4W7S
Crystal structure of the yeast DEAD-box splicing factor Prp28 at 2.54 Angstroms resolution
Summary for 4W7S
Entry DOI | 10.2210/pdb4w7s/pdb |
Descriptor | Pre-mRNA-splicing ATP-dependent RNA helicase PRP28, HEXAETHYLENE GLYCOL, GLYCEROL, ... (6 entities in total) |
Functional Keywords | splicing factor, dead-box protein, atpase, hydrolase |
Biological source | Saccharomyces cerevisiae (Baker's yeast) |
Total number of polymer chains | 2 |
Total formula weight | 108887.37 |
Authors | Jacewicz, A.,Smith, P.,Schwer, B.,Shuman, S. (deposition date: 2014-08-22, release date: 2014-10-29, Last modification date: 2024-10-23) |
Primary citation | Jacewicz, A.,Schwer, B.,Smith, P.,Shuman, S. Crystal structure, mutational analysis and RNA-dependent ATPase activity of the yeast DEAD-box pre-mRNA splicing factor Prp28. Nucleic Acids Res., 42:12885-12898, 2014 Cited by PubMed 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. PubMed: 25303995DOI: 10.1093/nar/gku930 PDB entries with the same primary citation |
Experimental method | X-RAY DIFFRACTION (2.542 Å) |
Structure validation
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