Functional and Structural Insights Revealed by Molecular Dynamics Simulations of an Essential RNA Editing Ligase in Trypanosoma brucei

RNA editing ligase 1 (TbREL1) is required for the survival of both the insect and bloodstream forms of Trypanosoma brucei, the parasite responsible for the devastating tropical disease African sleeping sickness. The type of RNA editing that TbREL1 is involved in is unique to the trypanosomes, and no close human homolog is known to exist. In addition, the high-resolution crystal structure revealed several unique features of the active site, making this enzyme a promising target for structure-based drug design. In this work, two 20 ns atomistic molecular dynamics (MD) simulations are employed to investigate the dynamics of TbREL1, both with and without the ATP substrate present. The flexibility of the active site, dynamics of conserved residues and crystallized water molecules, and the interactions between TbREL1 and the ATP substrate are investigated and discussed in the context of TbREL1's function. Differences in local and global motion upon ATP binding suggest that two peripheral loops, unique to the trypanosomes, may be involved in interdomain signaling events. Notably, a significant structural rearrangement of the enzyme's active site occurs during the apo simulations, opening an additional cavity adjacent to the ATP binding site that could be exploited in the development of effective inhibitors directed against this protozoan parasite. Finally, ensemble averaged electrostatics calculations over the MD simulations reveal a novel putative RNA binding site, a discovery that has previously eluded scientists. Ultimately, we use the insights gained through the MD simulations to make several predictions and recommendations, which we anticipate will help direct future experimental studies and structure-based drug discovery efforts against this vital enzyme

The F0F1-ATP Synthase Complex Contains Novel Subunits and Is Essential for Procyclic Trypanosoma brucei

The mitochondrial F0F1 ATP synthase is an essential multi-subunit protein complex in the vast majority of eukaryotes but little is known about its composition and role in Trypanosoma brucei, an early diverged eukaryotic pathogen. We purified the F0F1 ATP synthase by a combination of affinity purification, immunoprecipitation and blue-native gel electrophoresis and characterized its composition and function. We identified 22 proteins of which five are related to F1 subunits, three to F0 subunits, and 14 which have no obvious homology to proteins outside the kinetoplastids. RNAi silencing of expression of the F1 α subunit or either of the two novel proteins showed that they are each essential for the viability of procyclic (insect stage) cells and are important for the structural integrity of the F0F1-ATP synthase complex. We also observed a dramatic decrease in ATP production by oxidative phosphorylation after silencing expression of each of these proteins while substrate phosphorylation was not severely affected. Our procyclic T. brucei cells were sensitive to the ATP synthase inhibitor oligomycin even in the presence of glucose contrary to earlier reports. Hence, the two novel proteins appear essential for the structural organization of the functional complex and regulation of mitochondrial energy generation in these organisms is more complicated than previously thought

Novel Naphthalene-Based Inhibitors of Trypanosoma brucei RNA Editing Ligase 1

African sleeping sickness is a devastating disease that plagues sub-Saharan Africa. Neglected tropical diseases like African sleeping sickness cause significant death and suffering in the world's poorest countries. Current treatments for African sleeping sickness either have high costs, terrible side effects, or limited effectiveness. Consequently, new medicines are urgently needed. RNA editing ligase 1 is an important protein critical for the survival of Trypanosoma brucei, the unicellular parasite that causes African sleeping sickness. In this paper, we describe our recent efforts to use advanced computer techniques to identify chemicals predicted to prevent RNA editing ligase 1 from functioning properly. We subsequently tested our predicted chemicals and confirmed that a number of them inhibited the protein's function. Additionally, one of the chemicals was effective at stopping the growth of the parasite in culture. Although substantial work remains to be done in order to optimize these chemicals so they are effective and safe to use in human patients, the identification of these parasite-killing compounds is nevertheless a valuable step towards finding a better cure for this devastating disease

TbMP42 is a structure-sensitive ribonuclease that likely follows a metal ion catalysis mechanism

RNA editing in African trypanosomes is characterized by a uridylate-specific insertion and/or deletion reaction that generates functional mitochondrial transcripts. The process is catalyzed by a multi-enzyme complex, the editosome, which consists of approximately 20 proteins. While for some of the polypeptides a contribution to the editing reaction can be deduced from their domain structure, the involvement of other proteins remains elusive. TbMP42, is a component of the editosome that is characterized by two C2H2-type zinc-finger domains and a putative oligosaccharide/oligonucleotide-binding fold. Recombinant TbMP42 has been shown to possess endo/exoribonuclease activity in vitro; however, the protein lacks canonical nuclease motifs. Using a set of synthetic gRNA/pre-mRNA substrate RNAs, we demonstrate that TbMP42 acts as a topology-dependent ribonuclease that is sensitive to base stacking. We further show that the chelation of Zn2+ cations is inhibitory to the enzyme activity and that the chemical modification of amino acids known to coordinate Zn2+ inactivates rTbMP42. Together, the data are suggestive of a Zn2+-dependent metal ion catalysis mechanism for the ribonucleolytic activity of rTbMP42

Multiple evolutionary origins of Trypanosoma evansi in Kenya

Trypanosoma evansi is the parasite causing surra, a form of trypanosomiasis in camels and other livestock, and a serious economic burden in Kenya and many other parts of the world. Trypanosoma evansi transmission can be sustained mechanically by tabanid and Stomoxys biting flies, whereas the closely related African trypanosomes T. brucei brucei and T. b. rhodesiense require cyclical development in tsetse flies (genus Glossina) for transmission. In this study, we investigated the evolutionary origins of T. evansi. We used 15 polymorphic microsatellites to quantify levels and patterns of genetic diversity among 41 T. evansi isolates and 66 isolates of T. b. brucei (n = 51) and T. b. rhodesiense (n = 15), including many from Kenya, a region where T. evansi may have evolved from T. brucei. We found that T. evansi strains belong to at least two distinct T. brucei genetic units and contain genetic diversity that is similar to that in T. brucei strains. Results indicated that the 41 T. evansi isolates originated from multiple T. brucei strains from different genetic backgrounds, implying independent origins of T. evansi from T. brucei strains. This surprising finding further suggested that the acquisition of the ability of T. evansi to be transmitted mechanically, and thus the ability to escape the obligate link with the African tsetse fly vector, has occurred repeatedly. These findings, if confirmed, have epidemiological implications, as T. brucei strains from different genetic backgrounds can become either causative agents of a dangerous, cosmopolitan livestock disease or of a lethal human disease, like for T. b. rhodesiense