title: Crystal structure, mutational analysis and RNA-dependent ATPase activity of the yeast DEAD-box pre-mRNA splicing factor Prp28.
abstract: Yeast Prp28 is a DEAD-box pre-mRNA splicing factor implicated in displacing U1 snRNP from the 5' splice site. Here we report that the 588-aa Prp28 protein consists of a trypsin-sensitive 126-aa N-terminal segment (of which aa 1-89 are dispensable for Prp28 function in vivo) fused to a trypsin-resistant C-terminal catalytic domain. Purified recombinant Prp28 and Prp28-(127-588) have an intrinsic RNA-dependent ATPase activity, albeit with a low turnover number. The crystal structure of Prp28-(127-588) comprises two RecA-like domains splayed widely apart. AMPPNP•Mg2+ is engaged by the proximal domain, with proper and specific contacts from Phe194 and Gln201 (Q motif) to the adenine nucleobase. The triphosphate moiety of AMPPNP•Mg2+ is not poised for catalysis in the open domain conformation. Guided by the Prp28•AMPPNP structure, and that of the Drosophila Vasa•AMPPNP•Mg2+•RNA complex, we targeted 20 positions in Prp28 for alanine scanning. ATP-site components Asp341 and Glu342 (motif II) and Arg527 and Arg530 (motif VI) and RNA-site constituent Arg476 (motif Va) are essential for Prp28 activity in vivo. Synthetic lethality of double-alanine mutations highlighted functionally redundant contacts in the ATP-binding (Phe194-Gln201, Gln201-Asp502) and RNA-binding (Arg264-Arg320) sites. Overexpression of defective ATP-site mutants, but not defective RNA-site mutants, elicited severe dominant-negative growth defects.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
authors.forename: Agata
authors.initials: A
authors.lastname: Jacewicz
authors.affiliation: Microbiology and Immunology Department, Weill Cornell Medical College, New York, NY 10065, USA s-shuman@ski.mskcc.org.
authors.forename: Beate
authors.initials: B
authors.lastname: Schwer
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Smith
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA s-shuman@ski.mskcc.org.
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000737:chemistry; D000483:Alleles; D000818:Animals; D018360:Crystallography, X-Ray; D053487:DEAD-box RNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D029721:Drosophila Proteins; D029701:Saccharomyces cerevisiae Proteins; D000266:Adenylyl Imidodiphosphate; D012313:RNA; D000251:Adenosine Triphosphatases; C088939:PRP28 protein, S cerevisiae; C088269:vas protein, Drosophila; D053487:DEAD-box RNA Helicases