In eukaryotic cells, components of the 5′ to 3′ mRNA degradation machinery can undergo a rapid phase transition. The resulting cytoplasmic foci are referred to as processing bodies (P-bodies). The molecular details of the self-aggregation process are, however, largely undetermined. Herein, we use a bottom-up approach that combines NMR spectroscopy, isothermal titration calorimetry, X-ray crystallography, and fluorescence microscopy to probe if mRNA degradation factors can undergo phase transitions in vitro. We show that the Schizosaccharomyces pombe Dcp2 mRNA decapping enzyme, its prime activator Dcp1, and the scaffolding proteins Edc3 and Pdc1 are sufficient to reconstitute a phase-separation process. Intermolecular interactions between the Edc3 LSm domain and at least 10 helical leucine-rich motifs in Dcp2 and Pdc1 build the core of the interaction network. We show that blocking of these interactions interferes with the clustering behavior, both in vitro and in vivo.
Cellular liquid–liquid phase separation (LLPS) results in the formation of dynamic granules that play an important role in many biological processes. On a molecular level, the clustering of proteins into a confined space results from an indefinite network of intermolecular interactions. Here, we introduce and exploit a novel high-throughput bottom-up approach to study how the interactions between RNA, the Dcp1:Dcp2 mRNA decapping complex and the scaffolding proteins Edc3 and Pdc1 result in the formation of processing bodies. We find that the LLPS boundaries are close to physiological concentrations upon inclusion of multiple proteins and RNA. Within in vitro processing bodies the RNA is protected against endonucleolytic cleavage and the mRNA decapping activity is reduced, which argues for a role of processing bodies in temporary mRNA storage. Interestingly, the intrinsically disordered region (IDR) in the Edc3 protein emerges as a central hub for interactions with both RNA and mRNA decapping factors. In addition, the Edc3 IDR plays a role in the formation of irreversible protein aggregates that are potentially detrimental for cellular homeostasis. In summary, our data reveal insights into the mechanisms that lead to cellular LLPS and into the way this influences enzymatic activity.
The peptidoglycan of Staphylococcus aureus is highly amidated. Amidation of α-D-isoglutamic acid in position 2 of the stem peptide plays a decisive role in the polymerization of cell wall building blocks. S. aureus mutants with a reduced degree of amidation are less viable and show increased susceptibility to methicillin, indicating that targeting the amidation reaction could be a useful strategy to combat this pathogen. The enzyme complex that catalyzes the formation of α-D-isoglutamine in the Lipid II stem peptide was identified recently and shown to consist of two subunits, the glutamine amidotransferase-like protein GatD and the Mur ligase homolog MurT. We have solved the crystal structure of the GatD/MurT complex at high resolution, revealing an open, boomerang-shaped conformation in which GatD is docked onto one end of MurT. Putative active site residues cluster at the interface between GatD and MurT and are contributed by both proteins, thus explaining the requirement for the assembled complex to carry out the reaction. Site-directed mutagenesis experiments confirm the validity of the observed interactions. Small-angle X-ray scattering data show that the complex has a similar conformation in solution, although some movement at domain interfaces can occur, allowing the two proteins to approach each other during catalysis. Several other Gram-positive pathogens, including Streptococcus pneumoniae, Clostridium perfringens and Mycobacterium tuberculosis have homologous enzyme complexes. Combined with established biochemical assays, the structure of the GatD/MurT complex provides a solid basis for inhibitor screening in S. aureus and other pathogens.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.