Life Science: Structural Biology 22 Rotation mechanism of V 1 -ATPase Vacuolar ATPases (V-ATPases) function as proton pumps, which are involved in many processes such as bone resorption and cancer metastasis, and these membrane proteins represent attractive drug targets for osteoporosis and cancer. Their structures resemble those of F-ATPases, which function as ATP synthase in mitochondria, chloroplasts and bacteria. These ATPases consist of a hydrophilic portion (F 1 and V 1 ) and a membrane-embedded ion-transporting portion (F o and V o ), and have a similar reaction mechanism that occurs through rotation. F 1 -ATPase has been investigated in detail, and the molecular mechanism has been proposed on the basis of crystal structures of the complex [1], and extensive single-molecule observation of the rotation [2]. Similar V 1 -ATPase experiments have been conducted using the Thermus thermophiles enzyme, which functions physiologically as an ATP synthase. The crystal structures of the A 3 B 3 DF (V 1 ) complex at low resolution suggest differences in its structure and interactions compared with F 1 -ATPases [3]. Single-molecule analyses of V 1 - ATPase also suggest differences in torque generation and the coupling scheme of the rotation mechanism compared with F 1 [4]. Enterococcus hirae V-ATPase acts as a primary ion pump similar to eukaryotic V-ATPase, which transports Na + or Li + , instead of H + ions. Recently, we have succeeded in obtaining the crystal structures of A 3 B 3 and V 1 -ATPase complexes at high resolution, which enabled the generation of a model of the rotational mechanism. We determined the crystal structures of the nucleotide-free (2.8 Å) A 3 B 3 (denoted as eA 3 B 3 ) and the AMP-PNP-bound (3.4 Å) A 3 B 3 (denoted as bA 3 B 3 ) using beamline BL41XU (Fig. 1(a,c)) [5]. In eA 3 B 3 , one of the three A subunits adopts a closed conformation (denoted as A C ), which shifts the structure into the center of the A 3 B 3 ring, whereas the other two A subunits adopt similar open conformations (denoted as A O and A O’ ) (Fig. 1(b)). Similarly, one of the three B subunits shows a closed conformation (denoted as B C ) compared with the others (denoted as B O and B O’ ). The conserved nucleotide-binding sites are located between the three different combinations: A O B C , A O’ B O , and A C B O’ pairs. On the other hand, in bA 3 B 3 , AMP-PNP:Mg molecules bind at two A C B O’ each, and not at the other A O B C (Fig. 1(d)). We designated these A O B C pairs as the ‘empty’ form on the basis of their apparent very low affinity for AMP-PNP:Mg, and these A C B O’ pairs as the ‘bound’ form take the ATP-bound form. The A O’ B O in eA 3 B 3 seems to change to A C B O’ upon binding with AMPPNP:Mg. We designated this unique A O’ B O pair of eA 3 B 3 as the ‘bindable’ form. These asymmetries suggest that the formation of the A 3 B 3 hexamer ring imposes a restriction on the AB pair and induces conformational changes that cooperatively generate one empty (ATP-unbound form), one bindable (ATP- accessible form), and one bound (ATP-bound form) conformation, which in turn determine the order of nucleotide binding in the ring in the right-handed rotational orientation viewed from the top of the V 1 complex. Next, we crystallized and solved the crystal structure of the nucleotide-free (2.2 Å) V 1 -ATPase (denoted as eV 1 ) (Fig. 2(a)) and the AMP-PNP- bound (2.7 Å) V 1 -ATPase (denoted as bV 1 ). A and B subunits assembled asymmetrically and a central axis composed of D and F subunits penetrated into the cavity of the A 3 B 3 hexamer. The structures of eA 3 B 3 and eV 1 , which should have been induced by interaction with the DF complex, are compared. The eV 1 has an empty form (A O B C ) and a bound form (A C B O’ ), but the bound form of eV 1 is positioned as the site of the bindable form of eA 3 B 3 when both empty forms are superimposed. Therefore, the DF binding seems to induce a change from the bindable eA 3 B 3 to the bound form, similar to the conformational changes of the eA 3 B 3 induced by AMP- PNP binding. The remaining AB pair of eV 1 represents a more tightly packed structure composed of closer A and B subunit conformations approaching the center of the A 3 B3 ring. This is not observed in the structure of the A 3 B 3 complex (Fig. 2(b)). We designated the Fig. 1. Structure of the A 3 B 3 complex. (a ) Side view of the nucleotide-free A 3 B 3 structure (eA 3 B 3 ). (b) Top view of the C-terminal domain (shown in (a) as a transparent surface) of eA 3 B 3 from the N-terminal β -barrel side. Red triangles indicate the nucleotide-binding sites. (c) - (d) Structures of the AMP-PNP-bound A 3 B 3 complex (bA 3 B 3 ). (a) (b) (c) (d) Empty Empty Bound Bound Bound Bindable A O A O A O’ A O’ A C A C A C A C A C A C B O B O A O A O B O B O B O’ B O’ B O’ B O’ B C B C B C B C 23 new conformations as the ‘tight’ form (A CR B CR ), and DF complex binding seems to change the bound form of eA 3 B 3 to the tight form. In bV 1 , the overall structure of bV 1 was similar to that of eV 1 , although the binding sites of bound and tight forms were occupied with AMP-PNP:Mg. In the tight form, the position of the conserved Arg- finger (Arg350 of B CR ), which helps ATP hydrolysis, was closer to the nucleotide-binding site than that in the bound form (Fig. 2(c)). Thus, the ATP hydrolysis is stimulated by this approach triggered by the movement of the Arg-finger, which is induced by binding between the DF complex and A 3 B 3 . On the basis of these asymmetric structures, a rotation model of V 1 was proposed (Fig. 3). The V 1 - ATPase is bound to two ATP:Mg molecules in the bound and tight forms (Fig. 3(a)). Bound ATP in the tight form is awaiting ATP hydrolysis. When ATP is hydroid in the tight form, V 1 -ATPase starts the rotary reaction. The conformation of the A 3 B 3 part in V 1 - ATPase may return to eA 3 B3 in a cooperative manner (Fig. 3(b)). Thus, while the tight form changes to the empty form with the release of ADP and phosphate, the empty form changes to the bindable form. However, the interaction between the DF and the tight form might prevent these structural changes, and an intermediate state may exist instead of the state of eA 3 B 3 . After that, the new ATP molecule binds to the bindable form, and the conformation changes to the bound form. Thus, the state may be similar to that of bA 3 B 3 (Fig. 3(c)). Then, the DF rotates, and the bound form from the beginning changes to the next tight form vie interaction with the DF complex. In brief, the V 1 -ATPase returns to the initial state (Fig. 3(d)). Kano Suzuki a, * and Takeshi Murata a,b a Department of Chemistry, Chiba University b JST/PRESTO *E-mail: r2sd3103@chiba-u.jp References [1] J. P. Abrahams et al .: Nature 37 0 (1994) 621. [2] H. Noji et al .: Nature 386 (1997) 299. [3] N. Numoto et al .: EMBO Rep. 10 (2009) 1228. [4] H. Imamura et al .: Proc. Natl. Acad. Sci. USA 102 (2005) 17929. [5] S. Arai, S. Saijo, K. Suzuki, K. Mizutani, Y. Kakinuma, Y. Ishizuka-Katsura, N. Ohsawa, T. Terada, M. Shirouzu, S. Yokoyama, S. Iwata, I. Yamato and T. Murata: Nature 493 (2013) 703. Fig. 2. Structure of the A 3 B 3 DF complex. (a) Side view of the nucleotide-free A 3 B 3 DF structure (eV 1 ). (b) Top view of the C-terminal domain of eV 1 , as in Fig. 1(b), superimposed at the empty form onto that of transparent eA 3 B 3 (gray). (c) Nucleotide-binding sites. Tight form in eV 1 (color) and bound form in eA 3 B 3 (gray). Fig. 3. Rotation model of V 1 -ATPase. Top view of the C-terminal domain viewed as in Figs. 1(b, d) and Fig. 2(b). ATP with yellow "P" in (a) and (d) represents an ATP molecule that is committed to hydrolysis. The blue "P" in (b) represents a phosphate molecule after ATP hydrolysis. (a) The AMP-PNP-bound V 1 : first two ATPs are bound in the bound and tight forms. The reaction is triggered by the ATP hydrolysis in the tight form. (b) The nucleotide-free A 3 B3: by the conversion to ADP and phosphate, the conformation of the A 3 B 3 part in V 1 - ATPase may return to eA 3 B 3 (ground structure of A 3 B3 complex) in a cooperative manner. The tight form changes to the empty form with the release of ADP and phosphate and the empty form changes to the bindable form. (c) The AMP-PNP-bound A 3 B3 : by new ATP binding to the bindable form, the conformation changes to bA 3 B3 , which has two bound forms with two ATP, and then the DF rotates. (d) The bound form from the beginning changes to the next tight form, induced by DF binding, and the V 1 -ATPase returns to the initial state with 120° rotation. (a) (b) (c) Empty Tight Bound A O A O A C A C B O B O B CR B CR B C B C A CR A CR E261 K238 P-loop R262 B/R350 1.7Å 1.7Å T239 T239 (a) (b) (c) (d) Empty Tight Empty Bindable Bound Bound Bound Empty Empty Bound Tight Bound bV 1 bV 1 eA 3 B 3 bA 3 B 3 ATP access ADP and Pi release
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