The explosive epidemicity of amoebiasis caused by the facultative gastrointestinal protozoan parasite Entamoeba histolytica is a major public health problem in developing countries. Multidrug resistance and side effects of various available antiamoebic drugs necessitate the design of novel antiamobeic agents. The cysteine biosynthetic pathway is the critical target for drug design due to its significance in the growth, survival and other cellular activities of E. histolytica. Here, we have screened 0.15 million natural compounds from the ZINC database against the active site of the EhOASS enzyme (PDB ID. 3BM5, 2PQM), whose structure we previously determined to 2.4 Å and 1.86 Å resolution. For this purpose, the incremental construction algorithm of GLIDE and the genetic algorithm of GOLD were used. We analyzed docking results for top ranking compounds using a consensus scoring function of X-Score to calculate the binding affinity and using ligplot to measure protein-ligand interactions. Fifteen compounds that possess good inhibitory activity against EhOASS active site were identified that may act as potential high affinity inhibitors. In vitro screening of a few commercially available compounds established their biological activity. The first ranked compound ZINC08931589 had a binding affinity of ∼8.05 µM and inhibited about 73% activity at 0.1 mM concentration, indicating good correlation between in silico prediction and in vitro inhibition studies. This compound is thus a good starting point for further development of strong inhibitors.

Entamoeba histolytica is a protist parasite that is the causative agent of amoebiasis, and is a highly motile organism. The motility is essential for its survival and pathogenesis, and a dynamic actin cytoskeleton is required for this process. EhCoactosin, an actin-binding protein of the ADF/cofilin family, participates in actin dynamics, and here we report our studies of this protein using both structural and functional approaches. The X-ray crystal structure of EhCoactosin resembles that of human coactosin-like protein, with major differences in the distribution of surface charges and the orientation of terminal regions. According to in vitro binding assays, full-length EhCoactosin binds both F- and G-actin. Instead of acting to depolymerize or severe F-actin, EhCoactosin directly stabilizes the polymer. When EhCoactosin was visualized in E. histolytica cells using either confocal imaging or total internal reflectance microscopy, it was found to colocalize with F-actin at phagocytic cups. Over-expression of this protein stabilized F-actin and inhibited the phagocytic process. EhCoactosin appears to be an unusual type of coactosin involved in E. histolytica actin dynamics. E. histolytica is an important pathogen and a major cause of morbidity and mortality in developing nations. High level of motility and phagocytosis is responsible for the parasite invading different tissues of the host. Phagocytosis and motility depend on highly dynamic actin cytoskeleton of this organism. The mechanisms of actin dynamics is not well understood in E. histolytica. Here we report that coactosin like molecule from E. histolytica, EhCoactosin is involved in F-actin stabilization. The crystal structure obtained for the protein provides explanation for some functional differences observed with respect to the human homologue, such as ability to bind G-actin. Moreover, computational modelling along with crystal structure helps to explain the F-actin binding and stabilization by wild type protein. The mutational analysis further suggests that F-actin binding property does not depend on conserved Lys⁷⁵ residue as observed in Human coactosin like protein (HCLP) but other regions present in protein are involved in binding. Overexpression of this protein in trophozoites leads to stabilization of actin filaments which are not accessible to actin remodelling machinery thereby reducing the growth of parasite due to decreased rate of actin dependent endocytosis. Overall, EhCoactosin behaves as F-actin stabilizing protein in vitro and it also participates in processes like phagocytosis and pseudopod formation.

Cysteine (Cys) plays a major role in growth and survival of the human parasite Entamoeba histolytica. We report here the crystal structure of serine acetyltransferase (SAT) isoform 1, a cysteine biosynthetic pathway enzyme from E. histolytica (EhSAT1) at 1.77 Å, in complex with its substrate serine (Ser) at 1.59 Å and inhibitor Cys at 1.78 Å resolution. EhSAT1 exists as a trimer both in solution as well as in crystal structure, unlike hexamers formed by other known SATs. The difference in oligomeric state is due to the N-terminal region of the EhSAT1, which has very low sequence similarity to known structures, also differs in orientation and charge distribution. The Ser and Cys bind to the same site, confirming that Cys is a competitive inhibitor of Ser. The disordered C-terminal region and the loop near the active site are responsible for solvent-accessible acetyl-CoA binding site and, thus, lose inhibition to acetyl-CoA by the feedback inhibitor Cys. Docking and fluorescence studies show that EhSAT1 C-terminal-mimicking peptides can bind to O-acetyl serine sulfhydrylase (EhOASS), whereas native C-terminal peptide does not show any binding. To test further, C-terminal end of EhSAT1 was mutated and found that it inhibits EhOASS, confirming modified EhSAT1 can bind to EhOASS. The apparent inability of EhSAT1 to form a hexamer and differences in the C-terminal region are likely to be the major reasons for the lack of formation of the large cysteine synthase complex and loss of a complex regulatory mechanism in E. histolytica.

Entamoeba histolytica is the etiological agent of human amoebic colitis and liver abscess, and causes a high level of morbidity and mortality worldwide, particularly in developing countries. There are a number of studies that have shown a crucial role for Ca²⁺ and its binding protein in amoebic biology. EhCaBP5 is one of the EF hand calcium-binding proteins of E. histolytica. We have determined the crystal structure of EhCaBP5 at 1.9 Å resolution in the Ca²⁺-bound state, which shows an unconventional mode of Ca²⁺ binding involving coordination to a closed yet canonical EF-hand motif. Structurally, EhCaBP5 is more similar to the essential light chain of myosin than to Calmodulin despite its somewhat greater sequence identity with Calmodulin. This structure-based analysis suggests that EhCaBP5 could be a light chain of myosin. Surface plasmon resonance studies confirmed this hypothesis, and in particular showed that EhCaBP5 interacts with the IQ motif of myosin 1B in calcium independent manner. It also appears from modelling of the EhCaBP5-IQ motif complex that EhCaBP5 undergoes a structural change in order to bind the IQ motif of myosin. This specific interaction was further confirmed by the observation that EhCaBP5 and myosin 1B are colocalized in E. histolytica during phagocytic cup formation. Immunoprecipitation of EhCaBP5 from total E. histolytica cellular extract also pulls out myosin 1B and this interaction was confirmed to be Ca²⁺ independent. Confocal imaging of E. histolytica showed that EhCaBP5 and myosin 1B are part of phagosomes. Overexpression of EhCaBP5 increases slight rate (∼20%) of phagosome formation, while suppression reduces the rate drastically (∼55%). Taken together, these experiments indicate that EhCaBP5 is likely to be the light chain of myosin 1B. Interestingly, EhCaBP5 is not present in the phagosome after its formation suggesting EhCaBP5 may be playing a regulatory role. Entamoeba histolytica is the etiologic agent of amoebiasis, a major cause of morbidity and mortality in developing countries. The genome of this organism encodes 27 EF-hand containing calcium binding proteins suggesting an intricate Ca²⁺ signalling system that plays crucial role in phagocytosis and pathogenesis. Calcium binding protein-5 (EhCaBP5) is one of these CaBPs that displays sequence similarity with Calmodulin (CaM) but has only two possible calcium binding EF-hand loops in two separate domains. Interestingly crystal structure of EhCaPB5 showed more structural similarity with essential light chain (ELC) of myosin than that of CaM. The binding studies of EhCaBP5 with IQ motif peptides of myosins, showed that it interacts with IQ motif of unconventional Myosin IB. A number of experiments were carried out to show that EhCaBP5 indeed binds myosin IB and that this binding is Ca²⁺ independent. We also show here that EhCaBP5 participates in erythrophagocytosis and that its role in phagocytosis is different from that of EhCaBP3, another myosin 1B interacting calcium binding protein of E. histolytica. Our results presented here and in a number of other reports point towards a unique phagocytic pathway involving a number of calcium binding proteins in E. histolytica.