Dissimilar Roles of the Four Conserved Acidic Residues in the Thermal Stability of Poly(A)-Specific Ribonuclease

Abstract

Divalent metal ions are essential for the efficient catalysis and structural stability of many nucleotidyl-transfer enzymes. Poly(A)-specific ribonuclease (PARN) belongs to the DEDD superfamily of 3′-exonucleases, and the active site of PARN contains four conserved acidic amino acid residues that coordinate two Mg2+ ions. In this research, we studied the roles of these four acidic residues in PARN thermal stability by mutational analysis. It was found that Mg2+ significantly decreased the rate but increased the aggregate size of the 54 kDa wild-type PARN in a concentration-dependent manner. All of the four mutants decreased PARN thermal aggregation, while the aggregation kinetics of the mutants exhibited dissimilar Mg2+-dependent behavior. A comparison of the kinetic parameters indicated that Asp28 was the most crucial one to the binding of the two Mg2+ ions, while metal B might be more important in PARN structural stability. The spectroscopic and aggregation results also suggested that the alterations in the active site structure by metal binding or mutations might lead to a global conformational change of the PARN molecule.

The N-terminus modulates human Caf1 activity, structural stability and aggregation

“…As temperature increased, all four proteins aggregated faster with an increase of the aggregation rate k and a decrease of the lag time t 0 . However, the maximum turbidity A lim reached its maximum at 53 • C. The k·A lim value, which reflects the initial velocity of the aggregation [33], has been suggested to be a better monitor than the single ones to evaluate the impact of mutations on aggregation [34]. Consistent with the exponential dependence of the Gibbs free energy changes on temperature, the log 10 (k·A lim ) value showed a linear dependence on temperature (Fig.…”

Deadenylation: enzymes, regulation, and functional implications

“…PARN follows a typical two‐metal‐ion catalytic mechanism, which may also adopt by the other deadenylases based on the high conservation of the active site in the catalytic domain. The existence of Mg 2+ in the active site of deadenylase is not only essential for its activity but also modulates its stability and/or interactions with the non‐nuclease domains . Besides Mg 2+ , the other divalent metal ions, such as Mn 2+ , Co 2+ , Ca 2+ , and Zn 2+ , may also be applicable for deadenylases and sometimes can modulate the substrate selectivity of the enzyme .…”

Biochemical and Biophysical Characterization of the Deadenylase CrCaf1 from Chlamydomonas reinhardtii

“…Previous results have shown that the coordination of Mg 2+ stabilizes the active site of PARN, but promotes the aggregation of the 54 kDa PARN isoform at high temperatures [23], [36]. The effects of divalent metal ions on CrCaf1 stability was studied by thermal denaturation experiments.…”

“…This suggested that the coordination of divalent metal ions in the active site might modulate the repulsion of the negative charges of the four acidic residues, which further stabilized the protein. It is worth noting that the turbidity, which reflects the size and amount of the aggregates, is not a good evaluator of the action of cosolutes in some cases [35], [36]. Thus a quantitative evaluation was achieved by measuring the aggregation kinetics at 55°C (Figure 6C).…”

“…The thermal aggregation of the samples was monitored by measuring the turbidity at 400 nm ( A 400 ) with an Ultraspec 4300 pro UV/Visible spectrophotometer using a 1 ml cuvette. The aggregation kinetics was obtained by heating the protein samples at a given temperature, and the turbidity at 400 nm was recorded every 2 s. The kinetic data were analyzed along a first-order aggregation reaction using the following equation 35,36:where t is the time of incubation at a given temperature, A lim is the A 400 value at the infinite time and k is the rate constant of the first order reaction. Data fitting was performed by nonlinear regression analysis using the software Prism (GraphPad Inc.).…”