Interaction Between Transcription Factor EhPC4 and Polyadenylation Factor EhCFIm25 in Entamoeba histolytica:...

The coordination between transcription and mRNA processing is essential for eukaryotic gene regulation, yet the structural basis of this coupling remains poorly understood in Entamoeba histolytica, the protozoan parasite responsible for amoebiasis. In this study, we characterized the interaction between the transcriptional coactivator EhPC4 and the polyadenylation factor EhCFIm25 through an integrated in vitro and in silico approach. Far-Western assays confirmed direct physical interaction between both recombinant proteins. To elucidate the molecular mechanism, we performed 500 ns Molecular Dynamics simulations of full-length EhPC4, identifying high flexibility in its N-terminal region. Protein-protein docking analysis revealed a stable EhPC4-EhCFIm25 complex (Cluster C4) with favorable binding energies (∆G = -11.4 kcal/mol). Notably, heatmap analysis of the interaction interface identified a conserved "hotspot" at the C-terminal end of EhCFIm25 (residues 249-255) that mediates the binding with PC4 without occluding DNA-binding domain (K127 in EhPC4) or RNA-recognition motifs in EhCFIm25. Our findings suggest that EhCFIm25 serves as a molecular scaffold that physically couples transcription and polyadenylation, providing a structural framework for the efficient regulation of virulence-related genes in this parasite.