The conformational landscape of the ribosomal protein S15 and its influence on the protein interaction with 16S RNA

Abstract

The interaction between the ribosomal protein S15 and its binding sites in the 16S RNA was examined from two points of view. First, the isolated protein S15 was studied by comparing NMR conformer sets, available in the PDB and recalculated using the CNS-ARIA protocol. Molecular dynamics (MD) trajectories were then recorded starting from a conformer of each set. The recalculation of the S15 NMR structure, as well as the recording of MD trajectories, reveals that several orientations of the N-terminal alpha-helix alpha1 with respect to the structure core are populated. MD trajectories of the complex between the ribosomal protein S15 and RNA were also recorded in the presence and absence of Mg(2+) ions. The Mg(2+) ions are hexacoordinated by water and RNA oxygens. The coordination spheres mainly interact with the RNA phosphodiester backbone, reducing the RNA mobility and inducing electrostatic screening. When the Mg(2+) ions are removed, the internal mobility of the RNA and of the protein increases at the interaction interface close to the RNA G-U/G-C motif as a result of a gap between the phosphate groups in the UUCG capping tetraloop and of the disruption of S15-RNA hydrogen bonds in that region. On the other hand, several S15-RNA hydrogen bonds are reinforced, and water bridges appear between the three-way junction region and S15. The network of hydrogen bonds observed in the loop between alpha1 and alpha2 is consequently reorganized. In the absence of Mg(2+), this network has the same pattern as the network observed in the isolated protein, where the helix alpha1 is mobile with respect to the protein core. The presence of Mg(2+) ions may thus play a role in stabilizing the orientation of the helix alpha1 of S15.

FIGURE 1

Secondary structure of the three-way junction RNA involved in the interaction with S15. The basepairing is displayed according to the ontology developed by Leontis and Westhof (59). The basepair between G51 and C53 was not displayed to improve the figure readability. This figure was realized with S2S (60).

FIGURE 2

Structures of the S15-RNA complex (x-ray (1dk1) (a)), and of S15 (NMR (1ab3) (b)), and x-ray (1a32) (c)). The structures are shown in cartoon; the RNA is brown, and S15 is green and red in the α-helices. The N- and C-terminal parts of the protein are shown as well as the 5′ and 3′ ends of RNA. The α-helices S15 and the helices H20, H21, and H22 of RNA are also shown in the figure. This figure was realized with PyMOL 0.98 (61).

FIGURE 3

Conformers of the NMR structures 1ab3 (a) and 2fkx (b) shown in cartoons. The conformers are superposed to minimize the RMSD on the complete structure (a) or on residues 24–70 (b). The figure was realized with PyMOL 0.98 (61).

FIGURE 4

Mean values of the φ (a and c) and ψ (b and d) angles in the loop connecting the α1 and α2 helices of S15. The angle values measured in the NMR and x-ray structures (a and b) and calculated during the 11–15-ns interval of the MD trajectories (c and d). These angle values are plotted along the residue numbers. (a and b) The color code is 1ab3 (red), 2fkx (blue), 1dk1 (pink), 1a32 (green), 2avy (cyan), and 2a7w (orange). (c and d) The color code is s15-pdb (red), s15-aria (blue), s15-rna-mg (pink), and s15-rna (green). The x axis describes the residue numbers, and the y axis the angle values in degrees.

FIGURE 5

Comparison of trajectories s15-pdb and s15-aria. Coordinate RMSD from the starting point calculated on (a) all atoms or (b) the loop connecting helices α2 and α3 (residues 41–57). (c) Atomic fluctuations by residues. Angles between helices α1,α2 (d), α2,α3 (e), and α3,α4 (f). The full line is for the trajectory s15-aria, and the dotted/dashed lines for the trajectory s15-pdb. The x axis describes the trajectory time (ns) and the residue numbers, and the y axis describes the angle values (degrees) or the RMSD value and atomic fluctuations (Å). The secondary structures of S15 are shown below c.

FIGURE 6

Contact plot of the violated NOEs during the trajectories of the isolated protein S15 (open circles, s15-pdb; solid circles, s15-aria). The x and y axes describe the residue numbers.

FIGURE 7

Comparison of the trajectories s15-rna-mg and s15-rna. Atomic fluctuations by residues in S15 (a) and in RNA (b), calculated on the time interval 11-15 ns. The RMSD values from the starting point are calculated on (c) all atoms in S15 or (d) all atoms in RNA. The full line is for the trajectory s15-rna, and the dotted/dashed lines for the trajectory s15-rna-mg. The x axis describes the trajectory time (ns) or the residue numbers, and the y axis describes the atomic fluctuations (Å) or the RMSD (Å). The secondary structures of S15 and RNA are shown below a and b.

FIGURE 8

Superposition of the S15-RNA conformations recorded at 7881, 8837, 9895, and 11,347 ps in the presence of Mg²⁺ ions. The RNA is in brown, and the S15 conformers are in cyan (7881 ps), green (8837 ps), magenta (9895 ps), and red (,347 ps). (a) The full complex structure is shown in cartoon. A blue arrow indicates the α1-α2 loop and a red arrow the N-terminal region. The residues Arg-16 and Lys-4 are drawn in sticks. (b) Zoom on the loop linking α1 and α2, with Arg-16 drawn in sticks. (c) Zoom on the N-terminal part of α1 with Lys-4 and Gln-8 drawn in sticks. This figure was realized with PyMOL 0.98 (61).

FIGURE 9

Variation of the angles (a) φ of Ala-15, (b) φ of Thr-21, and (c) ψ of Asp-20 in the loop connecting the helices α1 and α2 in S15. The full line is for the trajectory s15-rna, and the dotted line for the trajectory s15-rna-mg. The x axis describes the trajectory time (ns), and the y axis describes the angle values (degrees).

FIGURE 10

Angles of the RNA phosphodiester backbone ((a) α, (b) β, (c) γ, (d) δ, (e) ɛ, and (f) ζ). The mean values and the standard deviations, calculated on the 11–15-ns range, are given along the residue number (dashed line, s15-rna-mg; solid line, s15-rna). The angle values measured on the 1dk1 structure are shown by diamonds. The secondary structures of RNA are shown at the bottom.

FIGURE 11

Values of the phosphodiester backbone angles for the U1 (a), U2 (b), C3 (c), and G4 (d) residues in UUCG tetraloops. The values obtained for the PDB files (1dk1 (3), 1f7y (4), 1hlx (51), 1i6u (52), 1kuq) are shown with open circles. The values calculated from the 11–15-ns intervals in s15-rna-mg and s15-rna are shown with dots and crosses, respectively. For the PDB file 1hlx and the MD trajectories, the mean values of the angles are shown, along with the standard deviations as dotted lines.

FIGURE 12

Variation of the angles (a) between H22 and H20 and (b) between H22 and H21 in the RNA during the MD trajectories of the complex. The full line is for the trajectory s15-rna and the dotted line for the trajectory s15-rna-mg. The two angles formed by the atom triplets (107-C6, 114-N2, 142-O2) and (107-C6, 114-N2, 98-N1) are plotted in degrees, according to the trajectory time. The first angle describes the relative orientation of the helices H22 and H20, and the second angle describes the relative orientation of the helices H22 and H21. The x axis describes the trajectory time (ns), and the y axis describes the angle values (degrees).

FIGURE 13

Structures of the S15-RNA complex (x-ray (1dk1) with the Mg²⁺ ions drawn in cpk). The structures are shown in cartoon, the RNA backbone is brown, the RNA bases are cyan, and S15 is green. This figure was realized with PyMOL 0.99 (61).