Active site residues and amino acid specificity of the ubiquitin carrier protein-binding RING-H2 finger domain.
EL5 is a rice ubiquitin-protein isopeptide ligase (E3) containing a RING-H2 finger domain that interacts with Oryza sativa (Os) UBC5b, a rice ubiquitin carrier protein. We introduced point mutations into the EL5 RING-H2 finger so that residues that functionally interact with OsUBC5b could be identified when assayed for ubiquitination activity in vitro. The residue positions were selected based on the results of an EL5 RING-H2 finger/OsUBC5b NMR titration experiment. These RING-H2 finger residues form or are adjacent to a shallow groove that is recognized by OsUBC5b. The E3 activity of EL5 is shown to be dependent on a Trp located at the center of the groove. We classified rice RING fingers according to the type of metal-chelating motif, i.e. RING-H2 or RING-HC, and according to the presence or absence of a conserved EL5-like Trp. We discuss the probable relationship between E3 activity and the conserved Trp.
Katoh
Shizue
S
Biochemistry Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
Tsunoda
Yuki
Y
Murata
Katsuyoshi
K
Minami
Eiichi
E
Katoh
Etsuko
E
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017124:Conserved Sequence; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012275:Oryza / Q000737:chemistry / Q000201:enzymology*; D017354:Point Mutation; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014364:Tryptophan; D025801:Ubiquitin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers
D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; C046288:ubiquitin carrier proteins; D014364:Tryptophan; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases