Structure and biochemical function of a prototypical Arabidopsis U-box domain.
U-box proteins, as well as other proteins involved in regulated protein degradation, are apparently over-represented in Arabidopsis compared with other model eukaryotes. The Arabidopsis protein AtPUB14 contains a typical U-box domain followed by an Armadillo repeat region, a domain organization that is frequently found in plant U-box proteins. In vitro ubiquitination assays demonstrated that AtPUB14 functions as an E3 ubiquitin ligase with specific E2 ubiquitin-conjugating enzymes. The structure of the AtPUB14 U-box domain was determined by NMR spectroscopy. It adopts the betabetaalphabeta fold of the Prp19p U-box and RING finger domains. In these proteins, conserved hydrophobic residues form a putative E2-binding cleft. By contrast, they contain no common polar E2 binding site motif. Two hydrophobic cores stabilize the AtPUB14 U-box fold, and hydrogen bonds and salt bridges interconnect the residues corresponding to zinc ion-coordinating residues in RING domains. Residues from a C-terminal alpha-helix interact with the core domain and contribute to stabilization. The Prp19p U-box lacks a corresponding C-terminal alpha-helix. Chemical shift analysis suggested that aromatic residues exposed at the N terminus and the C-terminal alpha-helix of the AtPUB14 U-box participate in dimerization. Thus, AtPUB14 may form a biologically relevant dimer. This is the first plant U-box structure to be determined, and it provides a model for studies of the many plant U-box proteins and their interactions. Structural insight into these interactions is important, because ubiquitin-dependent protein degradation is a prevalent regulatory mechanism in plants.
Andersen
Pernille
P
Institute of Molecular Biology, University of Copenhagen, DK-1353 Copenhagen K, Denmark.
Kragelund
Birthe B
BB
Olsen
Addie N
AN
Larsen
Flemming H
FH
Chua
Nam-Hai
NH
Poulsen
Flemming M
FM
Skriver
Karen
K
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019281:Dimerization; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism
D029681:Arabidopsis Proteins; D025801:Ubiquitin; C492492:AtPUB14 protein, Arabidopsis; D044767:Ubiquitin-Protein Ligases; D015032:Zinc