Role of Coupled Dynamics in the Catalytic Activity of Prokaryotic-like Prolyl-tRNA Synthetases

Abstract

Prolyl-tRNA synthetases (ProRSs) have been shown to activate both cognate and some noncognate amino acids and attach them to specific tRNAPro substrates. For example, alanine, which is smaller than cognate proline, is misactivated by Escherichia coli ProRS. Mischarged Ala-tRNAPro is hydrolyzed by an editing domain (INS) that is distinct from the activation domain. It was previously shown that deletion of the INS greatly reduced cognate proline activation efficiency. In the present study, experimental and computational approaches were used to test the hypothesis that INS deletion alters the internal protein dynamics leading to reduce catalytic function. Kinetic studies with two ProRS variants, G217A and E218A, revealed decreased amino acid activation efficiency. Molecular dynamics studies showed motional coupling between the INS and protein segments containing the catalytically important proline-binding loop (PBL, residues 199–206). In particular, the complete deletion of INS, as well as mutation of G217 or E218 to alanine, exhibited significant effects on the motion of the PBL. The presence of coupled-dynamics between neighboring protein segments was also observed through in silico mutations and essential dynamics analysis. Taken together, the present study demonstrates that structural elements at the editing domain-activation domain interface participate in coupled motions that facilitate amino acid binding and catalysis by bacterial ProRSs, which may explain why truncated or defunct editing domains have been maintained in some systems, despite the lack of catalytic activity.

Multiple Pathways Promote Dynamical Coupling between Catalytic Domains in Escherichia coli Prolyl-tRNA Synthetase

“…The lack of activity of the path III variant suggests strong coupling between N305A and G412A since the effect of the double mutant (ΔΔ G > 3.7 based on level of detection of this assay) is greater than expected based on the single mutant effects (2.4 kcal/mol and 0.29 kcal/mol, for N305A and G412A, respectively). In a recent study, we showed that proline activation efficiency was reduced by ~ 50-fold in the E218A variant (16). Thus, the lack of activity of the path IV double mutants is not surprising.…”

“…Finally, the role of these selected residues in site-to-site communication was probed experimentally by conducting site-directed mutagenesis and kinetic studies. In addition, in silico mutations were performed and their impact on protein dynamics was examined by comparing root-mean-square (RMS) fluctuations of the WT and mutated variants (16). …”

“…All simulations were carried out with a 500 ps equilibration step followed by a 25 ns production MD run. The details of the MD simulation protocol were as described previously (16). To evaluate the statistical significance of the MD simulation analysis, three replicates were generated for each protein system, as described in the protein simulation studies by Roy and Laughton (28).…”

“…PCA is a procedure by which the low-frequency (high-amplitude) collective motions of a biomolecule, which are often more relevant for its functions, are extracted from a MD simulation trajectory (31). This method has been described in detail in our previous study with this enzyme (16). Briefly, a covariance matrix of the C α atoms was generated using the simulated MD data set.…”

Probing the global and local dynamics of aminoacyl-tRNA synthetases using all-atom and coarse-grained simulations

“…The details of the procedure have been described earlier [4, 7, 41]. The last 25 ns of the 30 ns MD simulation data was used to extract the principal modes of collective dynamics (called principal components) using the program CARMA [42].…”