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Dissimilar roles of the four conserved acidic residues in the thermal stability of poly(A)-specific ribonuclease.

He GJ, Liu WF, Yan YB - Int J Mol Sci (2011)

Bottom Line: It was found that Mg(2+) 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 Mg(2+)-dependent behavior.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing 100084, China; E-Mails: he-gj06@mails.tsinghua.edu.cn (G.-J.H.); liuwf@mail.tsinghua.edu.cn (W.-F.L.).

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 Mg(2+) ions. In this research, we studied the roles of these four acidic residues in PARN thermal stability by mutational analysis. It was found that Mg(2+) 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 Mg(2+)-dependent behavior. A comparison of the kinetic parameters indicated that Asp28 was the most crucial one to the binding of the two Mg(2+) 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.

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Structure of the active site of PARN (adapted from reference [18] and PDB ID 2A1S). The four conserved acidic residues are highlighted by ball-and-stick model. The Mg2+ binding sites are predicted on the basis of previous studies [16,18]. The dashed lines indicate the possible interaction network that stabilizes the two Mg2+ ions. For clarity, the substrate molecule was not shown.
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f1-ijms-12-02901: Structure of the active site of PARN (adapted from reference [18] and PDB ID 2A1S). The four conserved acidic residues are highlighted by ball-and-stick model. The Mg2+ binding sites are predicted on the basis of previous studies [16,18]. The dashed lines indicate the possible interaction network that stabilizes the two Mg2+ ions. For clarity, the substrate molecule was not shown.

Mentions: Poly(A)-specific ribonuclease (PARN, EC 3.1.13.4) is involved in the mRNA decay regulation by specifically catalyzing the shortening of the 3′-end poly(A) tail [14,15]. The full length PARN contains three well-defined domains: The catalytic domain, the R3H domain and the RNA-recognition motif (RRM). The catalytic nuclease domain belongs to the DEDD superfamily of 3′-exonucleases, and shares a similar conserved core structure to the other members in this superfamily [16]. The active site of PARN contains four conserved acidic amino acid residues, D28, E30, D292 and D382, which are essential for the catalytic activity of PARN [17,18]. These acidic residues form a negative charge cave that can bind with two Mg2+ ions (Figure 1). It has been proposed that PARN may utilize the general two-metal ion mechanism for catalysis based on biochemical [18] and structural analysis [16] although no metal ion is present in the crystal structures of ligand-free and substrate-bound PARN [16]. The cofactor Mg2+ is also important to PARN stability against inactivation induced by heat treatment, but promotes thermal aggregation at high temperatures [12]. Because mutation of any of the four conserved acidic residues in the active site will inactivate PARN, we evaluated the roles of the two Mg2+ ions in PARN stability by thermal aggregation of the wild type (WT) and mutated enzymes. The data indicated that all of the four mutants decreased PARN thermal aggregation. The aggregation kinetics of the mutants exhibited dissimilar Mg2+ dependence, suggesting that these four conserved acidic residues played differential roles in Mg2+ coordination and protein stability.


Dissimilar roles of the four conserved acidic residues in the thermal stability of poly(A)-specific ribonuclease.

He GJ, Liu WF, Yan YB - Int J Mol Sci (2011)

Structure of the active site of PARN (adapted from reference [18] and PDB ID 2A1S). The four conserved acidic residues are highlighted by ball-and-stick model. The Mg2+ binding sites are predicted on the basis of previous studies [16,18]. The dashed lines indicate the possible interaction network that stabilizes the two Mg2+ ions. For clarity, the substrate molecule was not shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3116163&req=5

f1-ijms-12-02901: Structure of the active site of PARN (adapted from reference [18] and PDB ID 2A1S). The four conserved acidic residues are highlighted by ball-and-stick model. The Mg2+ binding sites are predicted on the basis of previous studies [16,18]. The dashed lines indicate the possible interaction network that stabilizes the two Mg2+ ions. For clarity, the substrate molecule was not shown.
Mentions: Poly(A)-specific ribonuclease (PARN, EC 3.1.13.4) is involved in the mRNA decay regulation by specifically catalyzing the shortening of the 3′-end poly(A) tail [14,15]. The full length PARN contains three well-defined domains: The catalytic domain, the R3H domain and the RNA-recognition motif (RRM). The catalytic nuclease domain belongs to the DEDD superfamily of 3′-exonucleases, and shares a similar conserved core structure to the other members in this superfamily [16]. The active site of PARN contains four conserved acidic amino acid residues, D28, E30, D292 and D382, which are essential for the catalytic activity of PARN [17,18]. These acidic residues form a negative charge cave that can bind with two Mg2+ ions (Figure 1). It has been proposed that PARN may utilize the general two-metal ion mechanism for catalysis based on biochemical [18] and structural analysis [16] although no metal ion is present in the crystal structures of ligand-free and substrate-bound PARN [16]. The cofactor Mg2+ is also important to PARN stability against inactivation induced by heat treatment, but promotes thermal aggregation at high temperatures [12]. Because mutation of any of the four conserved acidic residues in the active site will inactivate PARN, we evaluated the roles of the two Mg2+ ions in PARN stability by thermal aggregation of the wild type (WT) and mutated enzymes. The data indicated that all of the four mutants decreased PARN thermal aggregation. The aggregation kinetics of the mutants exhibited dissimilar Mg2+ dependence, suggesting that these four conserved acidic residues played differential roles in Mg2+ coordination and protein stability.

Bottom Line: It was found that Mg(2+) 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 Mg(2+)-dependent behavior.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing 100084, China; E-Mails: he-gj06@mails.tsinghua.edu.cn (G.-J.H.); liuwf@mail.tsinghua.edu.cn (W.-F.L.).

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 Mg(2+) ions. In this research, we studied the roles of these four acidic residues in PARN thermal stability by mutational analysis. It was found that Mg(2+) 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 Mg(2+)-dependent behavior. A comparison of the kinetic parameters indicated that Asp28 was the most crucial one to the binding of the two Mg(2+) 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.

Show MeSH