correspond-ing to the inner-tube domains is completelyremoved, the F(30-461) ®lament (Figure 5e) has thesame spoke-like features remaining on the innerwall of the outer-tube, which look more likebumps exposed to the solvent in the large centralhole. The round feature of the spoke may be pro-duced by rather limited resolution of the map orby the inner part of the spoke-like structure re-

moved. In any case, the map in Figure 5e indicatesthat the spoke-like structure is fairly rigid, andabout 30 residues at each terminus, in total 60residues, construct the inner-tube structure. Thespoke-like structures become signi®cantly smallerwhen nine residues are further removed from theCOOH terminus of F(30-461), as shown in thestructure of the F(30-452) ®lament (Figure 5f). Thisindicates that the nine residues, from 453 to 461,compose the main part of the spoke. This is alsoshown in the radial density distribution (Figure 6b),in which the pro®les of the F(30-461) and F(30-452)®laments have a signi®cant difference in the radialregion from 20 to 35 AÊ (dot-dashed line for F(30-461) and broken line for F(30-452)).

Note that all the pro®les of the radial densitydistributions shown in Figure 6 are close to eachother in the region outside a radius of 35 AÊ , indi-cating that there are no structural changes in thisregion, as also shown in the side view of the ®la-ments in Figure 4 and in the end-on view inFigure 5; small deviations in this region merelyrepresent the noise level of the Fourier data havingrelatively low quality at high resolution. These con-®rm the overall folding of ¯agellin proposed in ourprevious studies (Mimori et al., 1995; Yamashitaet al., 1995; Mimori-Kiyosue et al. 1996), in whichthe outer part of the ®lament and about 80% of theouter-tube have been assigned to a part of the ¯a-gellin sequence corresponding to the F40 fragment,which is from Asn66 to Arg450, and therefore notmodi®ed by terminal truncation. The mass insidethe outer-tube is reduced more as larger terminalsegments are removed, and ®nally a large centralhole with a diameter a little larger than 60 AÊ ap-pears in the maps of the F(30-461) and F(30-452)®laments, showing a very good quantitative corre-lation between the sizes of removed segments andthe reconstructed maps, as also demonstrated bythe quantitative mass analysis of those maps de-scribed in the next section.

Mass analysis of the subunit domains

Based on the radial density distributions of the®laments, the molecular masses were calculated forthree radial segments corresponding to the mor-phological domains identi®ed in the structure: theinner-tube (D0) with the central channel region,0 � 33 AÊ ; the outer-tube (D1), 33 � 57 AÊ ; and theouter part (D2 and D3), 57 � 117 AÊ (Figure 7). Theresults are summarized in Table 2. The masses ofthe outer-tube and the outer part are more or lessconstant, from 17 to 18 kDa and from 25 to 27 kDa,respectively, in all the ®laments. Only the mass in-side the outer-tube (the second column of Table 2)shows systematic reduction according to the num-ber of amino acid residues removed from the ter-mini, indicating that the terminal regions roughlyfrom 1 to 30 and 460 to 494 construct the inner-tube, and about ten residues from 450 to 460occupy the spoke region.

Figure 7. Assignment of the subunit domains identi®ed in the map to the amino acid sequence. a, Contoured map ofthe ¯agellar ®lament. A 50 AÊ thick cross-section is shown in axial view, where four domains, D0 to D3, are labeled inthe two subunits neighboring along the left-handed ®ve-start helix. The resolution of the map is 12 AÊ . The subunitboundary lines in the core region (broken lines) are arbitrarily drawn using radial lines, because the subunits are sowell packed in the core region that the boundary cannot be identi®ed unambiguously at this resolution. The scale barrepresents 100 AÊ . b, Positions on the sequence of the major proteolytic fragments (F40 and F27), predicted secondarystructures, assigned domains and positions of point mutations that affect the polymorphic form of the ®lament. Pre-dicted secondary structures are: open box, a-helix; closed box, b-sheet; small open box, turn (Vonderviszt et al., 1990).The shaded bars represent the conserved a-helical coiled-coil forming regions (Yamashita et al., 1995). The 11 sites ofmutations determined by Kanto et al. (1991) are indicated with shaded circles and the polymorphic form of eachmutant is labeled by a letter right below each circle: L, the L-type straight; R, the R-type straight; u, curly; o, coiled.c, Sequence positions of ¯agellin fragments. The mutation sites of the L- and R-type ®laments (SJW1660 and SJW1655,respectively) are indicated with shaded circles and the replaced amino acid is also indicated above each circle. Thepolymorphic forms of the resulting ®laments are indicated on the right side. All the straight ®laments formed byterminally truncated fragments have been identi®ed to have the Lt-type symmetry, and most of these ®lament struc-tures have been analyzed in the present study and by Mimori-Kiyosue et al. (1996).


Table 2. Molecular mass of domains from radial densitydistributions

Radial range (AÊ )Flagellin fragment 0±33 33±57 57±117

1655 ¯agellin 7.7 18.0 25.81103F(1-486) 7.1 17.2 26.31660F(1-486) 6.1 17.2 27.31655F(20-494) 5.9 16.8 26.21655F(1-461) 5.8 18.2 24.41655F(30-461) 1.4 17.5 26.01655F(30-452) 0.4 18.4 25.0

The molecular masses of the domains were calculated from theradial density distributions shown in Figure 5 and are givenin kDa units. Domains are: the radial range from 0 to 33 AÊ , D0;from 33 to 57 AÊ , D1; from 57 to 117 AÊ , D2 and D3 (the outerpart). For the CTF correction, the ratio of 0.047 was used forthe amplitude and phase contrast, except that the ratio of 0.055was used for the F(30-452) ®lament to adjust the density levelat the axis of the ®lament to be the solvent level. The boundarypositions of the radial segments are almost the same as thoseused in the X-ray equatorial analysis (Yamashita et al., 1995),but the numbers are simply multiples of the 3 AÊ grid unit inthis case.


Discussion

Quality of the Fourier data

Mainly two factors made it dif®cult to recordgood images of frozen-hydrated ®laments reconsti-tuted from truncated ¯agellins. One of them is therelatively high salt concentrations needed to keepthe ®lament structure stable, which resulted in alow image contrast, and therefore relatively largeamounts of defocus were required to obtain en-ough contrast for image analysis. This then re-sulted in a rather large decay in the amplitudedistributions and an increase in the phase-reversalfrequency of the phase contrast transfer function(CTF) toward high resolution as compared withthe case of intact ®laments, and therefore errors inthe CTF corrections would be large. The other fac-tor is some disorder in the ®lament images due tothe intrinsic instability of the ®laments. This is re-¯ected in broadening of layer-line widths or dif-fuse amplitudes around layer-lines. As a result, theaveraged amplitudes show a marked decline in thesignal to background-noise ratio beyond 1/20 AÊ ÿ1,which naturally reduces the effective resolutionand quality of the map, as compared to those ofthe intact ®laments.

As described in Results, phase residuals in thestatistics obtained during the alignment of many ®-laments for averaging show values as small asthose of the intact ®laments, but this is simply be-cause limited regions of layer-lines are included inthe alignment procedure to maximize the ®tting ac-curacy. Up-down differences of the phase residualsthat determine the polarities of the ®laments actu-ally show somewhat worse statistics for ®lamentsof truncated ¯agellins than those for the intact ®la-ments. The quality of the Fourier data is mostclearly demonstrated in the density maps (Figure 5)and radial density distributions (Figure 6), where

the differences in the outer part of the ®lamentsrepresent the noise level. Although they both showa certain amount of noise, it is small enough forthe deduced density distributions to reveal withsuf®cient reliability that the structural differencesbetween the ®laments are only inside the outer-tube. It is worth noting that even at this resolutionremoval of only nine residues (453 to 461) can bedetected fairly quantitatively.

Sequence assignment tomorphological domains

The amino acid sequence of ¯agellin has beenroughly assigned to the morphological domains of¯agellin subunit identi®ed in the map of image re-construction at 9 AÊ resolution, based on the vo-lumes of the domains (Mimori et al., 1995; Mimori-Kiyosue et al., 1996) and more precisely on themasses of the domains (Yamashita et al., 1995), to-gether with other structural information available.

The whole sequence of 494 amino acid residuesis divided into two parts, the terminal regions,from Ala1 to Arg65 and from Ser451 to Arg494,which have no ordered tertiary structure in themonomeric state (Vonderviszt et al., 1989), and therest, the 40 kDa fragment (F40), which consists ofthree compact domains (Vonderviszt et al., 1990).A central part of F40 from Tyr178 to Arg422 formstwo compact domains of 24.7 kDa (F27), which isthe most stable portion against proteolysis, and theremainder forms the third domain composed oftwo discontinuous sequences from Asn66 toLys177 and from Ser423 to Arg450 (Vondervisztet al., 1989, 1990). F27 contains the sequence knownto be exposed on the ®lament surface(Trachtenberg & DeRosier, 1988; Fedorov et al.,1988), and the molecular mass corresponds to thatof domains D2 and D3 (Mimori et al., 1995;Yamashita et al., 1995). The structure of the ¯agel-lar ®lament isolated from a straight-type mutant ofSJW46, whose ¯agellin lacks 89 amino acid resi-dues from Ala204 to Lys292, has actually shownthat this region contributes to the outermost sub-domain, the outer two thirds of D3 (Y.M.-K., I.Yamashita, Y. Fujiyoshi, S. Yamaguchi & K.N., un-published results). Therefore, the third domain ofF40 with a molecular mass of 15.3 kDa has been as-signed to occupy about 80% of D1, and terminalregions of 11.5 kDa that are disordered in themonomeric state have been assigned to D0 andabout 20% of D1. This assignment is also consistentwith the domain masses estimated from the radialdensity distributions obtained in this study, assummarized in Table 2.

Now, with more detailed information availablein the present study, the previous assignment iscon®rmed, and even ®ner assignment can be givenin the terminal regions (Figure 7). The structure ofthe F(30-461) ®lament indicates that the removedparts, from 1 to 29 and from 462 to 494, togetherconstruct the inner-tube structure, while the F(30-452) ®lament reveals that the residues from 453 to


462 contribute to a major part of the spoke-likestructure. The region assigned to the spoke is pre-dicted to be an a-helix (Figure 7b), suggesting thatthe spoke is an a-helical rod structure, although itis possible that a few residues in the NH2-terminalregion connecting D0 to D1 also contribute to partof the spoke density.

The folding of ¯agellin subunit in the ®lamentcan be traced as follows (Figure 7): about 30 NH2-terminal residues fold into an a-helical structure inD0 (the inner-tube); then the chain goes out to D1(the outer-tube), where about 150 residues foldmainly into a-helical coiled-coils and some b-struc-tures; the chain goes further out, passing throughD2 (the vertical column), ®rst into D3 (the outerprojection) and then back to D2, where the chainfolds into mainly anti-parallel b-sheets, until itreaches to residues around 420; the chain comesback to D1 and about 30 residues contribute againto the a-helical coiled-coils; the residues fromaround 450 to 460 form an a-helix to be the majorpart of the spoke; then ®nally the COOH-terminalsegment of about 30 residues folds into an a-helicalstructure in D0 and interacts with the NH2-term-inal segment of the neighboring subunit.

Intersubunit terminal interactions for filamentformation and stability

Flagellin fragments with terminal truncations ofvarious sizes can be polymerized into straight ®la-ments (Vonderviszt et al., 1991), and all the ®la-ments have been found to have the same helicalsymmetry, the Lt-type. Polymerization and stab-ility of those ®laments require relatively high con-centrations of (NH4)2SO4, where the concentrationdepends on the number of amino acid residues re-moved and the terminal side of truncation (Von-derviszt et al., 1991). Removal of COOH-terminalsegments mainly affects the polymerization ability,while the lack of NH2-terminal portions signi®-cantly reduces the ®lament stability as well, and itstruncation size dependence is much higher thanthat observed by COOH-terminal truncation. TheF(30-461) and F(30-452) fragments, deprived oflarge segments at both termini, and the F(59-494)fragment, lacking most of the disordered NH2-terminal portion, can be polymerized and stabil-ized only at very high salt concentrations. TheF(37-450) fragment can be polymerized only atvery low yield because of the competing micro-crystallization, while the F(53-450) fragment formsonly microcrystals.

These behaviors of the fragments can be ex-plained by the ®lament structures deduced in thepresent study, which give important insights intothe role of the disordered terminal regions in theformation of the ®lament structure. As alreadypointed out in our previous work (Mimori-Kiyosueet al., 1996), the interaction of the termini is likelyto be intersubunit, because the inner-tube structureis not formed and the inner-tube domains are mis-folded when a small terminal segment is removed

from either side. The resulting ®lament structure,the Lt-type, has a signi®cantly deformed subunitlattice at the inner-tube radius as compared to thatof intact ®laments, indicating a loss of the correctinner-tube domain interactions between the neigh-boring subunits (Namba & Vonderviszt, 1997).This is why the stabilities of the Lt-type ®lamentsare lower than those of the intact ®laments. How-ever, even when the inner-tube domains are mis-folded by small terminal truncation, intersubunitaggregations of those domains are formed in a par-tially ordered way around the axis to contribute tothe stability of the ®laments. This is probably whythe stability of the ®laments requires relativelylower salt concentrations than those necessary forpolymerization. The F(20-494) ®lament missing 19NH2-terminal residues requires higher salt concen-tration (0.3 M (NH4)2SO4) for its stability than theF(1-461) ®lament lacking 33 COOH-terminalresidues, probably because the distributions ofthe terminal segments are quite different in theaggregated inner-tube domains, as indicated inFigures 5c, d and 6a, making the amount of inter-actions less for the former. The F(30-461) and F(30-452) ®laments missing both terminal segments re-quire high salt concentrations for their stabilities(0.5 M and 0.6 M, respectively), because the inner-tube domain interactions are all gone. The F(59-494) ®lament requires a salt concentration almosttwice higher (1.0 M) than those, since it lacks mostof the disordered NH2-terminal portion and there-fore some part of the outer-tube structure isremoved. This also demonstrates that the role ofthe outer-tube structure is more important forthe structural stability than that of the inner-tubestructure.

For various sizes of truncation at either side ofthe termini, the salt concentrations required for ef-®cient polymerization are fairly uniform (around0.5 M (NH4)2SO4), as far as the sizes of truncationup to around 40 residues. However, when bothterminal segments are removed, the salt concen-trations required are signi®cantly increased (0.8 to0.9 M), which implies that the presence of eitherterminus can help to stabilize the initial subunitinteractions that start the ®lament formation. Anextremely high salt concentration (1.4 M) is necess-ary to polymerize the F(59-494) fragment,suggesting a very important role of the segmentfrom around 40 to 60 in the initial subunit inter-actions forming the outer-tube structure.

From the maps of the F(30-461) and F(30-452) ®-laments, the COOH-terminal portion disordered inthe monomeric state does not appear to contributesigni®cantly to the outer-tube structure. However,the polymerization ability is severely reducedwhen the whole disordered COOH-terminal regionis removed as well as large NH2-terminal segments(F(37-450) and F(53-450)). This suggests that asmall segment in the very inner part of the disor-dered COOH-terminal region, probably a few resi-dues around 450, and the inner part of thedisordered NH2-terminal region, namely the


segment from 40 to 65, are together involved incompletion of the outer-tube domain folding, andtherefore responsible for regulating initiation andstabilization of the outer-tube domain interactionsbetween the subunits.

The structures of the misfolded and aggregatedinner-tube domains found in the F(1-486), F(20-494) and F(1-461) ®laments seem to show thatthese are not completely random aggregations buthave some symmetry, because the maps of thoseportions clearly show signi®cant distributions ofmasses that follow the symmetry of the ®lamentsthat has been imposed in helical image reconstruc-tion. The structural details of the terminal inter-actions in the aggregations, however, remain to beseen by higher resolution structure analysis.

Sites of mutations responsible forpolymorphic forms

Mutants of Salmonella typhimurium have beenfound to have various polymorphic forms of ¯agel-lar ®laments, such as straight, curly, semi-coiled,coiled and small amplitude. From ¯agellins of 17such mutant strains studied, 15 independent mu-tations at 11 independent sites have been identi®ed(Kanto et al., 1991). These sites are summarized inFigure 7b. There are ®ve L-type straight mutants atfour sites (F53V, D107E, R124A, R124S, G426A)and one R-type straight mutant (A449V), and allthese sites are located in the outer-tube domain(D1). Three curly mutations (A427V, N433D,A449T) and one f1-type mutation (G426A/A449T)are also located in the outer-tube. One curly mu-tation (A414V) is located very close to the outer-tube, suggesting that this portion is probably partof the coiled coil. On the other hand, all three coiledmutations at two sites (Q472L, Q481L, Q481S) arefound in the inner-tube domain (D0). One curlymutation (D313Y) is the only site found in theouter part of the ®lament (D2 or D3). Thus, poly-morphic mutations are concentrated in the inner-and outer-tubes, while the domains of the outerpart may also have some in¯uence on the poly-morphic behavior of the ®lament, as suggested bypolymorphic behaviors of mutant ¯agellins withlarge central deletions (Yoshioka et al., 1995). TheCOOH-terminal region has a higher concentrationof mutation sites than the other. There seems to bea strong correlation between the polymorphictypes of the ®laments and the domains of mutationsites, although there is no simple way of interpret-ing this in terms of the subunit interactions at themoment. In any case, these mutation sites andpolymorphic forms indicate that the inner- andouter-tubes are major parts responsible for the ®neregulation of the polymorphic shape. Althoughboth domains are important, the outer-tube seemsto be mainly responsible for the two distinct sub-unit conformations, since the coiled conformationcan be produced with the outer-tube domain inter-actions alone when fragments missing the inner-tube domain are polymerized onto normal seeds.

This will be discussed in more detail in the last sec-tion of Discussion.

Filaments with unexpected symmetries

During the analysis of various types of straight¯agellar ®laments, we found three unusual ®la-ments having unexpected types of symmetries: oneLt-type ®lament reconstituted from intact ¯agellinof SJW1655, which usually forms the R-type; oneR-type ®lament from the F(30-461) fragment ofSJW1103 and one R-type from the F(1-486) frag-ment of SJW1655. In all cases, the symmetry waspreserved through the whole length of the ®la-ments, and the inner-tube domains were misfoldedand aggregated or missing in the same way as allthe other Lt-type ®laments reconstituted from frag-ments with terminal truncation.

It is reasonable to expect that the Lt-type can beformed by intact ¯agellins if somehow in the nu-cleation process the termini failed to interact prop-erly to form the inner-tube structure; once thepolymerization is nucleated in this way the struc-ture grows with the same symmetry because theLt-type can be formed only by a reconstructivetransformation from the R-type (Mimori-Kiyosueet al., 1996). Although the helical lattices of the R-and Lt-type ®laments are almost identical at the ra-dius of the outer-tube, the lattices at the inner-tubeare quite different, showing about 8 AÊ axial displa-cement of the subunits neighboring along the one-start helix, which would keep and propagate thefailure of the terminal interaction all the waythrough the length of the ®lament.

In the case of terminally truncated fragments,the R-type ®laments can be polymerized even inthe absence of the inner-tube structure, once theouter-tube lattice is formed accidentally in the R-type symmetry in the nucleation process, becausethe subunit conformations of the R- and Lt-typeare very similar with only slight subunit rotationsinvolved in the difference between the two(Mimori-Kiyosue et al., 1996).

The presence of ®laments with those exceptionalsymmetries demonstrates that once the polymeriz-ation nuclei are assembled in a certain symmetry,added subunits follow the symmetry. The structureof the ¯agellar ®lament is designed in such a waythat this strong cooperativity in the conformationaldetermination along the ®lament is one of its in-trinsic structural properties that play an importantrole in its polymorphic transitions.

Formation of coiled filament

In the case of the F(1-486) fragment of SJW1103¯agellin, which is produced by carboxypeptidaseA and B treatment, coiled ®laments were some-times observed to co-exist with the straight ®la-ments, while the probability of ®nding themvaried from preparation to preparation. A pre-liminary mass spectrometry measurement of afragment sample that showed a signi®cant ten-


dency of forming coiled ®laments revealed thatthe digestion was incomplete and a large portionof the fragments had the COOH terminus atGly487 instead of Pro486. This suggests that theF(1-487) fragments can form coiled ®laments aswell as Lt-type straight ®laments, while the F(1-486) fragments form mostly Lt-type ®laments.The axial projections of short segments of thecoiled ®laments show three peaks, in the sameway as the Lt-type ®lament of the F(1-486) frag-ment (Figure 1b), indicating that the inner-tubedomains are misfolded and aggregated aroundthe ®lament axis. And yet, in Fourier transformsof short segments of the coiled ®laments, the axialpositions of layer-lines indicate that the proto®la-ments have a very small tilt, showing that thesubunit lattice is identical to that of coiled ®la-ment formed by intact ¯agellins.

Terminally truncated fragments are known toform coiled ®laments when normal ®laments areused as seeds for polymerization (Vonderviszt et al.,1991). In those coiled ®laments, the fragments areassembled in the intact lattice as opposed to the Lt-type lattice. So, a plausible explanation of the pre-sent observation would be that, in the nucleationprocess, the F(1-487) fragment of SJW1103 ¯agellincan still form normal seeds at a signi®cant prob-ability, and then most of the fragments polymerizeonto the normal seeds to form coiled ®laments.When the proteolytic digestion is complete andthere are only the F(1-486) fragments in solution,the formation probability of normal seeds is largelyreduced and therefore the Lt-type straight ®la-ments are mostly found in the polymerization sol-ution.

Other fragments of ¯agellin from SJW1103 withlarger sizes of truncation occasionally show for-mation of coiled ®laments, but neither fragmentsfrom SJW1660 nor SJW1655 do so. This observationindicates that even if the inner-tube structure is notproperly formed, SJW1103 ¯agellin still shows arelatively strong tendency to form the intact latticerather than the Lt-type lattice, which results in thecoiled ®lament formation.

Distinct roles of the inner- and outer-tubein polymerization

The structures of the Lt-type ®laments presentedin this study as well as by Mimori-Kiyosue et al.(1996) have shown that the inner-tube structure isquite independent in its folding from the outer-tube structure. The inner and outer-tubes are con-nected to each other through the spoke-like struc-tures (Mimori et al., 1995; Morgan et al., 1995), andthere is a gap of at least a few aÊngstroÈm units be-tween them (Yamashita et al., 1995). The inner-tubedomains can be misfolded and aggregated, or evencompletely removed, without disturbing the struc-ture of the outer-tube. This strongly suggests dis-tinct roles of the two structures in polymerizationand polymorphism.

Based on the chain folding of the ¯agellin sub-unit in the ®lament described in Figure 7, the F40part of ¯agellin, which is already well folded in themonomeric state, corresponds to D2, D3 andalmost 80% of D1. This means that only 20% of theouter-tube domain has to fold up to form the baseof the ®lament structure in the initial stage of pol-ymerization. Therefore, it is likely that the initialprocess of polymerization involves subunit inter-actions between the preformed portions of theouter-tube domains, which would trigger the fold-ing of the remaining 20%, residues from around 30to 65, to complete the outer-tube structure, whichthen stabilizes the spoke structures. Then the inter-action of the termini causes the correct folding ofthe inner-tube domains into the inner-tube struc-ture. The basic structure of the ¯agellar ®lamentcan be formed with the interactions in the outer-tube alone, but the inner-tube structure modi®esthem from the relaxed Lt-type interactions to theR-type ones by putting some structural strainsthrough the spoke-like connections and stabilizesthem, as discussed later.

Flagellar-®lament assembly in vivo occurs at thedistal end of the ®lament (Iino, 1969). The assem-bly process is regulated so that it occurs only atthe distal end of the ®lament even in vitro(Asakura et al., 1964). Spontaneous polymeriz-ation is observed neither in vivo nor in vitrounder physiological conditions; it occurs only atrelatively high salt concentrations (Ada et al.,1963; Wakabayashi et al., 1969). The mechanismfor this regulation of ®lament formation can bedescribed as follows. In vivo, ¯agellin moleculesare synthesized in the cell body, and transportedthrough the central channel of the ®lament to thedistal end, where polymerization occurs. Flagellinsubunits composing the distal end of the ®lamentare thought to be well folded. Newly transported¯agellin subunits recognize those folded struc-tures to bind and become folded themselves.Since 80% of the outer-tube domain is alreadyfolded in the monomeric state, a large portion ofthe outer-tube domain bonding can easily beformed by having multiple bonding partnersproperly oriented and positioned. This could pro-mote folding of the small disordered terminal re-gions in the outer-tube domain and then foldingof the whole inner-tube domain by increasing thechance of terminal interactions. However, whenonly monomers are present in a solution ofphysiological salt concentration, no polymeriz-ation occurs because pairwise interactions be-tween the outer-tube domains alone would notbe stable enough to keep the proper orientationand mutual positioning to promote the folding ofthe terminal regions and to support the terminalinteractions. The process is highly cooperative.Thus, for the ef®cient regulation of assembly, it isimportant to have only a small portion of theouter-tube domain to be disordered while havingthe whole inner-tube domain disordered.


Distinct roles of the inner- and outer-tubein polymorphism

Polymorphic ability is lost in the Lt-type®lament, suggesting important roles of theinner-tube structure in ¯agellar polymorphism.Although the polymorphic ability is lost in theLt-type ®laments, in which the subunit inter-actions in the outer-tube strongly prefer to be ofthe R-state (Mimori-Kiyosue et al., 1996), ¯agellinfragments can be polymerized into coiled formwhen normal seeds are used as polymerizationnuclei (Vonderviszt et al., 1991), and both of thetwo states co-exist in coiled ®lament. Those coiled®laments formed by fragments should have theintact subunit lattice identical to that of coiled ®la-ments formed by intact ¯agellin. Thus, the differ-ent behaviors of the subunit conformations in theLt-type and coiled ®lament appear to be due totheir different subunit lattices, which can betransformed into each other only by a reconstruc-tive procedure, requiring complete break and re-formation of the subunit bonding. This impliesthat in the intact lattice, even in the absence ofthe inner-tube structure, the outer-tube domaininteractions switch into the L-state as well asstaying in the R-state under the in¯uence of ex-ternal perturbations, such as those imposed bythe normal ®lament structure of the polymeriz-ation nucleus.

Flagellin fragments from SJW1103 and its mu-tants, SJW1660 and SJW1655, all form Lt-type ®-laments, and both mutation sites are located inthe outer-tube domain as shown in Figure 7c,indicating that the mutations in the outer-tubedo not affect the subunit lattice of the outer-tube structure in the absence of the inner-tubestructure. Therefore, the Lt-type subunit packingis considered to be the most relaxed state ofthe outer-tube interactions without having anystrains from the terminal interactions in theinner-tube (Mimori-Kiyosue et al., 1996). The Lt-type lattice being almost identical to the R-type,while having largely different proto®lament tilt,implies that the 11 proto®laments that form theouter-tube can be twisted about the ®lamentaxis over a wide range of angles when theinner-tube domain interactions are missing. Thistwisting ¯exibility is actually a necessary featureof the ®lament structure to form the observedsupercoils, but the twist angle in the Lt-type isfar out of the range observed in the poly-morphic forms, which is between those of theL- and R-type. This suggests that the main roleof the inner-tube structure is to restrain thetwist angle of the proto®laments in the outer-tube within a narrow range centered somewherebetween those of the L- and R-type. This isthought to be the way by which a speci®cform of the intersubunit terminal interactions inthe inner-tube relative to the outer-tube domainmakes the ®lament stable in a speci®c poly-morphic form.

Characteristics of the conformational changesinvolved in polymorphism

Many lines of evidence have strongly indicatedthat the double-tubular structure in the core of the®lament is composed of axially aligned a-helicalcoiled coils: a Patterson analysis of the layer-lineintensity distributions obtained by X-ray ®ber dif-fraction indicates the presence of a-helical bundlesin the core region (Namba et al., 1989); densitymaps of the ®lament deduced by helical image re-construction show many rod-like densities alignednearly parallel to the ®lament axis in the core(Mimori et al., 1995); secondary structure andcoiled-coil predictions (Vonderviszt et al., 1990;Yamashita et al., 1995) indicate mostly a-helicalstructures in the terminal regions, which have beenassigned to the core region (Mimori et al., 1995;Yamashita et al., 1995), and are proved in the presentstudy to form the inner- and outer-tube structure.Comparison of the ®lament structures of the L- andR-type have shown that there is only a small changein the subunit orientation and the orientations rela-tive to the proto®lament are almost the same. Closecomparison of the two lattices shows that only asmall amount of sliding of the subunits along theneighboring proto®lament is involved in the poly-morphic switching (Mimori-Kiyosue et al., 1996).Implications of these results are that a small butdistinct distance of relative sliding between theaxially aligned a-helical bundles is the main featureof the switching in the subunit conformation andpacking responsible for the polymorphism.

When a speci®c species of ¯agellin forms the ®-lament structure, relative dispositions of the inner-and outer-tube domains in the local subunit in-teractions determine the proto®lament tilt, asdiscussed in the previous section. However, at thedetermined tilt, the tubular structure made of the11 proto®laments cannot necessarily be closed if allthe proto®laments are in one of the two states. Clo-sure of the tube can be achieved only by adjustingthe number ratio of the proto®laments in the twostates. Although the free energy is increased byhaving two conformational states in one structure,and by having elastic strains from bending of thetube caused by the L-state strand being slightlylonger than the R-state, a large negative free en-ergy is gained by interproto®lament bonding at theclosure of the tube. This makes the ®lament struc-ture stable in a supercoiled form with a speci®c he-lical pitch and diameter, which are determined bythe proto®lament tilt and the length difference ofthe proto®laments in the L- and R-states. Thispolymorphic mechanism is described in more de-tail by Namba & Vonderviszt (1997), who also givea plausible explanation as to how polymorphictransitions occur.

Polymorphic forms of the ®lament can be trans-formed from one into others by external pertur-bations, such as a twisting torque produced by aquick reversal of the ¯agellar motor rotation orchanges in the electrostatic interactions at the sub-


unit interfaces caused by changes in the solventpH and salt concentration. The twisting torquedirectly changes the proto®lament tilt. Ionic per-turbations could modify the relative disposition ofthe inner- and outer-tube domains, which thenchanges the proto®lament tilt. The change in theproto®lament tilt forces the number ratio of the L-and R-state proto®laments to alter and transformsthe ®lament into other polymorphic forms.

Materials and Methods

Preparation of filaments of flagellin fragments withterminal truncation

Flagellins were isolated from a wild-type strain,SJW1103, and two mutant strains, SJW1660 andSJW1655, and puri®ed as described by Vonderviszt et al.(1989). Fragments of ¯agellins missing terminal segmentsof various sizes within the disordered terminal regionswere prepared by limited proteolysis of monomeric ¯a-gellins and puri®ed by ionic-exchange column chroma-tography (Vonderviszt et al., 1991). The F(1-486)fragments, which lack the eight COOH-terminal resi-dues, were prepared from all three types of ¯agellin,while all the other fragments, F(20-494), F(1-461), F(30-461), F(30-452) and F(59-494), were made from SJW1655¯agellin. Filaments were reconstituted by adding(NH4)2SO4 to solutions containing the truncated ¯agel-lins to appropriate ®nal concentrations (0.2 to 1.5 M), asdescribed by Vonderviszt et al. (1991).

Electron cryomicroscopy

The experimental procedure of electron cryomicro-scopy, including the preparation of frozen hydratedsamples, is fully described by Mimori et al. (1995). Elec-tron micrographs were taken at a magni®cation of50,000� with a JEOL JEM-3000SFF (300 kV) electronmicroscope equipped with a cryo-stage cooled to 4 Kwith liquid helium (Fujiyoshi et al., 1991) and a ®eldemission electron source (Tuggel & Swanson, 1985). Theimages were recorded over a wide range of underfocus,from 1 to 5.5 mm. Relatively strong underfocus wasnecessary to record images at high enough contrast toobserve major layer-lines in the Fourier transforms, be-cause the density contrasts between the ®laments andsolvents were signi®cantly lower in the case of high saltconcentration required to stabilize the ®laments reconsti-tuted from fragments with relatively large terminal trun-cation.

Helical image reconstruction

The helical image reconstruction procedure was fol-lowed in the exactly same way as fully described byMimori et al. (1995), including correction for the contrasttransfer function for phases and amplitudes. Althoughthe intervals between the layer-lines are relativelynarrow in the Fourier transform of the Lt-type ®lamentas shown in Figure 3a and the intervals became evennarrower when relatively short lengths of the ®lamentimages were used to calculate Fourier transforms, theamplitude distributions on the neighboring layer-linesshowed a distinct pattern in the averaged layer-line data.This indicates that the layer-line structure factors werecollected without signi®cant in¯uence of diffuse ampli-

tudes from closely located layer-lines. Fourier data out to12 AÊ resolution were included for calculating the densitymaps and radial density distributions in all cases, exceptthat data out to 14 AÊ resolution were used in theF(1-461) and F(30-452) ®laments because of the lowerquality of the data at high resolution.

Acknowledgements

We thank K. Murata and Y. Fujiyoshi for technicalhelp in electron cryomicroscopy, H. Taniguchi for pre-liminary mass spectrometry of the F(1-486) fragments, K.Yonekura and C. Toyoshima for helical image recon-struction programs. We also thank I. Yamashita for valu-able discussions and comments, and T. Nitta and F.Oosawa for support and encouragement. This work waspartially supported by Special Coordination Funds of theScience and Technology Agency of Japan to K.N. andthe Hungarian OTKA T006307 grant to F.V.

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Edited by M. F. Moody

(Received 4 February 1997; received in revised form 18 April 1997; accepted 22 April 1997)

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