Functional link between DEAH/RHA helicase Prp43 activation and ATP base binding.
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.
Laboratoire de Cristallographie et RMN Biologiques, UMR CNRS 8015, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Pharmacie, Paris, France.
Julien
J
Robert-Paganin
Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative, Université de Toulouse, CNRS, UPS, France.
Maral
M
Halladjian
Laboratoire de Cristallographie et RMN Biologiques, UMR CNRS 8015, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Pharmacie, Paris, France.
Magali
M
Blaud
Laboratoire de Cristallographie et RMN Biologiques, UMR CNRS 8015, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Pharmacie, Paris, France.
Simon
S
Lebaron
Laboratoire de Cristallographie et RMN Biologiques, UMR CNRS 8015, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Pharmacie, Paris, France.
Lila
L
Delbos
Laboratoire de Cristallographie et RMN Biologiques, UMR CNRS 8015, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Pharmacie, Paris, France.
Florian
F
Chardon
Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative, Université de Toulouse, CNRS, UPS, France.
Régine
R
Capeyrou
Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative, Université de Toulouse, CNRS, UPS, France.
Odile
O
Humbert
Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative, Université de Toulouse, CNRS, UPS, France.
Yves
Y
Henry
Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative, Université de Toulouse, CNRS, UPS, France.
Anthony K
AK
Henras
Laboratoire de Cristallographie et RMN Biologiques, UMR CNRS 8015, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Pharmacie, Paris, France.
Stéphane
S
Réty
Laboratoire de Cristallographie et RMN Biologiques, UMR CNRS 8015, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Pharmacie, Paris, France.
Nicolas
N
Leulliot
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D019943:Amino Acid Substitution; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D054730:Protein Interaction Domains and Motifs; D011742:Pyrimidine Nucleotides / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C000590779:Gno1 protein, S cerevisiae; C546353:PFA1 protein, S cerevisiae; D011742:Pyrimidine Nucleotides; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; C109034:PRP43 protein, S cerevisiae; D053487:DEAD-box RNA Helicases