Functional link between DEAH/RHA helicase Prp43 activation and ATP base binding

• Published in: Nucleic acids research 2017-02, Vol.45 (3), p.1539-1552
• Main Authors: Robert-Paganin, Julien, Halladjian, Maral, Blaud, Magali, Lebaron, Simon, Delbos, Lila, Chardon, Florian, Capeyrou, Régine, Humbert, Odile, Henry, Yves, Henras, Anthony K, Réty, Stéphane, Leulliot, Nicolas
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - chemistry; Adenosine Triphosphate - metabolism; Amino Acid Substitution; Biochemistry, Molecular Biology; Catalytic Domain - genetics; Crystallography, X-Ray; DEAD-box RNA Helicases - chemistry; DEAD-box RNA Helicases - genetics; DEAD-box RNA Helicases - metabolism; Enzyme Activation; Kinetics; Life Sciences; Models, Molecular; Mutagenesis, Site-Directed; Protein Interaction Domains and Motifs; Pyrimidine Nucleotides - chemistry; Pyrimidine Nucleotides - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Structural Biology
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkw1233

The DEAH box helicase Prp43 is a bifunctional enzyme from the DEAH/RHA helicase family required both for the maturation of ribosomes and for lariat intron release during splicing. It interacts with G-patch domain containing proteins which activate the enzymatic activity of Prp43 in vitro by an unknown mechanism. In this work, we show that the activation by G-patch domains is linked to the unique nucleotide binding mode of this helicase family. The base of the ATP molecule is stacked between two residues, R159 of the RecA1 domain (R-motif) and F357 of the RecA2 domain (F-motif). Using Prp43 F357A mutants or pyrimidine nucleotides, we show that the lack of stacking of the nucleotide base to the F-motif decouples the NTPase and helicase activities of Prp43. In contrast the R159A mutant (R-motif) showed reduced ATPase and helicase activities. We show that the Prp43 R-motif mutant induces the same phenotype as the absence of the G-patch protein Gno1, strongly suggesting that the processing defects observed in the absence of Gno1 result from a failure to activate the Prp43 helicase. Overall we propose that the stacking between the R- and F-motifs and the nucleotide base is important for the activity and regulation of this helicase family.