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Solution structure and DNA-binding properties of the phosphoesterase domain of DNA ligase D.

Natarajan A, Dutta K, Temel DB, Nair PA, Shuman S, Ghose R - Nucleic Acids Res. (2011)

Bottom Line: PE exemplifies a new family of DNA end-healing enzymes found in all phylogenetic domains.PaePE has a disordered N-terminus and a well-folded core that differs in instructive ways from the crystal structure of a PaePE•Mn(2+)• sulfate complex, especially at the active site that is found to be conformationally dynamic.Spectral perturbations measured in the presence of weakly catalytic (Cd(2+)) and inhibitory (Zn(2+)) metals provide evidence for significant conformational changes at and near the active site, compared to the relatively modest changes elicited by Mn(2+).

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, The City College of New York, 160 Convent Avenue, New York, NY 10031, USA.

ABSTRACT
The phosphoesterase (PE) domain of the bacterial DNA repair enzyme LigD possesses distinctive manganese-dependent 3'-phosphomonoesterase and 3'-phosphodiesterase activities. PE exemplifies a new family of DNA end-healing enzymes found in all phylogenetic domains. Here, we determined the structure of the PE domain of Pseudomonas aeruginosa LigD (PaePE) using solution NMR methodology. PaePE has a disordered N-terminus and a well-folded core that differs in instructive ways from the crystal structure of a PaePE•Mn(2+)• sulfate complex, especially at the active site that is found to be conformationally dynamic. Chemical shift perturbations in the presence of primer-template duplexes with 3'-deoxynucleotide, 3'-deoxynucleotide 3'-phosphate, or 3' ribonucleotide termini reveal the surface used by PaePE to bind substrate DNA and suggest a more efficient engagement in the presence of a 3'-ribonucleotide. Spectral perturbations measured in the presence of weakly catalytic (Cd(2+)) and inhibitory (Zn(2+)) metals provide evidence for significant conformational changes at and near the active site, compared to the relatively modest changes elicited by Mn(2+).

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(A) Residues important for the catalytic activity of PaePE. Gln40 and Glu82 (along with Arg14 and Glu15 on the disordered N-terminus, not shown) important for the 3′-phosphomonoesterase activity are colored magenta; Lys66 and Arg76 that influence the 3′-phosphodiesterase activity are colored blue; His42, His48, Asp50, His84 and Trp88 influence both activities, are colored cyan. (B) Schematic representation of PaePE showing the secondary structural elements in the same orientation (looking down into the catalytic cavity) as in (a) and the left panels of (C–E). The location of the catalytic residues as in (a) is indicated by the dotted oval. The strands comprising the β3–β4–β7–β8 face that shows the largest perturbations in the presence of oligonucleotide is indicated by the red dots. (c) Influence of D18 on PaePE. Residues that are broadened out beyond detection during the titration course are colored blue and residues that display chemical shift changes [Δδ, Equation (1)] >0.11 ppm at an equimolar protein:oligonucleotide ratio, are colored red. The circled patches represent perturbations involving residues near pockets of positive charges on the face opposite to that bearing the catalytic residues. These perturbations are likely due to non-productive binding events. Residues for which data were not analyzed due to missing assignments, spectral overlap or weak peaks in the reference state, are colored green. Residues that show no significant spectral perturbations in the presence of oligonucleotide are shown in gray. The effects of D18p are quite similar to those of D18. (d) While the overall spectral perturbations on the catalytic face, in the presence of D16F1R1, are similar as in D18 and D18p, the perturbations on the face opposite to that bearing the catalytic residues, are not seen. (e) Sequence conservation in bacterial and archaeal PE domains are depicted on the PaePE surface using a cyan (least conserved) to maroon (most conserved) gradient. The catalytic residues and a large part of the putative oligonucleotide binding surface are well conserved.
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gkr950-F5: (A) Residues important for the catalytic activity of PaePE. Gln40 and Glu82 (along with Arg14 and Glu15 on the disordered N-terminus, not shown) important for the 3′-phosphomonoesterase activity are colored magenta; Lys66 and Arg76 that influence the 3′-phosphodiesterase activity are colored blue; His42, His48, Asp50, His84 and Trp88 influence both activities, are colored cyan. (B) Schematic representation of PaePE showing the secondary structural elements in the same orientation (looking down into the catalytic cavity) as in (a) and the left panels of (C–E). The location of the catalytic residues as in (a) is indicated by the dotted oval. The strands comprising the β3–β4–β7–β8 face that shows the largest perturbations in the presence of oligonucleotide is indicated by the red dots. (c) Influence of D18 on PaePE. Residues that are broadened out beyond detection during the titration course are colored blue and residues that display chemical shift changes [Δδ, Equation (1)] >0.11 ppm at an equimolar protein:oligonucleotide ratio, are colored red. The circled patches represent perturbations involving residues near pockets of positive charges on the face opposite to that bearing the catalytic residues. These perturbations are likely due to non-productive binding events. Residues for which data were not analyzed due to missing assignments, spectral overlap or weak peaks in the reference state, are colored green. Residues that show no significant spectral perturbations in the presence of oligonucleotide are shown in gray. The effects of D18p are quite similar to those of D18. (d) While the overall spectral perturbations on the catalytic face, in the presence of D16F1R1, are similar as in D18 and D18p, the perturbations on the face opposite to that bearing the catalytic residues, are not seen. (e) Sequence conservation in bacterial and archaeal PE domains are depicted on the PaePE surface using a cyan (least conserved) to maroon (most conserved) gradient. The catalytic residues and a large part of the putative oligonucleotide binding surface are well conserved.

Mentions: Positively charged residues distributed over the surface of PaePE (Supplementary Figure S7) could potentially act as loci for DNA binding. In order to determine the DNA-binding surface of PaePE, we analyzed spectral perturbations induced in 15N,1H TROSY spectra of uniformly 15N,2H-labeled protein by increasing concentrations of the 18-mer primer–template duplex, D18 (Figure 4a). Many spectral perturbations were seen in the presence of D18 (Figure 4b) reflecting the alteration of the chemical environment of the main chain nuclei due the combined effects of direct binding and induced conformational changes. Many residues were broadened beyond the threshold of detection early in the titration course (Supplementary Table S1). This extensive line broadening indicates exchange on the intermediate timescale (33). The broadened resonances correspond to residues situated near the active site, on the β3–β4 loop, on strands β4, β5 and β7, and on the β7–β8 loop. The positively charged residues His127, Lys136 and Arg140 were also completely broadened out early during the titration. From this group of perturbed residues, those on β5 (Asp109 and Gly111 flank the positively charged Arg110), and the set of positively charged residues that show localized perturbations (Lys136, Arg140) are quite distant from the catalytic site, all lying on the opposite face of the protein (Figure 5c). If one or more of these residues were to take part in a productive binding event, a substantial distortion would be required of the duplex bound to this patch to access the catalytic site. A conformational change in this region in response to DNA binding at the opposite face, while also possible, is difficult to rationalize without further evidence. It is therefore likely that the spectral perturbations seen in the presence of the D18 primer–template result from both productive and non-productive binding events. In addition to these extensive line-broadening effects, significant chemical shift changes (Supplementary Figure S8) were also seen for several residues in and around strands β4 and β8.Figure 4.


Solution structure and DNA-binding properties of the phosphoesterase domain of DNA ligase D.

Natarajan A, Dutta K, Temel DB, Nair PA, Shuman S, Ghose R - Nucleic Acids Res. (2011)

(A) Residues important for the catalytic activity of PaePE. Gln40 and Glu82 (along with Arg14 and Glu15 on the disordered N-terminus, not shown) important for the 3′-phosphomonoesterase activity are colored magenta; Lys66 and Arg76 that influence the 3′-phosphodiesterase activity are colored blue; His42, His48, Asp50, His84 and Trp88 influence both activities, are colored cyan. (B) Schematic representation of PaePE showing the secondary structural elements in the same orientation (looking down into the catalytic cavity) as in (a) and the left panels of (C–E). The location of the catalytic residues as in (a) is indicated by the dotted oval. The strands comprising the β3–β4–β7–β8 face that shows the largest perturbations in the presence of oligonucleotide is indicated by the red dots. (c) Influence of D18 on PaePE. Residues that are broadened out beyond detection during the titration course are colored blue and residues that display chemical shift changes [Δδ, Equation (1)] >0.11 ppm at an equimolar protein:oligonucleotide ratio, are colored red. The circled patches represent perturbations involving residues near pockets of positive charges on the face opposite to that bearing the catalytic residues. These perturbations are likely due to non-productive binding events. Residues for which data were not analyzed due to missing assignments, spectral overlap or weak peaks in the reference state, are colored green. Residues that show no significant spectral perturbations in the presence of oligonucleotide are shown in gray. The effects of D18p are quite similar to those of D18. (d) While the overall spectral perturbations on the catalytic face, in the presence of D16F1R1, are similar as in D18 and D18p, the perturbations on the face opposite to that bearing the catalytic residues, are not seen. (e) Sequence conservation in bacterial and archaeal PE domains are depicted on the PaePE surface using a cyan (least conserved) to maroon (most conserved) gradient. The catalytic residues and a large part of the putative oligonucleotide binding surface are well conserved.
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gkr950-F5: (A) Residues important for the catalytic activity of PaePE. Gln40 and Glu82 (along with Arg14 and Glu15 on the disordered N-terminus, not shown) important for the 3′-phosphomonoesterase activity are colored magenta; Lys66 and Arg76 that influence the 3′-phosphodiesterase activity are colored blue; His42, His48, Asp50, His84 and Trp88 influence both activities, are colored cyan. (B) Schematic representation of PaePE showing the secondary structural elements in the same orientation (looking down into the catalytic cavity) as in (a) and the left panels of (C–E). The location of the catalytic residues as in (a) is indicated by the dotted oval. The strands comprising the β3–β4–β7–β8 face that shows the largest perturbations in the presence of oligonucleotide is indicated by the red dots. (c) Influence of D18 on PaePE. Residues that are broadened out beyond detection during the titration course are colored blue and residues that display chemical shift changes [Δδ, Equation (1)] >0.11 ppm at an equimolar protein:oligonucleotide ratio, are colored red. The circled patches represent perturbations involving residues near pockets of positive charges on the face opposite to that bearing the catalytic residues. These perturbations are likely due to non-productive binding events. Residues for which data were not analyzed due to missing assignments, spectral overlap or weak peaks in the reference state, are colored green. Residues that show no significant spectral perturbations in the presence of oligonucleotide are shown in gray. The effects of D18p are quite similar to those of D18. (d) While the overall spectral perturbations on the catalytic face, in the presence of D16F1R1, are similar as in D18 and D18p, the perturbations on the face opposite to that bearing the catalytic residues, are not seen. (e) Sequence conservation in bacterial and archaeal PE domains are depicted on the PaePE surface using a cyan (least conserved) to maroon (most conserved) gradient. The catalytic residues and a large part of the putative oligonucleotide binding surface are well conserved.
Mentions: Positively charged residues distributed over the surface of PaePE (Supplementary Figure S7) could potentially act as loci for DNA binding. In order to determine the DNA-binding surface of PaePE, we analyzed spectral perturbations induced in 15N,1H TROSY spectra of uniformly 15N,2H-labeled protein by increasing concentrations of the 18-mer primer–template duplex, D18 (Figure 4a). Many spectral perturbations were seen in the presence of D18 (Figure 4b) reflecting the alteration of the chemical environment of the main chain nuclei due the combined effects of direct binding and induced conformational changes. Many residues were broadened beyond the threshold of detection early in the titration course (Supplementary Table S1). This extensive line broadening indicates exchange on the intermediate timescale (33). The broadened resonances correspond to residues situated near the active site, on the β3–β4 loop, on strands β4, β5 and β7, and on the β7–β8 loop. The positively charged residues His127, Lys136 and Arg140 were also completely broadened out early during the titration. From this group of perturbed residues, those on β5 (Asp109 and Gly111 flank the positively charged Arg110), and the set of positively charged residues that show localized perturbations (Lys136, Arg140) are quite distant from the catalytic site, all lying on the opposite face of the protein (Figure 5c). If one or more of these residues were to take part in a productive binding event, a substantial distortion would be required of the duplex bound to this patch to access the catalytic site. A conformational change in this region in response to DNA binding at the opposite face, while also possible, is difficult to rationalize without further evidence. It is therefore likely that the spectral perturbations seen in the presence of the D18 primer–template result from both productive and non-productive binding events. In addition to these extensive line-broadening effects, significant chemical shift changes (Supplementary Figure S8) were also seen for several residues in and around strands β4 and β8.Figure 4.

Bottom Line: PE exemplifies a new family of DNA end-healing enzymes found in all phylogenetic domains.PaePE has a disordered N-terminus and a well-folded core that differs in instructive ways from the crystal structure of a PaePE•Mn(2+)• sulfate complex, especially at the active site that is found to be conformationally dynamic.Spectral perturbations measured in the presence of weakly catalytic (Cd(2+)) and inhibitory (Zn(2+)) metals provide evidence for significant conformational changes at and near the active site, compared to the relatively modest changes elicited by Mn(2+).

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, The City College of New York, 160 Convent Avenue, New York, NY 10031, USA.

ABSTRACT
The phosphoesterase (PE) domain of the bacterial DNA repair enzyme LigD possesses distinctive manganese-dependent 3'-phosphomonoesterase and 3'-phosphodiesterase activities. PE exemplifies a new family of DNA end-healing enzymes found in all phylogenetic domains. Here, we determined the structure of the PE domain of Pseudomonas aeruginosa LigD (PaePE) using solution NMR methodology. PaePE has a disordered N-terminus and a well-folded core that differs in instructive ways from the crystal structure of a PaePE•Mn(2+)• sulfate complex, especially at the active site that is found to be conformationally dynamic. Chemical shift perturbations in the presence of primer-template duplexes with 3'-deoxynucleotide, 3'-deoxynucleotide 3'-phosphate, or 3' ribonucleotide termini reveal the surface used by PaePE to bind substrate DNA and suggest a more efficient engagement in the presence of a 3'-ribonucleotide. Spectral perturbations measured in the presence of weakly catalytic (Cd(2+)) and inhibitory (Zn(2+)) metals provide evidence for significant conformational changes at and near the active site, compared to the relatively modest changes elicited by Mn(2+).

Show MeSH