Free Energy Simulations of a GTPase: GTP and GDP Binding to Archaeal Initiation Factor 2

International audienceArchaeal initiation factor 2 (aIF2) is a protein involved in the initiation of protein biosynthesis. In its GTP-bound, "ON" conformation, aIF2 binds an initiator tRNA and carries it to the ribosome. In its GDP-bound, "OFF" conformation, it dissociates from tRNA. To understand the specific binding of GTP and GDP and its dependence on the ON or OFF conformational state of aIF2, molecular dynamics free energy simulations (MDFE) are a tool of choice. However, the validity of the computed free energies depends on the simulation model, including the force field and the boundary conditions, and on the extent of conformational sampling in the simulations. aIF2 and other GTPases present specific difficulties; in particular, the nucleotide ligand coordinates a divalent Mg(2+) ion, which can polarize the electronic distribution of its environment. Thus, a force field with an explicit treatment of electronic polarizability could be necessary, rather than a simpler, fixed charge force field. Here, we begin by comparing a fixed charge force field to quantum chemical calculations and experiment for Mg(2+):phosphate binding in solution, with the force field giving large errors. Next, we consider GTP and GDP bound to aIF2 and we compare two fixed charge force fields to the recent, polarizable, AMOEBA force field, extended here in a simple, approximate manner to include GTP. We focus on a quantity that approximates the free energy to change GTP into GDP. Despite the errors seen for Mg(2+):phosphate binding in solution, we observe a substantial cancellation of errors when we compare the free energy change in the protein to that in solution, or when we compare the protein ON and OFF states. Finally, we have used the fixed charge force field to perform MDFE simulations and alchemically transform GTP into GDP in the protein and in solution. With a total of about 200 ns of molecular dynamics, we obtain good convergence and a reasonable statistical uncertainty, comparable to the force field uncertainty, and somewhat lower than the predicted GTP/GDP binding free energy differences. The sign and magnitudes of the differences can thus be interpreted at a semiquantitative level, and are found to be consistent with the experimental binding preferences of ON- and OFF-aIF2

An ancient family of SelB elongation factor-like proteins with a broad but disjunct distribution across archaea

<p>Abstract</p> <p>Background</p> <p>SelB is the dedicated elongation factor for delivery of selenocysteinyl-tRNA to the ribosome. In archaea, only a subset of methanogens utilizes selenocysteine and encodes archaeal SelB (aSelB). A SelB-like (aSelBL) homolog has previously been identified in an archaeon that does not encode selenosysteine, and has been proposed to be a pyrrolysyl-tRNA-specific elongation factor (EF-Pyl). However, elongation factor EF-Tu is capable of binding archaeal Pyl-tRNA in bacteria, suggesting the archaeal ortholog EF1A may also be capable of delivering Pyl-tRNA to the ribosome without the need of a specialized factor.</p> <p>Results</p> <p>We have phylogenetically characterized the aSelB and aSelBL families in archaea. We find the distribution of aSelBL to be wider than both selenocysteine and pyrrolysine usage. The aSelBLs also lack the carboxy terminal domain usually involved in recognition of the selenocysteine insertion sequence in the target mRNA. While most aSelBL-encoding archaea are methanogenic Euryarchaea, we also find aSelBL representatives in Sulfolobales and Thermoproteales of Crenarchaea, and in the recently identified phylum Thaumarchaea, suggesting that aSelBL evolution has involved horizontal gene transfer and/or parallel loss. Severe disruption of the GTPase domain suggests that some family members may employ a hitherto unknown mechanism of nucleotide hydrolysis, or have lost their GTPase ability altogether. However, patterns of sequence conservation indicate that aSelBL is still capable of binding the ribosome and aminoacyl-tRNA.</p> <p>Conclusions</p> <p>Although it is closely related to SelB, aSelBL appears unlikely to either bind selenocysteinyl-tRNA or function as a classical GTP hydrolyzing elongation factor. We propose that following duplication of aSelB, the resultant aSelBL was recruited for binding another aminoacyl-tRNA. In bacteria, aminoacylation with selenocysteine is essential for efficient thermodynamic coupling of SelB binding to tRNA and GTP. Therefore, change in tRNA specificity of aSelBL could have disrupted its GTPase cycle, leading to relaxation of selective pressure on the GTPase domain and explaining its apparent degradation. While the specific role of aSelBL is yet to be experimentally tested, its broad phylogenetic distribution, surpassing that of aSelB, indicates its importance.</p

Exploring the Na+/K+-ATPase:Src kinase complex

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Pharmacological activation of NRF2 has protective effects during Mycobacterium abscessus infection by promoting host defences and reducing inflammatory damage in the context of cystic fibrosis

International audienceObjectives: Lung disease is the leading cause of mortality in cystic fibrosis(CF), a disorder caused by mutations in the CFTR gene, and is characterisedby a repetitive circle of infection and inflammation. Among the pathogenicbacteria found in CF airway, the multidrug-resistant Mycobacteriumabscessus (Mabs) is associated with accelerated inflammatory lungdamage and is often refractory to antibiotic therapies. Therefore, weexamined whether curcumin, an activator of the transcription factor NRF2,might provide a beneficial effect against Mabs infection in the context of CF.Methods: Herein, using CFTR-depleted zebrafish larvae as an innovativemodel of inflammation and infection, we sought to determine the effects ofcurcumin on host immune responses to Mabs infections in CF condition.Results: Firstly, we show that treatment with curcumin exerts anti-inflammatory effects in neutrophilic inflammation induced by Mabsinfection in CFTR-depleted zebrafish. As a consequence, the reducednumber of neutrophils at infected sites prevents tissue damage. Our findings show that curcumin alleviates Mabs-induced inflammatorydamage by restoring NRF2 activity in CF animals. Next, while curcuminhas no direct antibacterial activity against Mabs, our results indicated thatcurcumin improves the control of Mabs infections in CF zebrafish,correlating with reduced larval mortality and lower bacterial loads. Inparticular, we found that NRF2 activation with curcumin enhances Mabskilling in macrophages in vivo, demonstrating that NFR2 is instrumental toefficiently restrict the intracellular growth of Mabs, thereby preventingbacterial spread and more severe infection in CF.Conclusion: Our findings bring new understanding of the immune-targeted action of curcumin, and show that therapeutic strategies tonormalise NRF2 activity might simultaneously enhance bacterial killingand promote inflammation resolution thus prevent infectious andinflammatory lung damage in CF

L’hème, un nouveau ligand du récepteur des produits de glycation avancée (RAGE)

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Structure of the ternary initiation complex aIF2-GDPNP-methionylated initiator tRNA.

International audienceEukaryotic and archaeal translation initiation factor 2 (e/aIF2) is a heterotrimeric GTPase that has a crucial role in the selection of the correct start codon on messenger RNA. We report the 5-Å resolution crystal structure of the ternary complex formed by archaeal aIF2 from Sulfolobus solfataricus, the GTP analog GDPNP and methionylated initiator tRNA. The 3D model is further supported by solution studies using small-angle X-ray scattering. The tRNA is bound by the α and γ subunits of aIF2. Contacts involve the elbow of the tRNA and the minor groove of the acceptor stem, but not the T-stem minor groove. We conclude that despite considerable structural homology between the core γ subunit of aIF2 and the elongation factor EF1A, these two G proteins of the translation apparatus use very different tRNA-binding strategies

Structure of an archaeal heterotrimeric initiation factor 2 reveals a nucleotide state between the GTP and the GDP states

Initiation of translation in eukaryotes and in archaea involves eukaryotic/archaeal initiation factor (e/aIF)1 and the heterotrimeric initiation factor e/aIF2. In its GTP-bound form, e/aIF2 provides the initiation complex with Met–tRNAiMet. After recognition of the start codon by initiator tRNA, e/aIF1 leaves the complex. Finally, e/aIF2, now in a GDP-bound form, loses affinity for Met–tRNAiMet and dissociates from the ribosome. Here, we report a 3D structure of an aIF2 heterotrimer from the archeon Sulfolobus solfataricus obtained in the presence of GDP. Our report highlights how the two-switch regions involved in formation of the tRNA-binding site on subunit γ exchange conformational information with α and β. The zinc-binding domain of β lies close to the guanine nucleotide and directly contacts the switch 1 region. As a result, switch 1 adopts a not yet described conformation. Moreover, unexpectedly for a GDP-bound state, switch 2 has the “ON” conformation. The stability of these conformations is accounted for by a ligand, most probably a phosphate ion, bound near the nucleotide binding site. The structure suggests that this GDP–inorganic phosphate (Pi) bound state of aIF2 may be proficient for tRNA binding. Recently, it has been proposed that dissociation of eIF2 from the initiation complex is closely coupled to that of Pi from eIF2γ upon start codon recognition. The nucleotide state of aIF2 shown here is indicative of a similar mechanism in archaea. Finally, we consider the possibility that release of Pi takes place after e/aIF2γ has been informed of e/aIF1 dissociation by e/aIF2β