Structural insights into the U-box, a domain associated with multi-ubiquitination

Abstract

The structure of the U-box in the essential Saccharomyces cerevisiae pre-mRNA splicing factor Prp19p has been determined by NMR. The conserved zinc-binding sites supporting the crossbrace arrangement in RING-finger domains are replaced by hydrogen-bonding networks in the Ubox. These hydrogen-bonding networks are necessary for the structural stabilization and activity of the U-box. A conservative Val→Ile point mutation in the Prp19p U-box domain leads to premRNA splicing defects in vivo. NMR analysis of this mutant shows that the substitution disrupts structural integrity of the U-box domain. Furthermore, comparison of the Prp19p U-box domain with known RING-E2 complex structures demonstrates that both U-box and RING-fingers contain a conserved interaction surface. Mutagenesis of residues at this interface, while not perturbing the structure of the U-box, abrogates Prp19p function in vivo. These comparative structural and functional analyses imply that the U-box and its associated ubiquitin ligase activity are critical for Prp19p function in vivo.Ubiquitin (Ub) targeting of proteins for degradation by the proteosome involves polyubiquination of substrate proteins via an enzyme cascade consisting of activating (E1), conjugating (E2) and ligating (E3) enzymes. E3 ubiquitin ligases vary widely in size, composition and enzymology, reflecting their regulatory role in substrate recognition 1 . HECT and RING finger domain-containing proteins constitute two classes of E3 ligases. HECT domains bind Ub through a thioester bond and transfer Ub directly to substrate. RING finger E3s facilitate the transfer of Ub from the E2 to the substrate, rather than binding Ub directly.Correspondence should be addressed to K.L.G. kathy.gould@mcmail.vanderbilt.edu or W.J.C. walter.chazin@vanderbilt.edu. 3 These authors contributed equally to this work. Competing interests statementThe authors declare that they have no competing financial interests. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptA third class of E3 ubiquitin ligases has been recently identified 2,3 . This class of proteins contains a U-box motif, first identified in Saccharomyces cerevisiae Ufd2p. Ufd2p promotes the elongation of poly-ubiquitin chains in a U-box-dependent manner (recently termed an 'E4' activity) 4 . An alignment of U-boxes and RING motifs indicated that U-boxes lack the strictly conserved histidine and cysteine Zn 2+ -chelating residues found in RING fingers, but they share a similar pattern of hydrophobic and polar amino acids (Fig. 1a), raising the possibility that they have similar folds 5 .S. cerevisiae Prp19p is an essential pre-mRNA splicing factor that contains an N-terminal Ubox 6,7 . Interestingly, a mutation within the Prp19p U-box that results in the substitution of an isoleucine for a conserved valine (prp19-1) leads to pre-mRNA splicing defects and the disruption of several key protein-protein interactions within the spliceosome 8,9 . The profound physiological consequences that result from th...

The Prp19/Pso4 core complex undergoes ubiquitylation and structural alterations in response to DNA damage

“…S2A). Mutation of conserved residues including C5 and D40 have been shown to inactivate the Ub E3 ligase function of the Prp19 U box domain [17]. We therefore prepared alanine mutants at these sites and examined the mutant proteins for oligomerization.…”

“…The only known catalytic center in any of the four Pso4 complex members is a U-box domain located in the amino terminus of Prp19. U-box domains have been shown to contain an E3 ubiquitin (Ub) ligase activity [15,16], and such activity has been demonstrated for Prp19 in vitro [14,17,18], and this function is required for pre-mRNA splicing in vivo [17,18]. …”

Ubiquitination-mediated protein degradation and modification: an emerging theme in plant-microbe interactions

“…The U-box is a novel E3 ubiquitin ligase activity-related protein domain that was first identified in the yeast ubiquitination factor UFD2 [35]. The U-box contains ~75 amino acids and possesses a tertiary structure resembling that of the RING finger [36,37]. The major difference between the U-box and RING domains is that the U-box lacks the hallmark Zn 2+ -chelating cysteine and histidine residues characteristic of RING fingers.…”

“…The major difference between the U-box and RING domains is that the U-box lacks the hallmark Zn 2+ -chelating cysteine and histidine residues characteristic of RING fingers. Consequently, the conserved zinc-binding residues supporting the cross-brace arrangement in RING-finger domains are replaced by hydrogen-bonding networks in the U-box [37].…”

Heterologous Expression and Molecular and Cellular Characterization of CaPUB1 Encoding a Hot Pepper U-Box E3 Ubiquitin Ligase Homolog

“…As was found in other U-boxcontaining proteins, CaPUB1 possessed a single U-box domain near the N-terminal region. The U-box motif of CaPUB1 was 56% to 73% conserved with regard to the Arabidopsis AtPUBs and rice (Oryza sativa) spotted leaf11 (SPL11) proteins (Zeng et al, 2004), and was 27% to 37% identical to the corresponding domain in human Hs_Prp19 and hCHIP (Ohi et al, 2003;Fig. 1C).…”