Evidence that a catalytic glutamate and an 'Arginine Toggle' act in concert to mediate ATP hydrolysis and mechanochemical coupling in a viral DNA packaging motor

Abstract

ASCE ATPases include ring-translocases such as cellular helicases and viral DNA packaging motors (terminases). These motors have conserved Walker A and B motifs that bind Mg2+-ATP and a catalytic carboxylate that activates water for hydrolysis. Here we demonstrate that Glu179 serves as the catalytic carboxylate in bacteriophage λ terminase and probe its mechanistic role. All changes of Glu179 are lethal: non-conservative changes abrogate ATP hydrolysis and DNA translocation, while the conservative E179D change attenuates ATP hydrolysis and alters single molecule translocation dynamics, consistent with a slowed chemical hydrolysis step. Molecular dynamics simulations of several homologous terminases suggest a novel mechanism, supported by experiments, wherein the conserved Walker A arginine 'toggles' between interacting with a glutamate residue in the 'lid' subdomain and the catalytic glutamate upon ATP binding; this switch helps mediate a transition from an 'open' state to a 'closed' state that tightly binds nucleotide and DNA, and also positions the catalytic glutamate next to the γ-phosphate to align the hydrolysis transition state. Concomitant reorientation of the lid subdomain may mediate mechanochemical coupling of ATP hydrolysis and DNA translocation. Given the strong conservation of these structural elements in terminase enzymes, this mechanism may be universal for viral packaging motors.

Figure 1.

The Lambda Genome Packaging Pathway. One TerL subunit (cyan) and two TerS subunits (blue) tightly associate in the terminase protomer. TerL maturation (Nuc) and packaging (ATP) domains are indicated. Pathway details are provided in the text. Putative Walker B motif (VAGYD) and the conserved glutamate (E179) are indicated.

Figure 2.

Structural characterization of the mutant terminase enzymes. (A) Far-UV CD spectra of wild type and mutant terminase enzymes indicate that they all possess similar secondary structures. (B) Thermal denaturation of WT and mutant terminases. Solid lines are the best fits of the data to obtain T_m presented in Supplementary Table S3. (C) Sedimentation Velocity Analytical Ultracentrifugation analysis. The WT enzyme assembles into a protomer species (6S) that is in slow equilibrium with a ring-like tetramer of protomers (16S). The E179A and E179Q enzymes show native-like assembly behavior but the E179D mutant displays significant defects. The novel 3.3 species is likely the isolated TerL subunit which indicates dissociation of the mutant terminase protomer. The isolated TerS subunit is not observed in the sedimentation experiment because it avidly aggregates (73) and it has pelleted from solution during the AUC run.

Figure 3.

Biochemical Characterization of the Mutant Terminase Enzymes. (A) cos-cleavage endonuclease activity was quantified as described in Methods. The TerL^λ-E179D enzyme possesses a strong, non-specific nuclease activity that precludes accurate quantitation of specific cleavage at the cos site (not shown). Each bar represents the normalized average activity of at least three independent experiments with standard deviations indicated with error bars. (B) Steady state ATPase activity quantified as described in Methods. Each bar represents the normalized average activity of at least three independent experiments with standard deviation indicated. (C) Single turnover ATP hydrolysis quantified as described in Methods: black •, wild type terminase; green ♦, E179A mutant terminase; red ◊, E179Q mutant terminase. Each data point represents the average of at least three independent experiments with standard deviations indicated (in some cases the error bars are obscured by the data point). The solid lines represent the best fit of the data which affords the rate constants presented in Table 2. (D) DNA packaging activity was quantified as described in Methods. Each bar represents the normalized average activity of at least three independent experiments with standard deviations indicated with error bars.

Figure 4.

Single DNA Molecule Translocation Measurements. (A) Examples of single DNA molecule translocation measurements for wild type (WT) terminase with saturating ATP (500 μM) (top left), wildtype terminase with 2.5 μM ATP (top right) and E179D mutant terminase with 500 μM ATP (bottom). (B) Slipping frequency vs. motor velocity for WT and mutant terminases. (C) Pausing frequency vs. motor velocity for WT and mutant terminases. The results for WA V80A and R79K mutants published previously (41) are shown for comparison. Unless otherwise indicated, measurements were with saturating ATP (500 μM).

Figure 5.

Conformations of the ATP-Binding Pocket in WT TerL obtained from MD Simulations. Representative conformations of the apo binding pocket of TerL^T4 (A), TerL^P74-26 (B) and TerL^Sf6 (C). Walker motif residues and lid subdomain glutamate (see Table 1) are labeled and depicted as sticks. The WA arginine, catalytic glutamate and lid subdomain glutamate are highlighted in tan. Representative conformations of the ATP-bound binding pocket of TerL^T4 (D), TerL^P74-26 (E) and TerL^Sf6 (F). ATP is depicted as sticks, and Mg²⁺ is a green sphere. The WA arginine, catalytic glutamate and lid subdomain glutamate are labeled and are highlighted in cyan. In all three terminases, the conserved WA arginine (R162, R39, and R24 in TerL^T4, TerL^P74-26, and TerL^Sf6, respectively) interacts with the catalytic glutamate (E256, E150, E119 in TerL^T4, TerL^P74-26, and TerL^Sf6) near the γ-phosphate of ATP.

Figure 6.

Principal Component Analysis of TerL^T4 Reveals Lid Subdomain Motion Upon ATP Binding. (A) Principal component analysis (PCA) of TerL^T4 apo simulation shows little concerted motion of the lid subdomain (also called the NII-subdomain in TerL^T4 literature), circled by black dashed circle. Residues are colored based on the magnitude of root-mean-square fluctuations during the simulation, with red representing least mobile residues and blue representing most mobile residues. Arrows represent the projection of the individual residue motions onto the first principal component. (B) Principal component analysis of the first 10 ns of TerL^T4 ATP-bound simulation shows concerted rotation of the lid subdomain towards the ATPase active site consistent with prior experimental studies of Sf6 and P74-26 terminases. (C) Principal component analysis of the last 10 ns of TerL^T4 ATP-bound simulation shows little lid subdomain motion, indicating that the lid subdomain is stable in the closed conformation once the rotation is complete.

Figure 7.

Effects of the Conservative ASCE E→D Mutation Obtained from MD Simulations. Representative conformations of the ATP-binding pocket of ATP-bound WT TerLs (tan) superimposed onto the binding pocket conformations of the corresponding catalytic E→D mutant TerLs (cyan). Such analysis carried out for TerL^T4 (A) and TerL^P74-26 (B) structures show significant displacement of the labeled carboxylate functional group and miscoordination of the labeled γ-phosphate of ATP in both mutant structures. Separation distances, obtained from an equilibrium MD trajectory, between the carboxylate functional group and the γ-phosphate for WT (black) and catalytic E→D mutants (red) of TerL^T4 (C) and TerL^P74-26 (D). In both cases, the E→D change results in a significant increase this distance and the hindered ATP hydrolysis observed experimentally in the E→D mutants is attributed to this increased distance.