Functional importance of crenarchaea-specific extra-loop revealed by an X-ray structure of a heterotetrameric crenarchaeal splicing endonuclease.
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.
Yoshinari
Shigeo
S
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
Shiba
Tomoo
T
Inaoka
Daniel-Ken
DK
Itoh
Takashi
T
Kurisu
Genji
G
Harada
Shigeharu
S
Kita
Kiyoshi
K
Watanabe
Yoh-ichi
Y
D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry; D041543:Pyrobaculum / Q000201:enzymology*; D017384:Sequence Deletion; D013379:Substrate Specificity
D021122:Protein Subunits; D004722:Endoribonucleases; C051012:splicing endonuclease