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

Prolyl-tRNA synthetases (ProRSs) have been shown to activate both cognate and some noncognate amino acids and attach them to specific tRNA^Pro substrates. For example, alanine, which is smaller than cognate proline, is misactivated by <i>Escherichia coli</i> ProRS. Mischarged Ala-tRNA^Pro 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 this study, experimental and computational approaches were used to test the hypothesis that deletion of the INS alters the internal protein dynamics leading to reduced 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. Altogether, this 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

Strictly Conserved Lysine of Prolyl-tRNA Synthetase Editing Domain Facilitates Binding and Positioning of Misacylated tRNA^Pro

To ensure high fidelity in translation, many aminoacyl-tRNA synthetases, enzymes responsible for attaching specific amino acids to cognate tRNAs, require proof-reading mechanisms. Most bacterial prolyl-tRNA synthetases (ProRSs) misactivate alanine and employ a post-transfer editing mechanism to hydrolyze Ala-tRNA^Pro. This reaction occurs in a second catalytic site (INS) that is distinct from the synthetic active site. The 2′-OH of misacylated tRNA^Pro and several conserved residues in the <i>Escherichia coli</i> ProRS INS domain are directly involved in Ala-tRNA^Pro deacylation. Although mutation of the strictly conserved lysine 279 (K279) results in nearly complete loss of post-transfer editing activity, this residue does not directly participate in Ala-tRNA^Pro hydrolysis. We hypothesized that the role of K279 is to bind the phosphate backbone of the acceptor stem of misacylated tRNA^Pro and position it in the editing active site. To test this hypothesis, we carried out p<i>K</i>_a, charge neutralization, and free-energy of binding calculations. Site-directed mutagenesis and kinetic studies were performed to verify the computational results. The calculations revealed a considerably higher p<i>K</i>_a of K279 compared to an isolated lysine and showed that the protonated state of K279 is stabilized by the neighboring acidic residue. However, substitution of this acidic residue with a positively charged residue leads to a significant increase in Ala-tRNA^Pro hydrolysis, suggesting that enhancement in positive charge density in the vicinity of K279 favors tRNA binding. A charge-swapping experiment and free energy of binding calculations support the conclusion that the positive charge at position 279 is absolutely necessary for tRNA binding in the editing active site

Multiple Pathways Promote Dynamical Coupling between Catalytic Domains in <i>Escherichia coli</i> Prolyl-tRNA Synthetase

Aminoacyl-tRNA synthetases are multidomain enzymes that catalyze covalent attachment of amino acids to their cognate tRNA. Cross-talk between functional domains is a prerequisite for this process. In this study, we investigate the molecular mechanism of site-to-site communication in <i>Escherichia coli</i> prolyl-tRNA synthetase (Ec ProRS). Earlier studies have demonstrated that evolutionarily conserved and/or co-evolved residues that are engaged in correlated motion are critical for the propagation of functional conformational changes from one site to another in modular proteins. Here, molecular simulation and bioinformatics-based analysis were performed to identify dynamically coupled and evolutionarily constrained residues that form contiguous pathways of residue–residue interactions between the aminoacylation and editing domains of Ec ProRS. The results of this study suggest that multiple pathways exist between these two domains to maintain the dynamic coupling essential for enzyme function. Moreover, residues in these interaction networks are generally highly conserved. Site-directed changes of on-pathway residues have a significant impact on enzyme function and dynamics, suggesting that any perturbation along these pathways disrupts the native residue–residue interactions that are required for effective communication between the two functional domains. Free energy analysis revealed that communication between residues within a pathway and cross-talk between pathways are important for coordinating functions of different domains of Ec ProRS for efficient catalysis

Investigation of intrinsic dynamics of enzymes involved in metabolic pathways using coarse-grained normal mode analysis

<p>Intrinsic dynamics of proteins are known to play important roles in their function. In particular, collective dynamics of a protein, which are defined by the protein’s overall architecture, are important in promoting the active site conformation that favors substrate binding and effective catalysis. The primary sequence of a protein, which determines its three-dimensional structure, encodes unique dynamics. The intrinsic dynamics of a protein actually link protein structure to its function. In the present study, coarse-grained normal mode analysis was performed to examine the intrinsic dynamic patterns of 24 different enzymes involved in primary metabolic pathways. We observed that each metabolic enzyme exhibits unique patterns of motions, which are conserved across multiple species and functionally relevant. Dynamic cross-correlation matrices (DCCMs) are visibly identical for a given enzyme family but significantly different from DCCMs of other protein families, reinforcing that proteins with similar function exhibit a similar pattern of motions. The present work also reasserted that correct identification of unknown proteins is possible based on their intrinsic mobility patterns.</p