Structural and functional study of the regulation of the helicase Prp43

Les hélicases à ARN de la famille DEAH/RHA sont impliquées dans la plupart des processus essentiels à la vie tels que l'épissage, la biogenèse des ribosomes, la réplication, la transcription ou encore la détection d’ARN viraux. Ces enzymes sont capables de catalyser la dissociation de duplexes d'ARN, la réorganisation de structures secondaires ou de remodeler des complexes ARN-protéines. L'hélicase DEAH/RHA Prp43 présente la particularité d'être bifonctionnelle. Prp43 est impliquée dans l'épissage des Pré-ARNm, où elle assure le recyclage du spliceosome et du lasso, mais aussi dans la biogenèse des ribosomes où elle est impliquée dans la maturation des deux sous-unités. Prp43 est activée et régulée par cinq partenaires protéiques : Ntr1, Gno1, Pfa1, RBM5 et GPATCH2. Ces partenaires protéiques présentent tous un domaine G-patch et sont capables de stimuler les activités hélicase et ATPase de Prp43. La structure cristallographique de Prp43 en complexe avec l'ADP a été résolue au laboratoire. Cette structure a mis en évidence un mode de fixation du nucléotide inédit chez les autres hélicases, notamment au niveau de la base qui s'empile entre la phénylalanine 357 (F357) du domaine RecA2 et l'arginine 159 (R159) du domaine RecA1. Les déterminants de l'activation de Prp43 par les protéines à domaine G-patch demeurent méconnus. Dans ce travail, nous avons cherché à déterminer quel était le rôle de l’empilement de la base dans l’activation de Prp43. Nous présentons ici plusieurs structures cristallographiques de Prp43 en complexe avec tous les nucléotides diphosphates(NDP) et les désoxynucléotides triphosphates (dNDP). Ces structures ont permis de conclure qu'il y avait des différences dans l’empilement de la base selon le (d)NDP considéré. Des dosages d'activité NTPase de Prp43 avec et sans son partenaire protéique Pfa1 montrent que lorsque la base ne s'empile pas avec la F357 et la R159, l'activité de l'enzyme n'est pas correctement régulée par son partenaire protéique. Les dosages d’activité enzymatique sur les mutants ponctuels F357A et R159A révèlent que le résidu F357 permet de moduler l’activité de Prp43. Tous ces résultats nous ont permis de mettre en évidence un modèle de la régulation de Prp43 par les protéines à domaines G-patch et d'expliquer l'importance du mode de fixation de la base à l'enzyme dans cette régulation.RNA helicases from the DEAH/RHA family are involved in most of essential processes of life such as pre-mRNA splicing, ribosome biogenesis, replication, transcription or viral RNA sensing. These enzymes are able to catalyze RNA unwinding, secondary structures reorganization or RNA-protein complexes remodeling. The DEAH/RHA helicase Prp43 is remarkable because it is bifunctional, as it is involved both in pre-mRNA splicing, where it is responsible of spliceosome and lariat recycling and in the biogenesis of the two ribosomal subunits. Prp43 is activated by five protein partners: Ntr1, Gno1, Pfa1, RBM5 and GPATCH2. These protein partners all possess a G-patch domain and are able to stimulate helicase and ATPase activity of Prp43. The structure of Prp43 in complex with ADP has been solved by X-ray crystallography. The structure reveals that the nucleotide is bound to the enzyme in a novel mode that has never been observed in other known helicase structures. The specific feature of this binding mode is the base, stacked between phenylalanine (F357) from RecA2 domain and an arginine (R159) from RecA1 domain. Features of the activation of Prp43 by G-patch proteins are unclear. In this work, we investigated the role of base stacking in the activation of Prp43. We present several structures of Prp43 bound to all the nucleotide diphosphates (NDP) and deoxynucleotide diphosphates (dNTP). These results indicate that there are differences in stacking according to the (d)NDP bound to the enzyme. NTPase activity assays revealed that when stacking is weakened, Prp43 activity cannot be properly regulated by its protein partner Pfa1. Moreover, point mutations F357A and R159A show that stacking of F357 permits to modulate Prp43 activity. All these results allow us to propose a model of NTPase activity activation of Prp43 by G-patch proteins and to highlight the importance of base stacking in this regulation

Regulation of DEAH/RHA Helicases by G-Patch Proteins

RNA helicases from the DEAH/RHA family are present in all the processes of RNA metabolism. The function of two helicases from this family, Prp2 and Prp43, is regulated by protein partners containing a G-patch domain. The G-patch is a glycine-rich domain discovered by sequence alignment, involved in protein-protein and protein-nucleic acid interaction. Although it has been shown to stimulate the helicase’s enzymatic activities, the precise role of the G-patch domain remains unclear. The role of G-patch proteins in the regulation of Prp43 activity has been studied in the two biological processes in which it is involved: splicing and ribosome biogenesis. Depending on the pathway, the activity of Prp43 is modulated by different G-patch proteins. A particular feature of the structure of DEAH/RHA helicases revealed by the Prp43 structure is the OB-fold domain in C-terminal part. The OB-fold has been shown to be a platform responsible for the interaction with G-patch proteins and RNA. Though there is still no structural data on the G-patch domain, in the current model, the interaction between the helicase, the G-patch protein, and RNA leads to a cooperative binding of RNA and conformational changes of the helicase

Hypertrophic cardiomyopathy disease results from disparate impairments of cardiac myosin function and auto-inhibition

Hypertrophic cardiomyopathy (HCM) is caused by point mutations in sarcomeric proteins. Here the authors develop an optimized model of the sequestered state of cardiac myosin and define the features affecting the lever arm compliance, allowing them to group mutations in classes and to elucidate the molecular mechanisms leading to cardiac dysfunction in HCM

Crystal Structure of an EAL Domain in Complex with Reaction Product 5′-pGpG

<div><p>FimX is a large multidomain protein containing an EAL domain and involved in twitching motility in <em>Pseudomonas aeruginosa</em>. We present here two crystallographic structures of the EAL domain of FimX (residues 438–686): one of the apo form and the other of a complex with 5′-pGpG, the reaction product of the hydrolysis of c-di-GMP. In both crystal forms, the EAL domains form a dimer delimiting a large cavity encompassing the catalytic pockets. The ligand is trapped in this cavity by its sugar phosphate moiety. We confirmed by NMR that the guanine bases are not involved in the interaction in solution. We solved here the first structure of an EAL domain bound to the reaction product 5′-pGpG. Though isolated FimX EAL domain has a very low catalytic activity, which would not be significant compared to other catalytic EAL domains, the structure with the product of the reaction can provides some hints in the mechanism of hydrolysis of the c-di-GMP by EAL domains.</p> </div

Stereo view of the active site of FimX-EAL domain.

<p>(A) The residual density Fo-Fc calculated after molecular replacement with no ligand in the model is contoured in orange at 3 σ level (A). The FimX backbone is represented as green cartoon. (B) Interpretation of the density as the bound 5′-pGpG, shown as black sticks. The magnesium ion is shown as a magenta sphere and the catalytic water involved in its coordination as a red sphere. Side-chains of residues involved in ligand binding are shown. The final electron density map 2Fo-Fc contoured at 1.2 σ level is traced around the ligand.</p

Sequence alignments of EAL domains from various proteins.

<p>Alignement of EAL domains was done with ClustalW. Conserved residues important for the PDE-A activity are numbered #1 to #10. Residues conserved in FimX-EAL are boxed in red. The secondary structure is indicated for FimX-EAL structure.</p

Enzymatic activity of hydrolysis of BpNPP by FimX.

<p>(A) Kinetics parameters of full-length FimX. Initial velocities are expressed in arbitrary units. (B) Relative activity of B<i>p</i>NPP hydrolysis after 9 hours of full-length FimX (FimX-FL), GGDEF-EAL domains (Dual) and EAL domain (EAL). Catalytic activities are normalised against that of FimX-FL. Dual has 18.1% activity and the EAL domain alone retains 6.6% activity.</p

Structure of the homodimer of EAL domain of FimX with bound 5′-pGpG.

<p>(A) Structure of the homodimer of EAL domain of FimX with bound 5′-pGpG. The secondary structure elements are labelled on one monomer. (B) Stereo view of the superposition of homodimer of EAL domain of FimX in apo form crystallized in spacegroup P2₁2₁2₁ (dark blue and light blue) on the homodimer of FimX EAL in complex with 5′-pGpG crystallized in spacegroup P4₃2₁2 (red and green).</p

Representation of the cavity in the dimer of FimX-EAL bound to pGpG.

<p>(A) The cavity is presented as an orange surface inside the FimX-EAL dimer. It is connected to the outside by two channels of different size. Left and right views are rotated by 180°. The corresponding electrostatic surface is represented in (B). The edges of both channels are negatively charged and forbid the ligand to escape from the cavity.</p

Reverse NOE-pumping experiments.

<p>Expanded 1D NMR spectra recorded in D₂O at 600 MHz and 308 K, pH 6.5 of c-di-GMP and 5′-pGpG alone (respectively left panels A-B and E-F) and with FimX-EAL (right panels C-D and G-H). Panels A, C, E and G: reference 1D-esgp experiments. Panels B, D, F and H: RNP experiments. (S = signals arising from buffer).</p