34 Insights into the Stator Assembly of the Na + -Driven Vibrio Flagellar Motor from the Crystal Structure of MotY Life Science : Structural Biology Motile bacteria swim by rotating a helical filamentous organelle called flagellum. Each flagellum is driven by a reversible rotary motor embedded in the cell envelope. The flagellar motor is about 45 nm in diameter and divided into a rotating part (rotor) and a stationary part (stator) (Fig. 1). The energy source of the motor is a membrane gradient of a specific ion (H + or Na + ), and its flux through the stator channel is coupled with rotor-stator interactions to generate torque [1]. Marine bacterium Vibrio alginolyticus swims using a single polar flagellum in liquid environment. Its Na + -driven motor rotates surprisingly fast, up to 1700 rps at 300 mM NaCl, and requires unique proteins, MotX and MotY, in addition to the PomA/PomB stator proteins for torque generation (Fig. 1, right). MotX and MotY form a complex in the periplasm, and MotX affects the membrane localization of PomB, suggesting that MotX interacts with PomB. Recently, MotX and MotY have been found to form a ring-like structure (T-ring) beneath the P-ring of the flagellar basal body in V. alginolyticus . Furthermore, in the absence of MotX or MotY, the PomA/PomB stator does not localize to the flagellated cell pole, suggesting that these two proteins are involved in the incorporation and/or stabilization of the PomA/PomB complex within the motor [2]. To understand the role of MotY in the assembly and function of the Na + -driven motor, we determined the structure of MotY from V. alginolyticus at 2.9 Å resolution using diffraction data collected at beamline BL41XU [3]. The structure shows two distinct domains: an N-terminal domain (MotY-N) and a C-terminal domain (MotY-C) (Fig. 2(a)). MotY-N has a unique structure, composed of two β -sheets and two α -helices. The C-terminal end of helix α 2, which is followed by MotY-C, is anchored to sheet-B through a disulfide bridge between Cys-25 in β 3 and Cys-147 in α 2 (Fig. 2(b)). The two Cys residues are conserved in MotY from various bacteria with polar flagella. Therefore, MotY proteins containing the C25S, C147S, and C25S/C147S substitutions were susceptible to proteolysis and resulted in cells with reduced or lost motility, indicating that the disulfide stabilizes MotY-N [4]. MotY-C that contains a putative peptidoglycan-binding motif forms an α / β sandwich structure showing remarkable similarity to other OmpA/MotB-like proteins, such as the core domain of Pal and the OmpA-like domain of RmpM. The structural similarity suggests that MotY interacts with the peptidoglycan layer like those proteins. In the crystal structure, however, the putative peptidoglycan- binding site of MotY-C was more widely open than those of Pal and RmpM, and most of the loops forming the peptidoglycan-binding pocket were disordered (Fig. 2(c,d)). A disordered peptidoglycan-binding pocket may be an important property for free MotY since, after secretion into the periplasm, MotX and MotY presumably must diffuse around until they collide with the basal-body and form the T-ring structure. Therefore, MotY should not bind tightly to the peptidoglycan layer before it encounters the basal- body. Interaction with the basal-body may induce folding of the disordered chains into a functional peptidoglycan-binding pocket. On the basis of the structure and lines of evidence obtained from biochemical experiments using MotY fragments, we propose a model for stator assembly (Fig. 3). MotY may form a complex with MotX and diffuse around the periplasm with little or no affinity for the peptidoglycan layer (Fig. 3(a)). When the complex associates with the basal-body via MotY-N, conformational changes are induced that greatly increase the affinity of MotY-C for peptidoglycan. The Fig. 1. Cell body and flagellar basal body structure of the H + - and Na + -driven types. The stator consists of MotA and MotB for the H + -driven motor, and of PomA and PomB for the Na + -driven type. For Vibrio ’s Na + -driven polar flagellar motor, MotX and MotY, which form the T-ring in the basal body, are also essential for rotation. Salmonella cell ca. 50 nm Vibrio cell Na + -type T-ring L-ring P-ring FlgH Flgl (MotX,Y) PomB PomA FliG FliM,N Export apparatus MS-ring C-ring MotA MotB Rod Rod FliF FliM,N FliG H + -type 35 (a) (b) (c) OM PG IM IM MotY PomB PomA MotX PG PG OM IM PG OM OM PomA/PomB complex also forms before associating with the motor and diffuses around in the cytoplasmic membrane (Fig. 3(b)). When the PomA/PomB complex encounters the basal-body, the association of the periplasmic C-terminal domain of PomB with MotX positions PomB to bind to the peptidoglycan layer in the proper orientation to interact productively with the rotor (Fig. 3(c)). (a) (b) (c) (d) C C C N R73 D71 D37 F36 F34 R86 R168 H165 L21 R Q169 L82 Y78 N N Sheet-A Sheet-A Sheet-B C25 R262 C147 R262 Fig. 2. Structure of MotY. (a) Stereo view of the C α ribbon drawing of MotY. (b) The N-terminal and C- terminal domains are shown in blue and orange, respectively. The disulfide bond between Cys-25 and Cys-147 and the Arg-262 residue are displayed in a ball-and-stick format. (c) (d) Structure of Pal in complex with the peptidoglycan precursor (c) [5] and putative peptidoglycan-binding regions of MotY (d) . The residues contributing to the binding of the peptidoglycan precursor and the corresponding residues in MotY are indicated by blue balls. The peptidoglycan precursor is shown in a ball-and-stick representation. References [1] H. Terashima et al .: Int. Rev. Cell Mol. Biol. 270 (2008) 39. [2] H. Terashima et al .: Mol. Microbiol. 62 (2006) 1170. [3] S. Kojima, A. Shinohara, H, Terashima, T, Yakushi, M. Sakuma, M. Homma, K. Namba and K. Imada: Proc. Natl. Acad. Sci. USA 105 (2008) 7696. [4] J. Yagasaki et al .: J. Bacteriol. 188 (2006) 5308. [5] L.M. Parsons et al .: Biochemistry 45 (2006) 2122. Fig. 3. Model of stator assembly in the Na + -driven polar flagellar motor of Vibrio . MotY is shown as a space-filling model colored blue for MotY-N and yellow for MotY-C. MotX, PomA, and PomB are shown in orange, dark blue, and cyan, respectively. Details of the model are described in the text. Seiji Kojima a , Michio Homma a and Katsumi Imada b, * a Division of Biological Science, Nagoya University b Department of Frontier Biosciences, Osaka University *E-mail: kimada@fbs.osaka-u.ac.jp
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