Active site conformational dynamics are coupled to catalysis in the mRNA decapping enzyme Dcp2

Abstract

Removal of the 5' cap structure by Dcp2 is a major step in several 5'-3' mRNA decay pathways. The activity of Dcp2 is enhanced by Dcp1 and bound coactivators, yet the details of how these interactions are linked to chemistry are poorly understood. Here, we report three crystal structures of the catalytic Nudix hydrolase domain of Dcp2 that demonstrate binding of a catalytically essential metal ion, and enzyme kinetics are used to identify several key active site residues involved in acid/base chemistry of decapping. Using nuclear magnetic resonance and molecular dynamics, we find that a conserved metal binding loop on the catalytic domain undergoes conformational changes during the catalytic cycle. These findings describe key events during the chemical step of decapping, suggest local active site conformational changes are important for activity, and provide a framework to explain stimulation of catalysis by the regulatory domain of Dcp2 and associated coactivators.

Figure 1. Mg²⁺ coordination in the Dcp2 catalytic domain

(A) Ribbon diagram of Mg²⁺ coordination in the wild-type S. cerevisiae Dcp2 catalytic Nudix domain at the catalytic helix. Four conserved glutamates (E149, E152, E153, E198) and K135 are shown as sticks in green. Water molecules are shown as blue spheres, and the Mg²⁺ is shown as a black sphere. Colored in grey is the backbone carbonyl of N243 in a symmetry related molecule. Distances between atoms are shown in Å. (B) Fo−Fc difference electron density map of wild-type Dcp2 depicted as black mesh at an I/σ cutoff of 2.5. (C) Fo−Fc difference electron density map for the E198Q mutant at an I/σ cutoff of 2.0. (D) Fo−Fc difference electron density map of E153Q mutant at an I/sigma cutoff of 3.0.

Figure 2. Decapping rates for wild-type and mutant Dcp1/Dcp2 are affected by pH

(A) Representative time courses of the fraction m7GDP released and the corresponding first-order exponential fits to obtain k_obs over a range of pH values for wild-type Dcp1/Dcp2 decapping complex. (B) Plot of log (k_max) versus pH for wild-type decapping complex (green), K135A (yellow) and E153Q (purple). Symbols are mean of at least 3 independent experiments and error bars shown are standard deviation. Wild-type and K135A are fit using the 4-parameter equation used to model the dependence of k_max on pH (Harris et al., 2000). E153Q is fit to a line due to the linear dependence of k_max on pH.

Figure 3. Active site mutations in Dcp1/2 complex result in multiple decapping reaction products

(A) Representative TLC plate decapping assay data for Dcp1/2 K135A decapping complex at pH 8.0. Arrows point out the location of RNA substrate (black), m7GTP (green), m7GDP (blue) and m7GMP (red) as identified by incubation with NDPK (Fig. S2E). (B) Representative time course of the fraction of each K135A product formed as a function of time. m7GTP, m7GDP, and m7GMP are shown in green, blue, and red respectively. Total product formed is shown in black. Single exponential fits were used to obtain the individual k_obs for each product (see methods and Tables S1, S2). (C) Distribution of each product formed in the decapping reactions of wild-type, K135A, and E153Q variants, calculated by an average ratio of endpoints for each product across all pH values fit using a single exponential (Table S1). (D) Representative TLC plate for E153Q showing multiple products. Arrows point out the location of RNA substrate (black), m7GTP (green), m7GDP (blue) and m7GMP (red) as identified by incubation with NDPK (Fig. S2E). (E) Representative time course for the fraction of each E153Q product formed as a function of time. Coloring is the same as in (B). Single exponential fits were used to obtain the individual k_obs for each product (see methods and Tables S1, S2).

Figure 4. Methyl NMR pH titrations confirm E153 is the general base

(A) Select Ile, Leu and Val residues on the catalytic domain of S. cerevisiae Dcp2 are highlighted in blue with catalytic glutamates E149, E152, E153 and E198 highlighted in green and the Nudix helix in red. Residues are visualized on PDB 2JVB. (B) A superposition of ¹³C-¹H HSQC spectra of wild-type scDcp2 (100–245) at pH 4.5 (red), 5.5 (orange), 6.5 (lime), 7.1 (green), 7.6 (cyan), 8.0 (magenta) and 9.0 (purple). (C) Quantification of the normalized chemical shift change (Δδ) for L120, V121 and I199 across the pH titration. Note that the sidechain of L120 points in the opposite direction from V121. Solid lines are sigmoidal fits, with pKa_app values of 7.3 ± 0.02 and 7.2 ± 0.02 for I199 and V121, respectively. Standard error of the fit parameter is indicated. (D) ¹³C-¹H HSQC spectra of the E198Q mutant Dcp2 catalytic domain collected at pH values of 5 (red), 5.6 (orange), 6.0 (lime), 6.6 (green), 7.2 (cyan), 8.1 (magenta) and 8.9 (purple). Weak peaks in (B) are likely from residual contamination by the GB1 solubility tag. (E) ¹³C-¹H HSQC spectra of the E153Q mutant Dcp2 catalytic domain collected at pH values of 5.5 (orange), 6.6 (lime), 7.0 (green), 7.6 (cyan) and 8.5 (purple).

Figure 5. Change in pH Induces Changes in Dynamics

(A) The ¹³C linewidth (FWHM) for residues I199, V121 and L115 across the pH titration. (B–E) Example fitted ¹³C linewidths of I199 at pH values indicated. The experimental data is in points with the mixed Gaussian-Lorentzian fit as a line. Intensity is in units of signal-to-noise (S:N) with noise measured in NMRPipe.

Figure 6. Molecular dynamics simulations show qualitative and quantitative conformational changes between S. pombe Dcp2 with charged or protonated E147

(A) Simulations with protonated E147 to mimic the low pH state show relatively little flexibility in the 190’s loop. I193 (I199 in S. cerevisiae) is shown in blue sticks, V114 in red sticks, and the Nudix helix is colored green. (B) Simulations with charged E147 to mimic the high pH state show increased flexibility with I193 solvent exposed in the final state of two out of six simulations. Colors are the same as in (A). (C) Histograms of the distance between the terminal methyl groups of V114 and I193 when E147 is protonated to mimic the low pH state (C), when E147 is mutated to glutamine (D), or when E147 is charged to mimic the high pH state (E). The ordinate of all three histograms is probability density per bin; n ≈ 600,000 per histogram. Residues I193, V114 and E147 in S. pombe are S. cerevisiae I199, V121 and E153, respectively.