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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.

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Subunit assembly of the wild-type and engineered variants of PAE-EndA. (A) Gel filtration profiles. Arrowheads at the top of the figure indicate the elution position of the marker with corresponding molecular weights. Horizontal axis; retention time (min), vertical axis; Potential difference proportional to A280. (B) Analytical centrifugation profiles. The analysis were conducted at Research Institute of Biological Science, Katakura Industry Co. Ltd. Horizontal Axis; sedimentation coefficient, [S]; vertical axis, sedimentation distribution function, c(s). Abbreviations for the variants are the same as Figure 2.
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Figure 3: Subunit assembly of the wild-type and engineered variants of PAE-EndA. (A) Gel filtration profiles. Arrowheads at the top of the figure indicate the elution position of the marker with corresponding molecular weights. Horizontal axis; retention time (min), vertical axis; Potential difference proportional to A280. (B) Analytical centrifugation profiles. The analysis were conducted at Research Institute of Biological Science, Katakura Industry Co. Ltd. Horizontal Axis; sedimentation coefficient, [S]; vertical axis, sedimentation distribution function, c(s). Abbreviations for the variants are the same as Figure 2.

Mentions: To investigate subunit assembly, the wild-type PAE-EndA and the variants (fractions after the metal-affinity fractionation) were subjected to gel filtration analysis through a Superdex 200 10/300 (GE Healthcare) column. Retention times of the variants through the gel filtration column were almost the same as that of the wild-type endonuclease (29.14 min for wild type, 29.14 min for LP, 29.18 min for LPE and 29.06 min for LPEI, respectively, Figure 3A), indicating the formation of the heterotetrameric subunit structure for the wild-type endonuclease and the homodimeric structure for the variants in solution. Formation of the heterotetramer for the wild type and the homodimer for the variants was further confirmed by analytical ultracentrifugation (Figure 3B). The main peak of the wild-type endonuclease has a sedimentation coefficient of 3.85 S with a calculated molecular mass of 55.0 kDa. Meanwhile, the sedimentation coefficient values of the main peaks for the wild-type endonuclease and the variants were almost identical (3.84 S with a calculated molecular mass of 60.4 kDa for LP, 3.85 S with a calculated molecular mass of 62.0 kDa for LPE, and 3.89S with a calculated molecular mass of 59.3 kDa for LPEI, respectively). These data also suggest that the engineered linker variant proteins formed homodimers.


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)

Subunit assembly of the wild-type and engineered variants of PAE-EndA. (A) Gel filtration profiles. Arrowheads at the top of the figure indicate the elution position of the marker with corresponding molecular weights. Horizontal axis; retention time (min), vertical axis; Potential difference proportional to A280. (B) Analytical centrifugation profiles. The analysis were conducted at Research Institute of Biological Science, Katakura Industry Co. Ltd. Horizontal Axis; sedimentation coefficient, [S]; vertical axis, sedimentation distribution function, c(s). Abbreviations for the variants are the same as Figure 2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Subunit assembly of the wild-type and engineered variants of PAE-EndA. (A) Gel filtration profiles. Arrowheads at the top of the figure indicate the elution position of the marker with corresponding molecular weights. Horizontal axis; retention time (min), vertical axis; Potential difference proportional to A280. (B) Analytical centrifugation profiles. The analysis were conducted at Research Institute of Biological Science, Katakura Industry Co. Ltd. Horizontal Axis; sedimentation coefficient, [S]; vertical axis, sedimentation distribution function, c(s). Abbreviations for the variants are the same as Figure 2.
Mentions: To investigate subunit assembly, the wild-type PAE-EndA and the variants (fractions after the metal-affinity fractionation) were subjected to gel filtration analysis through a Superdex 200 10/300 (GE Healthcare) column. Retention times of the variants through the gel filtration column were almost the same as that of the wild-type endonuclease (29.14 min for wild type, 29.14 min for LP, 29.18 min for LPE and 29.06 min for LPEI, respectively, Figure 3A), indicating the formation of the heterotetrameric subunit structure for the wild-type endonuclease and the homodimeric structure for the variants in solution. Formation of the heterotetramer for the wild type and the homodimer for the variants was further confirmed by analytical ultracentrifugation (Figure 3B). The main peak of the wild-type endonuclease has a sedimentation coefficient of 3.85 S with a calculated molecular mass of 55.0 kDa. Meanwhile, the sedimentation coefficient values of the main peaks for the wild-type endonuclease and the variants were almost identical (3.84 S with a calculated molecular mass of 60.4 kDa for LP, 3.85 S with a calculated molecular mass of 62.0 kDa for LPE, and 3.89S with a calculated molecular mass of 59.3 kDa for LPEI, respectively). These data also suggest that the engineered linker variant proteins formed homodimers.

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