The internal and external motions of EF-G.

<p>The internal and external motions of EF-G shown from (a) side, (b) top and (c) front of EF-G. The structure of EF-G in the PRE state (<i>R₁</i> = 37 Å) (in white) superposed on that in the POST state (<i>R₁</i> = 15 Å) is depicted as a wire model in gray. The P- and E-tRNAs in the POST state are depicted as wire models in orange. The surrounding molecules, SRL (C2646-G2674), L14 and L12-CTD (L-chain, N69-P98) in the POST and PRE states are depicted as wire models in black and brown, respectively. GDP in the POST state is depicted as a space-filling model. Dynamic domains in the ribosome-bound EF-G for structures in the POST and PRE states were analyzed by DynDom3D program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951-Poornam1" target="_blank">[49]</a>. Each dynamic domain is colored: dynamic domain A (a large part of domain IV) in red, dynamic domain B (a large part of domain III) in yellow, dynamic domain C (a large part of domain II, and a part of domain G and subdomain G′) in thin blue and dynamic domain D (a large part of domain V and a part of domain G and subdomain G′) in green. The regions that were not assigned to a dynamic domain are shown in blue. The axis of dynamic domain X and Y is depicted as an arrow in color for dynamic domain X with a tip in color for dynamic domain Y. Dynamic domain X rotates anticlockwise around the axis of dynamic domains X and Y with respect to dynamic domain Y from the PRE to POST state. In the input parameter for the DynDom3D, the minimum ratio of external to internal displacement of 0.65 was used (default is 1.0). The ratio of internal to external displacement determines the acceptance criterion for a given domain pair. This lower value was required due to noise often seen from MD results <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951-Ishida1" target="_blank">[30]</a>. The axis of the external motion is shown in pink. As a reference, x-axis, y-axis (<b>e</b>_ratchet) and z-axis (<b>e</b>_tRNA) are depicted as thin black lines at the anticodon of P-tRNA. The length of these axes (1 Å) is magnified by a factor of 50. EF-G with the same orientation as (a–c) is shown as a reference on the right side in small scale, where structural domains of G, G′, II, III, IV and V of EF-G are colored in gray, black, thin blue, yellow, red and green, respectively.</p

Free-Energy Landscape of Reverse tRNA Translocation through the Ribosome Analyzed by Electron Microscopy Density Maps and Molecular Dynamics Simulations

<div><p>To understand the mechanism of reverse tRNA translocation in the ribosome, all-atom molecular dynamics simulations of the ribosome-tRNAs-mRNA-EFG complex were performed. The complex at the post-translocational state was directed towards the translocational and pre-translocational states by fitting the complex into cryo-EM density maps. Between a series of the fitting simulations, umbrella sampling simulations were performed to obtain the free-energy landscape. Multistep structural changes, such as a ratchet-like motion and rotation of the head of the small subunit were observed. The free-energy landscape showed that there were two main free-energy barriers: one between the post-translocational and intermediate states, and the other between the pre-translocational and intermediate states. The former corresponded to a clockwise rotation, which was coupled to the movement of P-tRNA over the P/E-gate made of G1338, A1339 and A790 in the small subunit. The latter corresponded to an anticlockwise rotation of the head, which was coupled to the location of the two tRNAs in the hybrid state. This indicates that the coupled motion of the head rotation and tRNA translocation plays an important role in opening and closing of the P/E-gate during the ratchet-like movement in the ribosome. Conformational change of EF-G was interpreted to be the result of the combination of the external motion by L12 around an axis passing near the sarcin-ricin loop, and internal hinge-bending motion. These motions contributed to the movement of domain IV of EF-G to maintain its interaction with A/P-tRNA.</p></div

Conformational changes around the P/E-gate and the tRNAs in the POST, INT and PRE states.

<p>Snapshots of the conformations around the P/E-gate and the tRNAs on the small subunit in the (a) POST (<i>R₁</i> = 0 Å), (b) POST (<i>R₁</i> = 15 Å), (c) INT (<i>R₁</i> = 29 Å) and (d) PRE (<i>R₁</i> = 37 Å) states in the first “r-translocation” simulation are shown. The atoms of G1338, A1339 and A790, of the P/E-gate are depicted as space-filling models in purple, dark blue and pink, respectively. The loop regions of the 16S nucleotides A780-G799 and C1328-G1347 are depicted as wire models in red and brown, respectively. Protein S13 is also shown in yellow as a reference.</p

The structural analysis of atomic models of the translocational ribosome.

1<p>In TI^POST(2tRNAs), two tRNAs are present in ap/P and pe/E hybrid states, where the lowercase and uppercase letters indicate tRNA contacts on the small and large subunit, respectively, in the following order: 30S head (a-site or p-site), 30S body/platform (p-site or e-site) and 50S subunit (P-site or E-site).</p>2<p>In TI^POST(Fus2), a single tRNA is present in a pe*/E hybrid state, where e* in pe*/E means that the anticodon stem-loop (ASL) of the tRNA lies between the p- and e-sites of the 30S body/platform.</p><p>TI^PRE(2tRNAs) consists of the ribosome, two tRNAs, mRNA, EF-G, GDP and viomycin. Each TI^PRE(Fus), TI^POST(Fus1) and TI^POST(Fus2) consists of the ribosome, a single tRNA, mRNA, EF-G and fusidic acid (Fus). Each TI^PRE(GDPCP1) and TI^PRE(GDPCP2) consists of the ribosome, a single tRNA, mRNA, EF-G, GDPCP. TI^POST(2tRNAs) consists of the ribosome, two tRNAs, mRNA, EF-G, GDP and Fus.</p><p>For 3J5X/3J5W and 3J5N/3J5O (<i>E. coli</i> ribosome), their superpositions on 2WRI/2WRI (<i>Thermus thermophiles</i> ribosome) were carried out using Chimera software's MatchMaker tool <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951-Pettersen1" target="_blank">[73]</a>.</p

The movements of the P-tRNA, E-tRNA and EF-G.

<p>(a) The paths of the centers of mass of the anticodons of the P-tRNA and E-tRNA, and of domain IV (residues 481–603 and the C-terminal region 675–690) of EF-G, the second reaction coordinates of <i>R₂</i>, are plotted against <i>R₁</i>. The paths obtained from the first and second “r-translocation” simulations are plotted with thick and thin lines, respectively.</p

The protocol of the EM-fitting and the umbrella sampling simulations.

<p>Schematic representation of the protocol of the EM-fitting and the umbrella sampling simulations to obtain the free-energy landscape is shown.</p

Model of r-translocation.

<p>Schematic representation of a model of r-translocation for (a) the ribosome in the PRE and locked states (tRNAs in the pp/E and aa/P states), (b) the ribosome in the PRE and unlocked states just after unlocking (tRNAs in the pp/E and aa/P states), (c) the ribosome in the INT and unlocked states (tRNAs in the ee*/E and pp*/P states) and (d) the ribosome in the POST and relocked states (tRNAs in the ee/E and pp/P states) are shown schematically. The large subunit of the ribosome is not shown for simplicity. (a) and (d) are in the locked and relocked states, respectively (closed state of the P/E-gate). In (a) and (d), the distance between A790 and G1338 of the P/E-gate is so narrow that the P/E-gate blocks the movement of the E-tRNA. (b) and (c) are in the unlocked state (open state of the P/E-gate). In (b) and (c), the distance between A790 and G1338 of the P/E-gate is wide enough to allow the E-tRNA to translocate from the E-stie to P-site.</p

The movement of domain IV of EF-G.

<p>The positions of the center of mass of Gln500 in loop I and His 573 in loop II of domain IV of EF-G are plotted against a plane whose normal axis (z-axis) coincides with the unit vector of tRNA translocation, <b>e</b><i>_tRNA</i>, in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951.e024" target="_blank">Eq. (14)</a>. The y-axis was set to coincide with the axis of the ratchet-like movement. The x-axis was set as a cross product of the z-axis and y-axis. The origin is at the center of mass of the anticodon of P-tRNA. (The figure of these axes is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone-0101951-g006" target="_blank">Fig. 6</a>.) The positions of a single EF-G is shown with diamonds, and the positions of EF-G bound to the ribosome is shown with squares and circles. Structures of EF-G are from, simulated structures in the POST (<i>R₁</i> = 15 Å), INT (<i>R₁</i> = 23 and 29 Å) and PRE (<i>R₁</i> = 37 Å) in the first “r-translocation” simulation, 3IZP (constructed by flexible fitting of a single EF-G into EMD-1365) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951-Li1" target="_blank">[65]</a>, 1PN6 (constructed by rigid-body fitting of a single EF-G into EMD-1365) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951-Valle1" target="_blank">[12]</a>, 2OM7 (constructed from EMD-1315, GMPPNP-bound) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951-Connell1" target="_blank">[14]</a>, 1JQM (constructed from a cryo-EM map, fusidic acid and GDP-bound) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951-Agrawal1" target="_blank">[66]</a>, TI^PRE(2tRNAs) (3J5X/3J5W constructed from EMD-5800, GDP and viomycin-bound) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951-Brilot1" target="_blank">[16]</a>, TI^PRE(Fus) (2XSY/2XTG constructed from EMD-1798, fusidic acid and GDP-bound) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951-Ratje1" target="_blank">[15]</a>, TI^{PRE(GDPCP1/2)} (4BTC/4BTD and 4JUW/4JUX, GDPCP-bound) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951-Chen1" target="_blank">[8] </a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951-Tourigny1" target="_blank">[9]</a>, TI^POST(2tRNAs) (3J5N/3J5O constructed from EMD-5775, fusidic acid and GDP-bound) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951-Ramrath1" target="_blank">[17]</a>, TI^POST(Fus1) (2XUY/2XUX constructed from EMD-1799, fusidic acid and GDP-bound) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951-Ratje1" target="_blank">[15]</a>, TI^POST(Fus2) (4KD8/4KD9, fusidic acid and GDP-bound) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951-Zhou1" target="_blank">[11]</a>, 2WRI/2WRJ (X-ray structure at <i>R₁</i> = 0 Å) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951-Gao1" target="_blank">[10]</a>, 1FNM (single EF-G mutant H573A, GDP-bound) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951-Laurberg1" target="_blank">[67]</a>, 2BM0 (single EF-G mutant T84A, GDP-bound) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951-Hansson1" target="_blank">[68]</a>, 2BV3 (single EF-G mutant T84A, GDPNP-bound) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951-Hansson2" target="_blank">[69]</a>, 2EFG (single EF-G wild-type, GDP-bound) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951-Czworkowski1" target="_blank">[70]</a>, 1ELO (single EF-G wild-type, nucleotide-free) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951-varsson1" target="_blank">[71]</a>, 1DAR (single EF-G wild-type, GDP-bound) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951-AlKaradaghi1" target="_blank">[72]</a>, 1WDT (single EF-2 homolog, GTP-bound) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951-Connell1" target="_blank">[14]</a>. These structures of EF-G were superposed on the structure of EF-G in 2WRI/2WRJ (at <i>R₁</i> = 0 Å) so as to minimize the structural differences in the main-chains of domains I and II (K4-L399). For 1WDT (single EF-2 homolog), Gln473 and His543 are used instead of Gln500 and His573, respectively. For 3J5X/3J5W and 3J5N/3J5O (<i>E. coli</i> EF-G), Gln508 and His584 are used instead of Gln500 and His573, respectively. The superpositions of 1WDT, 3J5X/3J5W and 3J5N/3J5O were carried out using Chimera software's MatchMaker tool <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101951#pone.0101951-Pettersen1" target="_blank">[73]</a>. The regression line for the positions of a single EF-G (except for 1WDT) is shown by broken line, and the regression line for the positions of EF-G bound to the ribosome (except for 1PN6) is shown by solid line.</p

The free-energy landscape of reverse tRNA translocation.

<p>The free-energy landscapes obtained from the first and second “r-translocation” simulations are plotted against <i>R₁</i> with thick and thin lines, respectively. The lowest free-energy was set at zero for each case. The average of the absolute difference between each value of their free-energies in the range of <i>R₁</i> = 17–41 Å was 2.2 kcal/mol. For the sake of convenience, the POST, INT and PRE states are leveled at <i>R₁</i> = ∼0–18 Å, ∼19–33 Å and ∼34–44 Å, respectively. The units of the reaction coordinate and the free-energy are Å and kcal/mol, respectively.</p

The movements of the head of the small subunit and the P/E-gate.

<p>(a) The angles of rotation of the head of the small subunit are plotted against <i>R₁</i>. For comparison, the values for the X-ray structure (2WRI/2WRJ), TI^PREs (TI^PRE(2tRNAs), TI^PRE(Fus) and TI^{PRE(GDPCP 1/2)}), TI^POSTs (TI^POST(2tRNAs), TI^POST(Fus1) and TI^POST(Fus2)) were plotted with triangles and circles, respectively. TI^POSTs are plotted at angle = −10.0° because they were outside the range of graph. (b) The widths of the P/E-gate are plotted against <i>R₁</i>. The width of the P/E-gate was defined as the distance between the center of mass of A790, and the center of mass of G1338 and A1339. For comparison, the widths for the X-ray structure (2WRI/2WRJ), TI^PREs and TI^POSTs were plotted with triangles and circles, respectively. The values from the first and second “r-translocation” simulations are plotted with thick and thin lines, respectively.</p