Methyl Relaxation Measurements Reveal Patterns of Fast Dynamics in a Viral RNA-Directed RNA Polymerase

Abstract

Molecular dynamics (MD) simulations combined with biochemical studies have suggested the presence of long-range networks of functionally relevant conformational flexibility on the nanosecond time scale in single-subunit RNA polymerases in many RNA viruses. However, experimental verification of these dynamics at a sufficient level of detail has been lacking. Here we describe the fast, picosecond to nanosecond dynamics of an archetypal viral RNA-directed RNA polymerase (RdRp), the 75 kDa P2 protein from cystovirus ϕ12, using analyses of (1)H-(1)H dipole-dipole cross-correlated relaxation at the methyl positions of Ile (δ1), Leu, Val, and Met residues. Our results, which represent the most detailed experimental characterization of fast dynamics in a viral RdRp until date, reveal a highly connected dynamic network as predicted by MD simulations of related systems. Our results suggest that the entry portals for template RNA and substrate NTPs are relatively disordered, while conserved motifs involved in metal binding, nucleotide selection, and catalysis display greater rigidity. Perturbations at the active site through metal binding or functional mutation affect dynamics not only in the immediate vicinity but also at remote regions. Comparison with the limited experimental and extensive functional and in silico results available for homologous systems suggests conservation of the overall pattern of dynamics in viral RdRps.

Figure 1

(A) The structural domains of the RdRps (P2) from cystoviruses ϕ12 (left) and ϕ6 (right). The domains are as follows – fingers (ϕ12: 35-78, 147-303, 358-420; ϕ6: 1-30, 104-276, 333-397; red), thumb (ϕ12: 82-134, 527-600; ϕ6: 37-91, 518-600; blue), palm (ϕ12: 304-357, 421-526; ϕ6: 277-332, 398-517) and the C-terminal domain (CTD; ϕ12: 601-659; ϕ6: 601-664; yellow). (B) The conserved sequence motifs A (ϕ12: 349-359; ϕ6: 324-334; orange-red), B (ϕ12: 416-436; ϕ6: 394-413; blue), C (ϕ12: 461-477, ϕ6: 445-461; coral green), D (ϕ12: 487-500; ϕ6: 475-488; purple), E (ϕ12: 505-517; ϕ6: 493-505; pink) and F (ϕ12: 295-299; ϕ6: 268-272; orange). (C) The methyl groups of Ile (δ1, both ϕ12 and ϕ6, red), Leu (ϕ12 only, coral green), Val (ϕ12 only, pink) and Met (ϕ12 only, orange) for which resonance assignments are available are shown on the three-dimensional structures of the cystoviral P2 proteins. Unassigned Ile (δ1 only), Leu, Val and Met methyl groups are shown as grey spheres. The arrows on the top panel depict the entrance to the template entry tunnel. The substrate NTPs enter through a tunnel at the back that is almost orthogonal to the template entry tunnel.

Figure 2

(A) Close-ups of the “allowed” (left) and “forbidden” spectra for M318 and I168 showing the change in peak intensities with relaxation delay (T). The “forbidden” spectra have been contoured at a 3.8-fold lower level than the “allowed” spectra. The forbidden spectrum for T = 1 ms uses double the number of scans as the corresponding allowed spectrum. (B) The experimental intensity ratios corresponding to the spectra shown in (B) (solid circles, M318, black; I168, red) and the fits (solid lines) to Equation 2 are shown on the bottom panel.

Figure 3

Residues that show statistically significant $\mathrm{\Delta }{S}_{\mathit{\text{axis}}}^2$ values in the presence of Mg²⁺ (compared to the unliganded state of ϕ12 P2; top panel), Mg²⁺/GMPCPP (compared to the Mg²⁺-loaded state of ϕ12 P2; middle panel) and for the ts-mutant (the T425I mutant compared to wild-type, unliganded ϕ12 P2; bottom panel). $\mathrm{\Delta }{S}_{\mathit{\text{axis}}}^2$ between states i and j are considered to be statistically significant when $\mathrm{\Delta }{S}_{\mathit{\text{axis}}}^2=|S_{\mathit{\text{axis}}}^2(i)-S_{\mathit{\text{axis}}}^2(j)|>\sigma S_{\mathit{\text{axis}}}^2(i)+\sigma S_{\mathit{\text{axis}}}^2(j)+0.03$; $\sigma S_{\mathit{\text{axis}}}^2(i)$ is the error in the $S_{\mathit{\text{axis}}}^2$ value for the i^th state. Remote residues (defined as those that do not have any heavy atoms within 15 Å of any other atom of the binding/mutation site) are labeled. The fonts are colored based on the domain that houses a particular residue – red for fingers, green for palm, blue for thumb and yellow for C-terminal. Residues that comprise the secondary Mg²⁺-binding site (G348, E503, V507 and D470) are shown and colored green in the top panel. The catalytic residues (D349, D469 and D470) are colored cyan and the two GTP molecules are colored purple and magenta in the middle panel. The site of mutation (T425I) is colored orange (and labeled in black) in the bottom panel.

Figure 4

(A) Chemical shift differences for methyl positions between wild-type ϕ12 P2 and the ts-mutant, are shown. Residues that display significant perturbations (Δδ > 0.04 ppm) are labeled. The magenta font indicates residues that do not have any heavy atoms within 10 Å of any heavy atoms of the mutation site. (B) Plot of the chemical shift changes (Δδ) against the renormalized closeness scores (Π) calculated using Equation 5 for wild-type P2 (black circles) and the ts-mutant (open red circles). Residues with significant Δδ values > (0.04 ppm) also have large Π values (> 0.5) as indicated by the shaded region on the top right quadrant bound by Π = 0.5 and Δδ = 0.04 ppm (indicated by the dashed lines).

Figure 5

(A) Distribution of Σ values (calculated using Equation 6) ≤ 0.25 in P2. (B) Distribution of Σ values ≥ 0.75. (C) Distribution of Σ values in the conserved RdRp sequence motifs (A-F) that form the catalytic site. In all cases the P2 proteins from ϕ12 and ϕ6 cystoviruses are shown in the left and right panels, respectively.