Conjugative plasmids are typically locked in intergenomic and sexual conflicts with coresident rivals, whose translocation they block using fertility inhibition factors (FINs). We describe here the first crystal structure of an enigmatic FIN Osa deployed by the proteobacterial plasmid pSa. Osa contains a catalytically active version of the ParB/Sulfiredoxin fold with both ATPase and DNase activity, the latter being regulated by an ATP-dependent switch. Using the Agrobacterium tumefaciens VirB/D4 type-IV secretion system (T4SS), a relative of the conjugative T4SS, we demonstrate that catalytically active Osa blocks T-DNA transfer into plants. With a partially reconstituted T4SS in vitro, we show that Osa degrades T-DNA in the T-DNA-VirD2 complex prior to its translocation. Further, we present evidence for conservation and interplay between ATPase and DNase activities throughout the ParB/Sulfiredoxin fold, using other members of the family, namely P1 ParB and RK2 KorB, which have general functional implications across diverse biological contexts.

Double-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here we describe biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage asccφ28. Size-exclusion chromatography, analytical ultracentrifugation, small angle x-ray scattering, and negative stain TEM indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Å and radius of gyration of ~54 Å. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other dsDNA phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high resolution structural studies and rigorous biophysical/biochemical analysis.

No abstract

Double-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here, we describe the biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage asccφ28. Size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), small angle X-ray scattering (SAXS), and negative stain transmission electron microscopy (TEM) indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Å and radius of gyration of ~54 Å. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other double-stranded DNA (dsDNA) phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high-resolution structural studies and rigorous biophysical/biochemical analysis.

Malaria remains a dreadful disease by putting every year about 3.4 billion people at risk and resulting into mortality of 627 thousand people worldwide. Existing therapies based upon Quinines and Artemisinin-based combination therapies have started showing resistance, pressing the need for search of anti-malarials with different mechanisms of action. In this respect erythrocyte invasion by Plasmodium is immensely crucial, as being obligate intracellular parasite it must invade host cells. This process is mediated by interaction between conserved Apical Membrane Antigen (AMA1) and Rhoptry Neck (RON2) protein, which is compulsory for successful invasion of erythrocyte by Plasmodium and manifestation of the disease Malaria. Here, using the physicochemical properties of the compounds available from a confirmatory high throughput screening, which were tested for their disruption capability of this crucial molecular interaction, we trained supervised classifiers and validated their robustness by various statistical parameters. Best model was used for screening new compounds from Traditional Chinese Medicine Database. Some of the best hits already find their use as anti-malarials and the model predicts that an essential part of their effectiveness is likely due to inhibition of AMA1-RON2 interaction. Pharmacophoric features have also been identified to ease further designing of possible leads in an effective way.