Investigating the interaction of Escherichia coli stringent starvation protein A (EcSspA) with indanyloxyacetic acid 94 and ethacrynic acid
Date
2020
Authors
Mokoena, Rethabile
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Abstract
Stringent starvation protein A (SspA) is a highly conserved protein found in Gram-negative bacteria, such as Escherichia coli (E. coli). SspA plays a critical role in the activation of defence systems in bacteria, such as acid tolerance, to aid in the survival of Gram-negative bacteria exposed to harmful conditions. E. coli SspA (EcSspA) is a bacterial homologue of human chloride intracellular channel 1 (hCLIC1) and both are members of the glutathione transferase (GST) superfamily. EcSspA and hCLIC1 exhibit similarities in structure and ion channel formation. Ion channel activity of membranous hCLIC1 and EcSspA is inhibited by indanyloxyacetic acid 94 (IAA-94) as well as the enzymatic activity of soluble hCLIC1, however, the effect that IAA-94 has on soluble EcSspA is unknown. Ethacrynic acid (EA), a structural homologue of IAA-94, is a known inhibitor of cytosolic GST enzymatic activity and its effect on EcSspA is also unknown. The aim of this study was to investigate the interaction of soluble EcSspA with IAA-94 and EA using 8-anilino-1-naphthalenesulfonic acid (ANS) displacement, isothermal titration calorimetry (ITC) and induced-fit molecular docking. EcSspA was purified to homogeneity using nickel ion immobilised metal affinity chromatography (Ni2+-IMAC). Structural analysis of soluble EcSspA using far-UV circular dichroism, intrinsic tryptophan fluorescence spectroscopy as well as size exclusion high performance liquid chromatography, confirmed that soluble EcSspA has a predominantly α-helical backbone, with tryptophan residues that are partially solvent accessible and that it is a dimeric protein. Data obtained from ITC, ANS displacement and induced-fit molecular docking experiments, indicated that there was weak binding between EcSspA and both ligands at the dimer interface. The data further showed no interaction occurred at the EcSspA surface pocket. Therefore indicating that the dimer interface is a potential target site on soluble EcSspA. However, it remains unknown how the binding of IAA-94 and EA to soluble EcSspA would influence EcSspA function
Description
A dissertation submitted in fulfilment of the requirements for the degree Master of Science
in Molecular and Cell Biology in the Faculty of Science, University of the Witwatersrand, 2020