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.

The Chaperone Activities of DsbG and Spy Restore Peptidoglycan Biosynthesis in the elyC 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 Escherichia coli factor ElyC has been shown to be important for peptidoglycan biosynthesis at low temperatures. PG production in ΔelyC 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 ΔelyC 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 ΔelyC 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 ΔelyC cells. In fact, we found that the amount of protein aggregates was greater in the ΔelyC mutant than in the wild type. Taken together, our results show a protein-folding defect in the envelope compartments of ΔelyC cells that blocks PG production, and they reveal a new physiological activity of DsbG.IMPORTANCE 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 Escherichia coli factor ElyC, which is important for PG biosynthesis at low temperatures. We revealed an important protein-folding defect in the ΔelyC 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 in vivo.

The biology of Mur ligases as an antibacterial target.

With antibiotic resistance mechanisms increasing in diversity and spreading among bacterial pathogens, the development of new classes of antibacterial agents against judiciously chosen targets is a high-priority task. The biochemical pathway for peptidoglycan biosynthesis is one of the best sources of antibacterial targets. Within this pathway are the Mur ligases, described in this review as highly suitable targets for the development of new classes of antibacterial agents. The amide ligases MurC, MurD, MurE and MurF function with the same catalytic mechanism and share conserved amino acid regions and structural features that can conceivably be exploited for the design of inhibitors that simultaneously target more than one enzyme. This would provide multi-target antibacterial weapons with minimized likelihood of target-mediated resistance development.