Application of bio-layer interferometry for the analysis of ribosome-protein interactions.

The ribosome, a ribonucleoprotein complex, performs the function of protein translation. While ribosomal RNA catalyzes polypeptide formation, several proteins assist the ribosome throughout the translation process. Studying the biochemical and kinetic properties of these proteins interacting with the ribosome is vital for elucidating their roles. Various techniques, such as zonal centrifugation, pull-down assays, dynamic light scattering (DLS), fluorescence polarization, and surface plasmon resonance (SPR) are employed for this purpose, each presenting unique advantages and limitations. We add to the repertoire of techniques by using Bio-Layer Interferometry (BLI) to examine interactions between the ribosome and translation factors. Our findings demonstrate that BLI can detect interactions of Escherichia coli ribosomes with two proteins: E. coli initiation factor 2 (IF2) and P. falciparum translation enhancing factor (PTEF). A protein (Green Fluorescent Protein; GFP) known not to bind to E. coli ribosomes, shows no binding in the BLI assay. We show that BLI could be used to study the ribosome-protein interactions as it has key advantages like label-free procedures, ease of assay performance, and ribosome sample reuse. Our results highlight the comprehensive use of BLI in studying the ribosome-protein interactions, in addition to studying protein-protein and protein-ligand interactions.

Toxoplasma Gondii Importin α Shows Weak Auto-Inhibition.

Importin α is a nuclear transporter that binds to nuclear localization signals (NLSs), consisting of 7-20 positively charged amino acids found within cargo proteins. In addition to cargo binding, intramolecular interactions also occur within the importin α protein due to binding between the importin ß-binding (IBB) domain and the NLS-binding sites, a phenomenon called auto-inhibition. The interactions causing auto-inhibition are driven by a stretch of basic residues, similar to an NLS, in the IBB domain. Consistent with this, importin α proteins that do not have some of these basic residues lack auto-inhibition; a naturally occurring example of such a protein is found in the apicomplexan parasite Plasmodium falciparum. In this report, we show that importin α from another apicomplexan parasite, Toxoplasma gondii, harbors basic residues (KKR) in the IBB domain and exhibits auto-inhibition. This protein has a long, unstructured hinge motif (between the IBB domain and the NLS-binding sites) that does not contribute to auto-inhibition. However, the IBB domain may have a higher propensity to form an α-helical structure, positioning the wild-type KKR motif in an orientation that results in weaker interactions with the NLS-binding site than a KRR mutant. We conclude that the importin α protein from T. gondii shows auto-inhibition, exhibiting a different phenotype from that of P. falciparum importin α. However, our data indicate that T. gondii importin α may have a low strength of auto-inhibition. We hypothesize that low levels of auto-inhibition may confer an advantage to these important human pathogens.

Conservation of Importin α Function in Apicomplexans: Ivermectin and GW5074 Target Plasmodium falciparum Importin α and Inhibit Parasite Growth in Culture.

Signal-dependent transport into and out of the nucleus mediated by members of the importin (IMP) superfamily of nuclear transporters is critical to the eukaryotic function and a point of therapeutic intervention with the potential to limit disease progression and pathogenic outcomes. Although the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii both retain unique IMPα genes that are essential, a detailed analysis of their properties has not been performed. As a first step to validate apicomplexan IMPα as a target, we set out to compare the properties of P. falciparum and T. gondii IMPα (PfIMPα and TgIMPα, respectively) to those of mammalian IMPα, as exemplified by Mus musculus IMPα (MmIMPα). Close similarities were evident, with all three showing high-affinity binding to modular nuclear localisation signals (NLSs) from apicomplexans as well as Simian virus SV40 large tumour antigen (T-ag). PfIMPα and TgIMPα were also capable of binding to mammalian IMPß1 (MmIMPß1) with high affinity; strikingly, NLS binding by PfIMPα and TgIMPα could be inhibited by the mammalian IMPα targeting small molecules ivermectin and GW5074 through direct binding to PfIMPα and TgIMPα to perturb the α-helical structure. Importantly, GW5074 could be shown for the first time to resemble ivermectin in being able to limit growth of P. falciparum. The results confirm apicomplexan IMPα as a viable target for the development of therapeutics, with agents targeting it worthy of further consideration as an antimalarial.

High-Throughput Screening to Identify Inhibitors of Plasmodium falciparum Importin α.

The global burden of malaria and toxoplasmosis has been limited by the use of efficacious anti-parasitic agents, however, emerging resistance in Plasmodium species and Toxoplasma gondii threatens disease control worldwide, implying that new agents/therapeutic targets are urgently needed. Nuclear localization signal (NLS)-dependent transport into the nucleus, mediated by members of the importin (IMP) superfamily of nuclear transporters, has shown potential as a target for intervention to limit viral infection. Here, we show for the first time that IMPα from P. falciparum and T. gondii have promise as targets for small molecule inhibitors. We use high-throughput screening to identify agents able to inhibit P. falciparum IMPα binding to a P. falciparum NLS, identifying a number of compounds that inhibit binding in the µM-nM range, through direct binding to P. falciparum IMPα, as shown in thermostability assays. Of these, BAY 11-7085 is shown to be a specific inhibitor of P. falciparum IMPα-NLS recognition. Importantly, a number of the inhibitors limited growth by both P. falciparum and T. gondii. The results strengthen the hypothesis that apicomplexan IMPα proteins have potential as therapeutic targets to aid in identifying novel agents for two important, yet neglected, parasitic diseases.