•PemIKSa toxin-antitoxin system is widespread in S. pseudintermedius.•PemIKSa exhibits sequence heterogeneity.•PemISa antitoxin seems pseudogenized.•PemKSa toxin is an active RNase, however nontoxic ...to host cells.•TA systems are regulatory entities rather than growth inhibitors.
Toxin-antitoxin (TA) systems are ubiquitous in bacteria and on numerous occasions have been postulated to play a role in virulence of pathogens. Some Staphylococcus aureus strains carry a plasmid, which encodes the highly toxic PemIKSa TA system involved in maintenance of the plasmid but also implicated in modulation of gene expression. Here we showed that pemIKSa1-Sp TA system, homologous to the plasmid-encoded PemIKSa, is present in virtually each chromosome of S. pseudintermedius strain, however exhibits sequence heterogeneity. This results in two length variants of the PemKSa1-Sp toxin. The shorter (96 aa), C-terminally truncated toxin is enzymatically inactive, whereas the full length (112 aa) variant is an RNase, though nontoxic to the host cells. The lack of toxicity of the active PemKSa-Sp2 toxin is explained by increased substrate specificity. The pemISa1-Sp antitoxin gene seems pseudogenized, however, the whole pemIKSa1-Sp system is transcriptionally active. When production of N-terminally truncated antitoxins using alternative start codons is assumed, there are five possible length variants. Here we showed that even substantially truncated antitoxins are able to interact with PemKSa-Sp2 toxin and inhibit its RNase activity. Moreover, the antitoxins can rescue bacterial cells from toxic effects of overexpression of plasmid-encoded PemKSa toxin. Collectively, our data indicates that, contrary to the toxic plasmid-encoded PemIKSa TA system, location of pemIKSa1-Sp in the chromosome of S. pseudintermedius results in the loss of its toxicity. Interestingly, the retained RNase activity of PemKSa1-Sp2 toxin and functionality of the putative, N-terminally truncated antitoxins suggest the existence of evolutionary pressure for alleviation/mitigation of the toxin’s toxicity and retention of the inhibitory activity of the antitoxin, respectively.
Chloroplasts were formed by uptake of cyanobacteria into eukaryotic cells ca. 1.6billion years ago. During evolution most of the cyanobacterial genes were transferred from the chloroplast to the ...nuclear genome. The rbcX gene, encoding an assembly chaperone required for Rubisco biosynthesis in cyanobacteria, was duplicated. Here we demonstrate that homologous eukaryotic chaperones (AtRbcX1 and AtRbcX2) demonstrate different affinities for the C-terminus of Rubisco large subunit and determine their crystal structures.
Three-dimensional structures of AtRbcX1 and AtRbcX2 were resolved by the molecular replacement method. Equilibrium binding constants of the C-terminal RbcL peptide by AtRbcX proteins were determined by spectrofluorimetric titration. The binding mode of RbcX–RbcL was predicted using molecular dynamic simulation.
We provide crystal structures of both chaperones from Arabidopsis thaliana providing the first structural insight into Rubisco assembly chaperones form higher plants. Despite the low sequence homology of eukaryotic and cyanobacterial Rubisco chaperones the eukaryotic counterparts exhibit surprisingly high similarity of the overall fold to previously determined prokaryotic structures. Modeling studies demonstrate that the overall mode of the binding of RbcL peptide is conserved among these proteins. As such, the evolution of RbcX chaperones is another example of maintaining conserved structural features despite significant drift in the primary amino acid sequence.
The presented results are the approach to elucidate the role of RbcX proteins in Rubisco assembly in higher plants.
► Structural details of eucaryotic Rubisco chaperones are shown for the first time. ► Crystal structures of two RbcX homologs from Arabidopsis thaliana are provided. ► Both AtRbcX share the same overall topology with their cyanobacterial equivalents. ► Structural details differ significantly in the case of AtRbcX1. ► High conservation of the binding pockets of distant RbcX chaperones is revealed.
The p53 pathway is inactivated in almost all types of cancer by mutations in the p53 encoding gene or overexpression of the p53 negative regulators, Mdm2 and/or Mdmx. Restoration of the p53 function ...by inhibition of the p53-Mdm2/Mdmx interaction opens up a prospect for a nongenotoxic anticancer therapy. Here, we present the syntheses, activities, and crystal structures of two novel classes of Mdm2-p53 inhibitors that are based on the 3-pyrrolin-2-one and 2-furanone scaffolds. The structures of the complexes formed by these inhibitors and Mdm2 reveal the dimeric protein molecular organization that has not been observed in the small-molecule/Mdm2 complexes described until now. In particular, the 6-chloroindole group does not occupy the usual Trp-23 pocket of Mdm2 but instead is engaged in dimerization. This entirely unique binding mode of the compounds opens new possibilities for optimization of the Mdm2–p53 interaction inhibitors.
Immune checkpoint‐targeting antibodies brought a recent breakthrough in cancer immunotherapy, but the development of small molecule checkpoint inhibitors is lagging behind. Here, the characterization ...of a macrocyclic peptide (p101) capable of blocking the PD‐L1/PD‐1 immune checkpoint is reported at the interface of cancer and immune cells. NMR and other physicochemical data demonstrate that the macrocycle binds to PD‐L1 to sterically hinder its interaction with PD‐1. The crystal structure of the complex highlights the detailed features of the binding site and demonstrates that the interaction is primarily guided by hydrophobic forces. A reporter PD‐L1/PD‐1 blockade bioassay demonstrates that p101 is able to inhibit the interaction in the cellular environment. Finally, it is shown that p101 releases the immune response of primary human T‐cells from PD‐L1 mediated inhibition.
The PD‐L1/PD‐1 immune checkpoint has proved to be a valid target for cancer immunotherapy, and small molecule inhibitors are now awaited as a next step. This study provides evidence of competitive inhibition of the PD‐L1/PD‐1 checkpoint by PD‐L1 lignad–macrocyclic peptide p101. A structural rationale for inhibition and proof‐of‐concept in human primary immune cells are provided.
Blockade of the immunoinhibitory PD‐1/PD‐L1 pathway using monoclonal antibodies has shown impressive results with durable clinical antitumor responses. Anti‐PD‐1 and anti‐PD‐L1 antibodies have now ...been approved for the treatment of a number of tumor types, whereas the development of small molecules targeting immune checkpoints lags far behind. We characterized two classes of macrocyclic‐peptide inhibitors directed at the PD‐1/PD‐L1 pathway. We show that these macrocyclic compounds act by directly binding to PD‐L1 and that they are capable of antagonizing PD‐L1 signaling and, similarly to antibodies, can restore the function of T‐cells. We also provide the crystal structures of two of these small‐molecule inhibitors bound to PD‐L1. The structures provide a rationale for the checkpoint inhibition by these small molecules, and a description of their small molecule/PD‐L1 interfaces provides a blueprint for the design of small‐molecule inhibitors of the PD‐1/PD‐L1 pathway.
Trennender Ring: Makrocyclische Peptid‐Inhibitoren können den PD‐1/PD‐L1‐Pfad blockieren, indem sie direkt an PD‐L1 binden und, ähnlich wie Anti‐PD‐L1‐Antikörper, die Funktion von T‐Zellen wiederherstellen. Strukturen der Grenzfläche zwischen Makrocyclus und PD‐L1 bilden die Grundlage für den Entwurf von niedermolekularen Inhibitoren mit Antitumoreigenschaften.
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