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Biology

ANNUAL REPORT OF OSAKA UNIVERSITY—Academic Achievement—2004-2005

Structure of the Bacterial Flagellar Hook and Implication for the Molecular Universal Joint MechanismPaper in journals: this is the first page of a paper published in Nature.[Nature] 431, 1062-1068 (2004)

� Reprinted with permission from Nature (Vol. 431, pp. 1062-1068, 2004). Copyright : Macmillan Magazines Ltd.

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Osaka University 100 Papers : 10 Selected Papers

ANNUAL REPORT OF OSAKA UNIVERSITY—Academic Achievement—2004-2005

The following is a comment on the published paper shown on the preceding page.

Structure of the Bacterial Flagellar Hook and Implication forthe Molecular Universal Joint MechanismIMADA Katsumi and NAMBA Keiichi(Graduate School of Frontier Biosciences)

Introduction

Bacteria swim by rotating long helical filaments called the fla-gellum. It is a complex molecular machine constructed by morethan 20 thousands of protein molecules including about 25 differ-ent kinds of protein1. The flagellum consists of three distinct regions:a long helical filament, a short highly curved segment called hookand a basal body2 (Fig. 1). The filament with typical length of 10- 15 µm is constructed by a single protein flagellin. The filamentis a helical propeller driven by the molecular motor in the basalbody, which is embedded in the cell wall. The filament is con-nected to the motor through the hook, whose length is 55 ± 6 nm3.The hook acts as a universal joint and transmits the torque gener-ated by the motor to the filament smoothly. For efficient trans-mission of torque, the hook is flexible in bending, but rigid againsttwist. The hook is a helical assembly of about 120 copies of chem-ically identical protein FlgE2, and can also be described as a tubu-lar structure comprising 11 protofilaments4. In every revolution ofthe motor rotating at 300 revolutions per second, each protofila-ment is thought to undergo a cycle of conformational changes froma state of shorter repeat to a state of longer repeat and from thelonger to the shorter. Thus, well-organized structural change and

molecular motion are required for smooth and continuous rotation.What is the molecular mechanism of such quick and harmonic motionin the hook? How is the mechanical property of the hook produced?The three dimensional structure of the hook determined by X-raycrystallography and electron cryomicroscopy, and computer sim-ulation based on the atomic structure have revealed the answers tothese questions.

Fig. 1. Schematic drawing of the architecture of the bacterialflagellum. Different colors represent different protein compo-nents.

44 ANNUAL REPORT OF OSAKA UNIVERSITY—Academic Achievement—2004-2005

3D-Structure of FlgE31The hook is such a huge and complex molecular assembly that

its structural determination was quite a challenge. We applied X-ray crystallographic method to solve the atomic structure of FlgEsubunit and electron cryomicroscopy to obtain a density map ofthe straight hook structure at medium resolution, and then, com-bined both results to determine the atomic structure of the hook.

The N-terminal 70 and C-terminal 33 residues of FlgE are native-ly unfolded in the monomeric form in solution5, but these regionsare important for polymerization to form the hook. Therefore, we

prepared a central core fragment lacking both the N- and C- ter-minal regions, named it FlgE31, and solved its structure by X-raycrystallography6. The structure of FlgE31, as shown in Fig. 2a, hastwo domains, D1 and D2, connected by a short stretch of two-stranded anti-parallel β-sheet. Both domains have an oval shapeand consist mostly of β-structures. Domain D2 is composed ofeight-stranded β-barrel with extra loops. Domain D1 has an unusu-al fold composed of complex β-hairpins and a unique triangularloop.

Partial Atomic model of the straight hookUsing electron cryomicroscopy and helical image reconstruc-

tion technique, we obtained a three-dimensional density map ofthe straight hook filament at 15 Å resolution. A partial atomic modelof the hook filament was, then, constructed by fitting the crystalstructure of FlgE31 into the density map followed by the real spacerefinement7 (Fig. 2b and 2c). The atomic model shows extensivecontacts between D2 domains along the 6-start helix (Fig. 2d). D2domain contacts are found neither along the 11-start helix, whichis nearly axial, nor the 5-start helix (Fig. 3a). Intersubunit interac-tions between D1 domains are very weak in any of these three heli-cal directions (Fig. 3a). The axial 11-start protofilament structureis mainly held by the D1-D2 interactions with these two domainsarranged radially (Fig. 3b). This is important for the hook func-tion as a molecular universal joint as described below.

Model of a curved hookThe wild type hook is highly curved2, and therefore hook protofil-

aments on the inner side of the curve must have shorter repeat dis-tances than those on the outer side. While acting as a universaljoint, each of hook protofilaments must continuously vary its repeatdistance and must be going through dynamic conformationalchanges during rotation. To see the amounts of axial extension andcompression and what types of structural changes would be respon-sible for them, we built a model of a curved hook based on theatomic model of the straight hook. We continuously deformed the

Fig. 2. Atomic models of FlgE31 and straighthook. (a) The Cα backbone trace of FlgE31.The chain is color coded from blue to red fromthe N to the C terminus. (b), (c) Docking of theatomic model of FlgE31 into the outer twodomains of the hook. (b) End-on view; (c) sideview. (d) The Cα backbone trace of the straighthook. Each subunit is color coded as (a). Threeof the 11-stranded protofilaments are removedin front part for the bottom half and in back forthe top half for clearer views of domain inter-actions.

Fig. 3. Magnified views of intermolecular interactions along various helical lines of the straighthook. (a) Seven subunits viewed from the inside. (b) Three subunits along the protofilament(11-start). Atomic models are represented with Cα backbone in stick and side chains in balland stick and color coded as in Fig. 2. Arrows in three different colors in (a) indicate the direc-tions of three representative helical lines: bottom three for D1 domains and top three for D2domains.

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Osaka University 100 Papers : 10 Selected Papers

ANNUAL REPORT OF OSAKA UNIVERSITY—Academic Achievement—2004-2005

helical lattice of the straight hook so that the hook axis conformsan observed right-handed helical line8. The model of the curvedhook is shown in Fig. 4a. A short segment of the curved hook ismagnified and two 11-start helical arrays, one on the inside andthe other on the outside, are shown in Fig. 4b. The near axial repeatdistances between D2 domains on the inside and outside of thecurved hook are 35 Å and 59 Å, respectively, while those betweenD1 domains are 39 Å and 54 Å, respectively. In the straight hook,the distance is 46 Å. Thus, the interdomain distances along the

References

1. Macnab, R.M. Annu. Rev. Microbiol. 57, 77-100 (2003).

2. DePamphilis, M.L. & Adler, J. J. Bacteriol. 105, 376-83 (1971).

3. Hirano, T., Yamaguchi, S., Oosawa, K. & Aizawa, S.-I. J. Bacteriol.176, 5439-5449 (1994).

4. Morgan, D.G., Macnab, R.M., Francis, N.R. & DeRosier, D.J. J. Mol. Biol.229, 79-84 (1993).

5. Vonderviszt, F., Ishima, R., Akasaka, K. & Aizawa, S. J. Mol. Biol. 226, 575-9 (1992).

6. Samatey, F.A., Matsunami, H., Imada, K., Nagashima, S. & Namba, K. ActaCryst. D60, 2078-2080 (2004).

7. Shaikh, T.R., Thomas, D.R., Chen, J.Z., Samatey, F.A., Matsunami, H., Imada,K., Namba, K. & DeRosier, D.J. Proc. Natl Acad. Sci. USA, 102, 1023-1028(2005).

8. Kato, S., Okamoto, M. & Asakura, S. J. Mol. Biol. 173, 463-476 (1984).

protofilament have to be compressed orextended by 6 to 8 Å for the D1 array and11 to 13 Å for the D2 array. Predictedchanges in the domain packing are shownin Fig. 4c and 4d. How could this large con-formational change of the hook protofila-ment possible?

Molecular dynamic simulation and implication for the universaljoint mechanism

To answer this question, we carried out a molecular dynamicsimulation of extension and compression of the protofilament usingthe atomic model of three subunits with surrounding water mole-cules. The simulation was done by shifting the reference coordi-nates of domain D2 of the subunit at the top and domain D1 of thesubunit at the bottom in the opposite direction and applying posi-tional restraints to the main chain atoms of these domains to com-press by 5 Å or extend by 15 Å (Fig. 5). The main axial intermol-ecular interactions that hold the protofilament structure are betweendomain D1 of the upper subunit and D2 of the lower subunitthrough the triangular loop of domain D1 (Fig. 3b and Fig. 4b).The conformation of the triangular loop shows relatively small changesupon axial compression or extension; instead, the bonding inter-actions between amino acid residues of the triangular loop and theinner face of D2 show multiple steps of changes in bonding part-ners (Fig. 5b), realizing a large slippage at this D1-D2 interface.The bending flexibility of the hook, which is essential for its uni-versal joint function, is probably due to this stepwise axial-slidingalong with flexibility in relative domain orientation.

Fig. 5. Simulated extension and compression of hook protofilament. Protofilament models atfive different stages at 5Å intervals are superimposed with different colors: dark blue, lightblue, green, yellow, and red, from the most compressed to the most extended state. (a) Thewhole three subunits. (b) Magnified view of upper half. D2 domains at the top and D1 domainsat the bottom have equal intervals (2.5Å) along the vertical axis. It should be noted that thetop corner of the triangular loop of domain D1 of the top subunit (arrow) shows stepwise move-ments (a jump from dark blue to light blue, very small movements from light blue to green andto yellow, and another jump to red), while domain D2 of the middle subunit stays more or lessin the same position. This indicates that the triangular loop of domain D1 and the surface ofdomain D2 can have distinct side chain bonding partners depending on the state of exten-sion or compression.

Fig. 4. Atomic model of the supercoiled hook. (a) Atomic model ofcoiled hook with a schematic diagram of the basal body spanningthe outer (OM) and cytoplasmic (CM) membranes. (b) Magnifiedimage of the coiled hook with the innermost and outermost protofil-aments on the left and right, respectively. The inner core domainsmade of both terminal chains and the central channel are repre-sented by dotted grey lines. (c), (d) Intermolecular packing arrange-ments of D2 domains on the inner side (c) and on the outer side(d) of the coiled hook surface. Only domain D2 is color coded asin Fig. 2, while domain D1 is colored light grey.