title: 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 cross-brace arrangement in RING-finger domains are replaced by hydrogen-bonding networks in the U-box. 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 pre-mRNA 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.
authors.affiliation: Howard Hughes Medical Institute School of Medicine, Vanderbilt University, Nashville, Tennessee 37232, USA.
authors.forename: Melanie D
authors.initials: MD
authors.lastname: Ohi
authors.forename: Craig W
authors.initials: CW
authors.lastname: Vander Kooi
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Rosenberg
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Chazin
authors.forename: Kathleen L
authors.initials: KL
authors.lastname: Gould
mesh_terms: D000595:Amino Acid Sequence; D004355:Drug Stability; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary; D012326:RNA Splicing; D000072260:RNA Splicing Factors; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D017381:Spliceosomes; D025801:Ubiquitin / Q000737:chemistry*
chemical_list: C079877:PRP19 protein, S cerevisiae; D000072260:RNA Splicing Factors; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin