How does the antimicrobial protein amoebapore-A dimerize and does this process play a role in function?

Abstract

Amoebapore A (APA-1) is a cytotoxic protein secreted by the parasite Entamoeba histolytica and is one of the causative agents of amoebic dysentery. APA-1 is believed to form an oligomeric pore-like structure that inserts into cells leading to cell lyses. APA- 1 exists as a dimer at pH 5, which is believed to be its intermediate oligomeric active state before further oligomerisation in the target cell membrane. Based on the structure of the monomer and preliminary NMR data, two models have been proposed to describe the structure of the dimer. The aim of this project was to determine which model is correct and which residues at the dimer interface are responsible for dimer formation. This was to be achieved by performing site-directed mutagenesis to generate mutants of APA-1 to test the two models. The mutants were designed to probe amino acids thought to play key roles at different dimerisation interfaces proposed by the two models. Due to the cytotoxicity of APA-1, it is not possible to overexpress this protein in living cells such as E. coli. Consequently, cell-free protein synthesis was optimised and used to produce the different mutants. Some of these then underwent preliminary purification using a three-step process of IMAC HPLC purification, protease cleavage to remove the His6-tag and concluded by Reverse Phase HPLC purification. Future work with the optimised cell free system will enable dimer formation categorisation through techniques such as Isothermal titration calorimetry, small angle X-ray scattering and NMR spectroscopy. Furthermore bioassays examining pore formation and bactericidal activity could be conducted in the future.