Structure and biochemical function of a prototypical Arabidopsis U-box domain

Pernille Andersen, Birthe B Kragelund, Addie N Olsen, Flemming H Larsen, Nam-Hai Chua, Flemming M Poulsen, Karen Skriver

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78 Citationer (Scopus)

Abstract

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.
OriginalsprogEngelsk
TidsskriftJournal of Biological Chemistry
Vol/bind279
Udgave nummer38
Sider (fra-til)40053-40061
Antal sider8
ISSN0021-9258
DOI
StatusUdgivet - 2004

Bibliografisk note

Keywords: Amino Acid Sequence; Arabidopsis; Arabidopsis Proteins; Dimerization; Hydrogen Bonding; Molecular Sequence Data; Protein Structure, Tertiary; Ubiquitin; Ubiquitin-Protein Ligases; Zinc

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