Molecular dynamics of ribosomal elongation factors G and Tu

Abstract

Translation on the ribosome is controlled by external factors. During polypeptide lengthening, elongation factors EF-Tu and EF-G consecutively interact with the bacterial ribosome. EF-Tu binds and delivers an aminoacyl-tRNA to the ribosomal A site and EF-G helps translocate the tRNAs between their binding sites after the peptide bond is formed. These processes occur at the expense of GTP. EF-Tu:tRNA and EF-G are of similar shape, share a common binding site, and undergo large conformational changes on interaction with the ribosome. To characterize the internal motion of these two elongation factors, we used 25 ns long all-atom molecular dynamics simulations. We observed enhanced mobility of EF-G domains III, IV, and V and of tRNA in the EF-Tu:tRNA complex. EF-Tu:GDP complex acquired a configuration different from that found in the crystal structure of EF-Tu with a GTP analogue, showing conformational changes in the switch I and II regions. The calculated electrostatic properties of elongation factors showed no global similarity even though matching electrostatic surface patches were found around the domain I that contacts the ribosome, and in the GDP/GTP binding region.

Fig. 1

Left: Secondary structure model of EF-G in the complex with GDP and Mg²⁺ (PDB code 1FNM); domain I(G), residues 1–280 (red); insert (G′), residues 158–253 (yellow); domain II, residues 289–391 (dark blue); domain III, residues 404–482 (orange); domain IV, residues 483–603, 675–689 (green); domain V, residues 604–673 (brown). Right: EF-Tu complexed with aa-tRNA, GDP, and Mg²⁺ (based on the PDB code 1TTT); domain I(G), residues 1–211 (red); domain II, residues 220–311 (dark blue); domain III, residues 312–405 (light blue); aa-tRNA is divided into the acceptor stem (orange), anticodon arm (green), and T-arm (brown). Domains I(G) and II of EF-Tu and EF-G are homologous and common for GTPases

Fig. 2

Radius of gyration (top) and RMSD (bottom) calculated for the C_α atoms of EF-G (black line) and for the C_α and P atoms of the EF-Tu:tRNA complex (grey line) plotted as a function of the simulation time

Fig. 3

RMSD matrix of EF-G (left) calculated for C_α and EF-Tu:tRNA complex (right) calculated for C_α and P atoms. The legend refers to the upper half of the matrices. Clusters resulting from the RMSD matrix are shown in blue below the diagonal

Fig. 4

Average structures of the three most numerous populations of EF-G (left) and EF-Tu:tRNA (right) derived on the basis of clustering of their RMSD matrices presented in Fig. 3

Fig. 5

Distances between the centres of masses (COM) of domains II and IV of EF-G (Fig. 1 left), domain II and tRNA of the EF-Tu:tRNA complex (Fig. 1, right), and domains I, II, and III of EF-Tu plotted as a function of the simulation time

Fig. 6

Graphical representation of extreme projections along first three eigenvectors illustrating collective motion of EF-G (top) and EF-Tu:tRNA (bottom). Arrows show the directions of the first three eigenvectors derived from PCA

Fig. 7

Conformational changes observed in the switch I (in green) and II (in yellow) regions of EF-Tu. Left: Secondary structures of the switch I region of the EF-Tu crystal structure (a) and the changes observed in MD simulations plotted as a function of the simulation time in EF-Tu (b) and EF-Tu:GDP (c). Secondary structure codes: cyan, turn; blue, 3–10 helix; yellow, extended conformation; pink, α helix; green, isolated bridge. Right: Representations of the X-ray structure of EF-Tu in the complex with a GTP analogue (shown in magenta as van der Waals spheres) (a), MD snapshots of EF-Tu (b) and EF-Tu:GDP (c); for clarity the ligand is not shown). Atom colouring: domain I(G), residues 1–211 (red); domain II, residues 220–311 (dark blue); domain III, residues 312–405 (light blue)

Fig. 8

The root mean square fluctuation of C_α and P atoms of a EF-G:GDP, EF-G, and b EF-Tu:tRNA, EF-Tu:GDP, EF-Tu

Fig. 9

The distances between the centres of masses of the CCA-end phenylalanine and histidine 67 rings observed in the crystal structure (blue) and in MD simulation (orange) of EF-Tu:tRNA

Fig. 10

The electrostatic potential of EF-G (left) and the EF-Tu:tRNA complex (right) projected on to their van der Waals surfaces (partially transparent). Interior black detail shows the secondary structure of EF-G and EF-Tu:tRNA

Fig. 11

The electrostatic potential in the GTP/GDP binding site (GDP is shown in purple) projected on to van der Waals surfaces of EF-G (left) and EF-Tu:tRNA (right)

Fig. 12

The electrostatic potential calculated for the isolated EF-Tu projected on to its van der Waals surface. The tRNA molecule (black ribbon) is also shown to present its binding mode