Alternative N-terminal regions of Drosophila myosin heavy chain II regulate communication of the purine binding loop with the essential light chain

We investigated the biochemical and biophysical properties of one of the four alternative exon-encoded regions within the Drosophila myosin catalytic domain. This region is encoded by alternative exons 3a and 3b and includes part of the N-terminal β–barrel. Chimeric myosin constructs (IFI-3a and EMB-3b) were generated by exchanging the exon 3-encoded areas between native slow embryonic body wall (EMB) and fast indirect flight muscle myosin isoforms (IFI). We found that this exchange alters the kinetic properties of the myosin S1 head. The ADP release rate (k-D) in the absence of actin is completely reversed for each chimera compared to the native isoforms. Steady-state data also suggest a reciprocal shift, with basal and actin-activated ATPase activity of IFI-3a showing reduced values compared to wild-type IFI, whereas for EMB-3b these values are increased compared to wild-type EMB. In the presence of actin, ADP affinity (KAD) is unchanged for IFI-3a, compared to IFI, but ADP-affinity for EMB-3b is increased, compared to EMB, and shifted towards IFI values. ATP-induced dissociation of acto-S1 (K1k+2) is reduced for both exon 3 chimeras. Homology modeling, combined with a recently reported crystal structure for Drosophila EMB, indicate that the exon 3 encoded region in the myosin head is part of the communication pathway between the nucleotide binding pocket (purine-binding loop) and the essential light chain, emphasizing an important role for this variable N-terminal domain in regulating acto-myosin cross-bridge kinetics, in particular with respect to the force-sensing properties of myosin isoforms

Dimerisation induced formation of the active site and the identification of three metal sites in EAL-phosphodiesterases

The bacterial second messenger cyclic di-3′,5′-guanosine monophosphate (c-di-GMP) is a key regulator of bacterial motility and virulence. As high levels of c-di-GMP are associated with the biofilm lifestyle, c-di-GMP hydrolysing phosphodiesterases (PDEs) have been identified as key targets to aid development of novel strategies to treat chronic infection by exploiting biofilm dispersal. We have studied the EAL signature motif-containing phosphodiesterase domains from the Pseudomonas aeruginosa proteins PA3825 (PA3825EAL) and PA1727 (MucREAL). Different dimerisation interfaces allow us to identify interface independent principles of enzyme regulation. Unlike previously characterised two-metal binding EAL-phosphodiesterases, PA3825EAL in complex with pGpG provides a model for a third metal site. The third metal is positioned to stabilise the negative charge of the 5′-phosphate, and thus three metals could be required for catalysis in analogy to other nucleases. This newly uncovered variation in metal coordination may provide a further level of bacterial PDE regulation

Cryo-EM and molecular docking shows myosin-S1 loop 4 contacts actin and tropomyosin on thin filaments

The motor protein, myosin, drives muscle and non-muscle motility by binding to and moving along actin of thin filaments. Myosin-binding to actin also modulates interactions of the regulatory protein, tropomyosin, on thin filaments, and conversely tropomyosin affects myosin-binding to actin. Insight into this reciprocity will facilitate a molecular level elucidation of tropomyosin regulation of myosin interaction with actin in muscle contraction, and in turn, promote better understanding non-muscle cell motility. Indeed, experimental approaches, such as fiber diffraction, cryo-electron microscopy and 3D reconstruction, have long been used to define regulatory interaction of tropomyosin and myosin on actin at a structural level. However, their limited resolution has not proven sufficient to determine tropomyosin and myosin contacts at an atomic-level and thus to fully substantiate possible functional contributions. To overcome this deficiency, we have followed a hybrid approach by performing new cryo-EM reconstruction of myosin-S1‒decorated F-actin-tropomyosin together with atomic-scale protein-protein docking of tropomyosin to the EM models. Here, cryo-EM data were derived from filaments reconstituted with α1-actin, cardiac αα-tropomyosin, and masseter muscle β-myosin complexes; masseter myosin, which shares sequence identity with β-cardiac myosin-heavy chain, was used because of its stability in vitro. The data were used to build an atomic model of the tropomyosin cable that fits onto the actin filament between the tip of the myosin head and a cleft on the innermost edge of actin subunits. The docking and atomic scale fitting showed multiple discrete interactions of myosin loop 4 and acidic residues on successive 39 to 42 residue-long tropomyosin pseudo-repeats. The contacts between S1 and tropomyosin on actin appear to compete with and displace ones normally found between actin and tropomyosin on myosin-free thin filaments in relaxed muscle, thus restructuring the filament during myosin-induced activation

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

A Perspective for Ménière’s Disease: In Silico Investigations of Dexamethasone as a Direct Modulator of AQP2

International audienceMénière’s disease is a chronic illness characterized by intermittent episodes of vertigo associated with fluctuating sensorineural hearing loss, tinnitus and aural pressure. This pathology strongly correlates with a dilatation of the fluid compartment of the endolymph, so-called hydrops. Dexamethasone is one of the therapeutic approaches recommended when conventional antivertigo treatments have failed. Several mechanisms of actions have been hypothesized for the mode of action of dexamethasone, such as the anti-inflammatory effect or as a regulator of inner ear water homeostasis. However, none of them have been experimentally confirmed so far. Aquaporins (AQPs) are transmembrane water channels and are hence central in the regulation of transcellular water fluxes. In the present study, we investigated the hypothesis that dexamethasone could impact water fluxes in the inner ear by targeting AQP2. We addressed this question through molecular dynamics simulations approaches and managed to demonstrate a direct interaction between AQP2 and dexamethasone and its significant impact on the channel water permeability. Through compartmentalization of sodium and potassium ions, a significant effect of Na+ upon AQP2 water permeability was highlighted as well. The molecular mechanisms involved in dexamethasone binding and in its regulatory action upon AQP2 function are described