Structures of the cGMP-dependent protein kinase in malaria parasites reveal a unique structural relay mechanism for activation.

The cyclic guanosine-3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) was identified >25 y ago; however, efforts to obtain a structure of the entire PKG enzyme or catalytic domain from any species have failed. In malaria parasites, cooperative activation of PKG triggers crucial developmental transitions throughout the complex life cycle. We have determined the cGMP-free crystallographic structures of PKG from Plasmodium falciparum and Plasmodium vivax, revealing how key structural components, including an N-terminal autoinhibitory segment (AIS), four predicted cyclic nucleotide-binding domains (CNBs), and a kinase domain (KD), are arranged when the enzyme is inactive. The four CNBs and the KD are in a pentagonal configuration, with the AIS docked in the substrate site of the KD in a swapped-domain dimeric arrangement. We show that although the protein is predominantly a monomer (the dimer is unlikely to be representative of the physiological form), the binding of the AIS is necessary to keep Plasmodium PKG inactive. A major feature is a helix serving the dual role of the N-terminal helix of the KD as well as the capping helix of the neighboring CNB. A network of connecting helices between neighboring CNBs contributes to maintaining the kinase in its inactive conformation. We propose a scheme in which cooperative binding of cGMP, beginning at the CNB closest to the KD, transmits conformational changes around the pentagonal molecule in a structural relay mechanism, enabling PKG to orchestrate rapid, highly regulated developmental switches in response to dynamic modulation of cGMP levels in the parasite

Caractérisation du rôle du facteur ElyC dans la biogenèse de l’enveloppe chez l’organisme modèle Escherichia coli

Le peptidoglycane (PG) est le composant principal de l’enveloppe bactérienne, il protège les cellules de la pression osmotique. Un défaut dans le PG cause la lyse de la bactérie. De ce fait, le mécanisme de biosynthèse du PG une excellente cible antibactérienne. Cependant, la biosynthèse du PG et les mécanismes de régulation qui assurent le maintien de l’intégrité de ce composant de l’enveloppe, ne sont pas totalement connus. Dans le but d’élargir nos connaissances quant à ces mécanismes, nous avons choisi d’étudier le rôle du facteur ElyC nouvellement identifié chez Escherichia coli. Ce facteur est important pour le maintien de l’intégrité du PG à 21 °C. Le mutant ΔelyC présente un arrêt de la biosynthèse du PG, suivi de la lyse de la bactérie à la fin de la phase exponentielle de croissance à 21 °C. Premièrement, nous avons identifié les protéines périplasmiques, DsbG et Spy, comme suppresseurs multicopies du phénotype de lyse du mutant ΔelyC à 21 °C. Nous avons démontré que ces protéines agissent par leur activité chaperonne pour corriger un défaut de repliement des protéines dans l’enveloppe, ce qui rétablissent totalement le défaut de PG chez le mutant ΔelyC à 21 °C. Ces résultats suggèrent que la biosynthèse du PG pourrait être bloquée en raison d’un défaut de repliement des protéines de l’enveloppe. Deuxièmement, nous avons étudié l’importance d’ElyC à 37 °C. Nous avons démontré que ElyC est aussi importante pour la biosynthèse du PG à 37 °C. En effet, l’analyse de la composition du PG a démontré une diminution de la quantité de ce composé à 21 °C et à 37 °C. Néanmoins, cette diminution était plus importante à 21 °C, Ce qui démontre que le taux de biosynthèse du PG diminue en absence du facteur ElyC et cette diminution devient plus sévère à basse température. Cela suggère que ElyC est impliquée dans un mécanisme dit « Cold sensitive » (Cs). Finalement, nous avons analysé la réponse transcriptomique induite en absence du facteur ElyC dans les deux conditions de températures. Cette réponse était proportionnelle au degré des dommages dans le PG. Le nombre de gènes différemment exprimés chez le mutant ΔelyC par rapport à une souche sauvage était plus élevé à 21 °C qu’à 37 °C. De plus, plusieurs systèmes de réponse aux stress dans l’enveloppe étaient activés chez le mutant à 21 °C, entraînant l’induction de plusieurs gènes impliqués dans des mécanismes de réparations et de résistances aux stress affectant l’enveloppe. Cependant, la réponse au stress dans l’enveloppe était plus modérée chez le mutant ΔelyC à 37 °C. Cette analyse a aussi démontré une diminution dans l’expression de plusieurs gènes impliqués dans des fonctions physiologiques au niveau de la membrane interne, aux deux températures, ce qui suggère une perturbation au niveau de cette membrane. En conclusion, ce travail a démontré que ElyC est un facteur important à la biosynthèse du PG à 21 °C et à 37 °C. Son rôle serait de réguler le mécanisme de la biosynthèse du PG.Peptidoglycan (PG) is the main structural component of bacterial envelope and it protects bacterial cells against osmotic pressure. Defects in PG cause bacterial cell lysis. Therefore, the pathway for PG synthesis is one of the best sources of antibacterial targets. However, the molecular assembly of PG and the regulatory pathways for the maintenance of this critical envelope layer are still not well understood. To broaden our understanding of these processes, we aimed to study the newly discovered Escherichia coli factor ElyC that is needed for maintaining PG integrity at 21°C. The ΔelyC mutant presents a PG biosynthesis arrest followed by bacterial cell lysis at the end of the logarithmic phase of growth at 21°C. First, we identified the periplasmic proteins, DsbG and Spy, as multi-copy suppressors of the ΔelyC lysis phenotype at 21°C. We showed that these suppressors act by a chaperone activity to restore protein folding defect in the envelope compartment of the mutant which fully correct the PG synthesis defect at 21°C. These results suggest that the PG biosynthesis is blocked due to a protein folding defect in the envelope. Secondly, we evaluated the requirement of ElyC for cell growth and PG synthesis at 37°C. We showed that ElyC is also important for PG biosynthesis at 37°C. In fact, the compositional analysis of PG revealed a decrease in the amount of this compound either at 21 or 37°C. Nevertheless, this decrease was greater at 21°C, which might explain the lysis of the mutant at this temperature. These results demonstrate that the rate of PG biosynthesis decreases in the absence of ElyC factor and this decrease becomes more severe at low temperature. This suggested that ElyC plays an important role in a cold sensitive process. Finally, we analysed the transcriptomic response in ElyC defective cells at both temperatures. The response was proportional to the degree of PG damages. The number of differentially expressed genes in the ΔelyC compared to wild-type strain was higher at 21°C than at 37°C. Furthermore, several envelope stress response systems were activated at 21°C, leading to the induction of several genes involved in mechanisms such as envelope stress resistance and damage repair. However, the envelope stress response was moderate at 37°C. The analysis of downregulated genes in the ΔelyC mutant showed that several of these genes are involved in mechanisms taking place in the inner membrane, which suggested a defect at the level of this compartment. In conclusion, this work demonstrated that ElyC is an important factor for PG biosynthesis at all tested temperatures. ElyC might be a direct or indirect regulator of PG biosynthesis mechanism

The Chaperone Activities of DsbG and Spy Restore Peptidoglycan Biosynthesis in the Mutant by Preventing Envelope Protein Aggregation.

Peptidoglycan (PG) is the main structural component of bacterial envelopes. It protects bacterial cells against variations in osmotic pressure and cell lysis. The newly discovered factor ElyC has been shown to be important for peptidoglycan biosynthesis at low temperatures. PG production in Δ mutant cells is totally blocked after a few hours of growth at 21°C, triggering cell lysis. In this study, we took a candidate approach to identify genetic suppressors of the Δ mutant cell lysis phenotype. We identified the periplasmic proteins DsbG and Spy as multicopy suppressors and showed that their overproduction restores PG biosynthesis in the Δ mutant. Interestingly, we found that DsbG acts by a novel mechanism, which is independent of its known reductase activity and substrates. DsbG, like Spy, acts as a chaperone to reduce the amounts of protein aggregates in the envelopes of Δ cells. In fact, we found that the amount of protein aggregates was greater in the Δ mutant than in the wild type. Taken together, our results show a protein-folding defect in the envelope compartments of Δ cells that blocks PG production, and they reveal a new physiological activity of DsbG. Peptidoglycan biosynthesis is a dynamic and well-controlled pathway. The molecular assembly of PG and the regulatory pathways ensuring its maintenance are still not well understood. Here we studied the newly discovered factor ElyC, which is important for PG biosynthesis at low temperatures. We revealed an important protein-folding defect in the Δ mutant and showed that overproduction of the periplasmic chaperone DsbG or Spy was sufficient to correct the protein-folding defect and restore PG biosynthesis. These results show that the PG defect in the absence of ElyC is caused, at least in part, by a protein-folding problem in the cell envelope. Furthermore, we showed, for the first time, that the periplasmic protein DsbG has chaperone activity