Crowder-Induced Conformational Ensemble Shift in Escherichia coli Prolyl-tRNA Synthetase.

The effect of molecular crowding on the structure and function of Escherichia coli prolyl-transfer RNA synthetase (Ec ProRS), a member of the aminoacyl-transfer RNA synthetase family, has been investigated using a combined experimental and theoretical method. Ec ProRS is a multidomain enzyme; coupled-domain dynamics are essential for efficient catalysis. To gain insight into the mechanistic detail of the crowding effect, kinetic studies were conducted with varying concentrations and sizes of crowders. In parallel, spectroscopic and quantum chemical studies were employed to probe the "soft interactions" between crowders and protein side chains. Finally, the dynamics of the dimeric protein was examined in the presence of crowders using a long-duration (70 ns) classical molecular dynamic simulations. The results of the simulations revealed a shift in the conformational ensemble, which is consistent with the preferential exclusion of cosolutes. The "soft interactions" model of the crowding effect also explained the alteration in kinetic parameters. In summary, the study found that the effects of molecular crowding on both conformational dynamics and catalytic function are correlated in the multidomain Ec ProRS, an enzyme that is central to protein synthesis in all living cells. This study affirmed that large and small cosolutes have considerable impacts on the structure, dynamics, and function of modular proteins and therefore must be considered for stabilizing protein-based pharmaceuticals and industrial enzymes.

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 Escherichia coli 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 pKa, 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 pKa 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.

The role of mitochondria in anchoring dynein to the cell cortex extends beyond clustering the anchor protein.

Organelle distribution is regulated over the course of the cell cycle to ensure that each of the cells produced at the completion of division inherits a full complement of organelles. In yeast, the protein Num1 functions in the positioning and inheritance of two essential organelles, mitochondria and the nucleus. Specifically, Num1 anchors mitochondria as well as dynein to the cell cortex, and this anchoring activity is required for proper mitochondrial distribution and dynein-mediated nuclear inheritance. The assembly of Num1 into clusters at the plasma membrane is critical for both of its anchoring functions. We have previously shown that mitochondria drive the assembly of Num1 clusters and that these mitochondria-assembled Num1 clusters serve as cortical attachment sites for dynein. Here we further examine the role for mitochondria in dynein anchoring. Using a GFP-αGFP nanobody targeting system, we synthetically clustered Num1 on eisosomes to bypass the requirement for mitochondria in Num1 cluster formation. Utilizing this system, we found that mitochondria positively impact the ability of synthetically clustered Num1 to anchor dynein and support dynein function even when mitochondria are no longer required for cluster formation. Thus, the role of mitochondria in regulating dynein function extends beyond simply concentrating Num1; mitochondria likely promote an arrangement of Num1 within a cluster that is competent for dynein anchoring. This functional dependency between mitochondrial and nuclear positioning pathways likely serves as a mechanism to order and integrate major cellular organization systems over the course of the cell cycle.