Limits...
Functional importance of crenarchaea-specific extra-loop revealed by an X-ray structure of a heterotetrameric crenarchaeal splicing endonuclease.

Yoshinari S, Shiba T, Inaoka DK, Itoh T, Kurisu G, Harada S, Kita K, Watanabe Y - Nucleic Acids Res. (2009)

Bottom Line: Meanwhile, a deletion of six amino acids in a Crenarchaea-specific loop abolished the endonuclease activity even on a substrate with canonical BHB motif.These results indicate that the subunit architecture is not a major factor responsible for the difference of substrate specificity between single- and two-subunit EndA systems.Rather, the structural basis for the broad substrate specificity is built into the crenarchaeal splicing endonuclease itself.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. yoshinas@m.u-tokyo.ac.jp

ABSTRACT
Archaeal splicing endonucleases (EndAs) are currently classified into three groups. Two groups require a single subunit protein to form a homodimer or homotetramer. The third group requires two nonidentical protein components for the activity. To elucidate the molecular architecture of the two-subunit EndA system, we studied a crenarchaeal splicing endonuclease from Pyrobaculum aerophilum. In the present study, we solved a crystal structure of the enzyme at 1.7-A resolution. The enzyme adopts a heterotetrameric form composed of two catalytic and two structural subunits. By connecting the structural and the catalytic subunits of the heterotetrameric EndA, we could convert the enzyme to a homodimer that maintains the broad substrate specificity that is one of the characteristics of heterotetrameric EndA. Meanwhile, a deletion of six amino acids in a Crenarchaea-specific loop abolished the endonuclease activity even on a substrate with canonical BHB motif. These results indicate that the subunit architecture is not a major factor responsible for the difference of substrate specificity between single- and two-subunit EndA systems. Rather, the structural basis for the broad substrate specificity is built into the crenarchaeal splicing endonuclease itself.

Show MeSH
Engineered homodimeric PAE-EndAs retain splicing endonuclease activity. (A) GelCode-Blue-stained 15% SDS–PAGE gel. In each lane, 2 μg of the fraction eluted from TALON resin was loaded. (B) Splicing endonuclease assay. Sulfolobus tokodaii tRNATrp precursor was used as the substrate. Lane assignments are; M, molecular mass marker in (A); mock, mock incubation with enzyme solvent; WT, wild-type 6xHis-PAE-EndA; LP, variant LP; LPE, variant LPE; LPEI, variant LPEI. (C) Accumulation of 5′-exon on the time course.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2724299&req=5

Figure 2: Engineered homodimeric PAE-EndAs retain splicing endonuclease activity. (A) GelCode-Blue-stained 15% SDS–PAGE gel. In each lane, 2 μg of the fraction eluted from TALON resin was loaded. (B) Splicing endonuclease assay. Sulfolobus tokodaii tRNATrp precursor was used as the substrate. Lane assignments are; M, molecular mass marker in (A); mock, mock incubation with enzyme solvent; WT, wild-type 6xHis-PAE-EndA; LP, variant LP; LPE, variant LPE; LPEI, variant LPEI. (C) Accumulation of 5′-exon on the time course.

Mentions: Figure 1I shows the superimposed X-ray structures for the interdomain loop region of AFU-EndA, the C-terminal residues of the structural subunit and the N-terminal residue of the catalytic subunit of PAE-EndA. An interesting observation about the crystal structure of PAE-EndA is that the C-terminal residues of the structural subunit form an extended arm toward the N-terminal residue of the catalytic subunit. This extension is not observed in MJA-EndA, and the extension seems to overlap with the interdomain loop connecting the N-terminal and C-terminal domain of AFU-EndA. We expected that the introduction of approximately three amino acid residues between the C-terminus of the structural subunit (PAE0789 protein) and the N-terminus of the catalytic subunit (PAE2269 protein) could connect the two subunits without a large change in the mutual organization of the subunits and could make the enzyme a homodimer. Therefore, we constructed plasmids that express variant PAE-EndAs in which the C-terminus of the structural subunit with 6×His-tag (6×His-PAE0789) was fused to the N-terminus of the catalytic subunit (PAE2269 protein) with the introduction of zero (variant NoAA), two (-Leu-Pro-, variant LP), three (-Leu-Pro-Glu-, variant LPE) or four (-Leu-Pro-Glu-Ile-, variant LPEI) amino acids between the subunits. We selected the introduced amino acid residues by comparing the missing part in PAE-EndA with the interdomain loop of AFU-EndA (Figure 1I). All of the variants could be expressed in E. coli. However, the variant NoAA could not be recovered as a soluble protein after incubation at 80°C (data not shown). The other variants (LP, LPE and LPEI) could be expressed and recovered as soluble proteins after the purification steps used for the co-expression system proteins (Figure 2A). Variants that remained soluble were metal-affinity purified and subjected to a 15-min time course endonuclease assay. All variants tested possessed splicing endonuclease activity toward a substrate with the canonical BHB motif, S. tokodaii tRNATrp precursor (Figure 2B). Then, the amount of accumulation for the 5′-exon was measured as shown in Figure 4C. Each variant exhibited accumulation of the 5′-exon with good linearity up to 10 min. Some differences could be observed for their activities. At the 5-min time point, each variant accumulated more 5′-exon as compared to the amount of 5′-exon accumulated by the wild-type endonuclease (LP; 1.3-fold, LPE; 2.9-fold, LPEI; 3.6-fold, Figure 2C).Figure 2.


Functional importance of crenarchaea-specific extra-loop revealed by an X-ray structure of a heterotetrameric crenarchaeal splicing endonuclease.

Yoshinari S, Shiba T, Inaoka DK, Itoh T, Kurisu G, Harada S, Kita K, Watanabe Y - Nucleic Acids Res. (2009)

Engineered homodimeric PAE-EndAs retain splicing endonuclease activity. (A) GelCode-Blue-stained 15% SDS–PAGE gel. In each lane, 2 μg of the fraction eluted from TALON resin was loaded. (B) Splicing endonuclease assay. Sulfolobus tokodaii tRNATrp precursor was used as the substrate. Lane assignments are; M, molecular mass marker in (A); mock, mock incubation with enzyme solvent; WT, wild-type 6xHis-PAE-EndA; LP, variant LP; LPE, variant LPE; LPEI, variant LPEI. (C) Accumulation of 5′-exon on the time course.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724299&req=5

Figure 2: Engineered homodimeric PAE-EndAs retain splicing endonuclease activity. (A) GelCode-Blue-stained 15% SDS–PAGE gel. In each lane, 2 μg of the fraction eluted from TALON resin was loaded. (B) Splicing endonuclease assay. Sulfolobus tokodaii tRNATrp precursor was used as the substrate. Lane assignments are; M, molecular mass marker in (A); mock, mock incubation with enzyme solvent; WT, wild-type 6xHis-PAE-EndA; LP, variant LP; LPE, variant LPE; LPEI, variant LPEI. (C) Accumulation of 5′-exon on the time course.
Mentions: Figure 1I shows the superimposed X-ray structures for the interdomain loop region of AFU-EndA, the C-terminal residues of the structural subunit and the N-terminal residue of the catalytic subunit of PAE-EndA. An interesting observation about the crystal structure of PAE-EndA is that the C-terminal residues of the structural subunit form an extended arm toward the N-terminal residue of the catalytic subunit. This extension is not observed in MJA-EndA, and the extension seems to overlap with the interdomain loop connecting the N-terminal and C-terminal domain of AFU-EndA. We expected that the introduction of approximately three amino acid residues between the C-terminus of the structural subunit (PAE0789 protein) and the N-terminus of the catalytic subunit (PAE2269 protein) could connect the two subunits without a large change in the mutual organization of the subunits and could make the enzyme a homodimer. Therefore, we constructed plasmids that express variant PAE-EndAs in which the C-terminus of the structural subunit with 6×His-tag (6×His-PAE0789) was fused to the N-terminus of the catalytic subunit (PAE2269 protein) with the introduction of zero (variant NoAA), two (-Leu-Pro-, variant LP), three (-Leu-Pro-Glu-, variant LPE) or four (-Leu-Pro-Glu-Ile-, variant LPEI) amino acids between the subunits. We selected the introduced amino acid residues by comparing the missing part in PAE-EndA with the interdomain loop of AFU-EndA (Figure 1I). All of the variants could be expressed in E. coli. However, the variant NoAA could not be recovered as a soluble protein after incubation at 80°C (data not shown). The other variants (LP, LPE and LPEI) could be expressed and recovered as soluble proteins after the purification steps used for the co-expression system proteins (Figure 2A). Variants that remained soluble were metal-affinity purified and subjected to a 15-min time course endonuclease assay. All variants tested possessed splicing endonuclease activity toward a substrate with the canonical BHB motif, S. tokodaii tRNATrp precursor (Figure 2B). Then, the amount of accumulation for the 5′-exon was measured as shown in Figure 4C. Each variant exhibited accumulation of the 5′-exon with good linearity up to 10 min. Some differences could be observed for their activities. At the 5-min time point, each variant accumulated more 5′-exon as compared to the amount of 5′-exon accumulated by the wild-type endonuclease (LP; 1.3-fold, LPE; 2.9-fold, LPEI; 3.6-fold, Figure 2C).Figure 2.

Bottom Line: Meanwhile, a deletion of six amino acids in a Crenarchaea-specific loop abolished the endonuclease activity even on a substrate with canonical BHB motif.These results indicate that the subunit architecture is not a major factor responsible for the difference of substrate specificity between single- and two-subunit EndA systems.Rather, the structural basis for the broad substrate specificity is built into the crenarchaeal splicing endonuclease itself.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. yoshinas@m.u-tokyo.ac.jp

ABSTRACT
Archaeal splicing endonucleases (EndAs) are currently classified into three groups. Two groups require a single subunit protein to form a homodimer or homotetramer. The third group requires two nonidentical protein components for the activity. To elucidate the molecular architecture of the two-subunit EndA system, we studied a crenarchaeal splicing endonuclease from Pyrobaculum aerophilum. In the present study, we solved a crystal structure of the enzyme at 1.7-A resolution. The enzyme adopts a heterotetrameric form composed of two catalytic and two structural subunits. By connecting the structural and the catalytic subunits of the heterotetrameric EndA, we could convert the enzyme to a homodimer that maintains the broad substrate specificity that is one of the characteristics of heterotetrameric EndA. Meanwhile, a deletion of six amino acids in a Crenarchaea-specific loop abolished the endonuclease activity even on a substrate with canonical BHB motif. These results indicate that the subunit architecture is not a major factor responsible for the difference of substrate specificity between single- and two-subunit EndA systems. Rather, the structural basis for the broad substrate specificity is built into the crenarchaeal splicing endonuclease itself.

Show MeSH