title: Solution structure of the catalytic domain of the mitochondrial protein ICT1 that is essential for cell vitality.
abstract: The ICT1 protein was recently reported to be a component of the human mitoribosome and to have codon-independent peptidyl-tRNA hydrolysis activity via its conserved GGQ motif, although little is known about the detailed mechanism. Here, using NMR spectroscopy, we determined the solution structure of the catalytic domain of the mouse ICT1 protein that lacks an N-terminal mitochondrial targeting signal and an unstructured C-terminal basic-residue-rich extension, and we examined the effect of ICT1 knockdown (mediated by small interfering RNA) on mitochondria in HeLa cells using flow cytometry. The catalytic domain comprising residues 69-162 of the 206-residue full-length protein forms a structure with a β1-β2-α1-β3-α2 topology and a structural framework that resembles the structure of GGQ-containing domain 3 of class 1 release factors (RFs). Half of the structure, including the GGQ-containing loop, has essentially the same sequence and structure as those in RFs, consistent with the peptidyl-tRNA hydrolysis activity of ICT1 on the mitoribosome, which is analogous to RFs. However, the other half of the structure differs in shape from the corresponding part of RF domain 3 in that in ICT1, an α-helix (α1), instead of a β-turn, is inserted between strand β2 and strand β3. A characteristic groove formed between α1 and the three-stranded antiparallel β-sheet was identified as a putative ICT1-specific functional site by a structure-based alignment. In addition, the structured domain that recognizes stop codons in RFs is replaced in ICT1 by a C-terminal basic-residue-rich extension. It appears that these differences are linked to a specific function of ICT1 other than the translation termination mediated by RFs. Flow cytometry analysis showed that the knockdown of ICT1 results in apoptotic cell death with a decrease in mitochondrial membrane potential and mass. In addition, cytochrome c oxidase activity in ICT1 knockdown cells was decreased by 35% compared to that in control cells. These results indicate that ICT1 function is essential for cell vitality and mitochondrial function.
authors.affiliation: Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu-shi, Gunma 376-8515, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Handa
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Hikawa
authors.forename: Naoya
authors.initials: N
authors.lastname: Tochio
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kogure
authors.forename: Makoto
authors.initials: M
authors.lastname: Inoue
authors.forename: Seizo
authors.initials: S
authors.lastname: Koshiba
authors.forename: Peter
authors.initials: P
authors.lastname: Güntert
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Inoue
authors.forename: Takanori
authors.initials: T
authors.lastname: Kigawa
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
authors.forename: Nobukazu
authors.initials: N
authors.lastname: Nameki
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D002470:Cell Survival; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010454:Peptide Termination Factors / Q000737:chemistry / Q000235:genetics; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012269:Ribosomal Proteins; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
chemical_list: D029968:Escherichia coli Proteins; C098021:MRPL58 protein, human; D024101:Mitochondrial Proteins; D010454:Peptide Termination Factors; D011506:Proteins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C489006:prfB protein, E coli