Understanding the inhibition and post-translational modification of soluble CLIC1 and its structural homologs

Abstract

The chloride intracellular channel 1 (CLIC1) protein is a unique membrane protein considering that it can reversibly transit between a soluble and membrane-bound conformation, whereby the ion channel activity of the latter becomes inhibited by indanyloxyacetic acid-94 (IAA-94). It therefore remained uncertain if the constraint in the ion channel activity of CLIC1 was due to IAA-94 binding to its soluble conformation in order to prevent its structural transition into a membrane competent state and/or IAA-94 binding to the membrane conformation of CLIC1. Therefore, this study aimed at understanding the interaction between IAA-94 and soluble CLIC1 by performing isothermal titration calorimetry (ITC) experiments on CLIC1, together with human glutathione transferase P1-1 (hGSTP1-1) and Escherichia coli stringent starvation protein A (EcSspA), the structural homologs of CLIC1. Based on ITC, it was shown that IAA-94 did not bind to CLIC1, but instead it was shown to interact with hGSTP1-1 and EcSspA, the latter being a metamorphic protein whose ion channel activity is also inhibited by IAA-94. Interestingly, although IAA-94 is a structural analogue of ethacrynic acid (EAA) (an inhibitor for hGSTP1-1), the latter was shown to bind to CLIC1, but not to EcSspA. However, based on structural sequence alignment, it was apparent that the residues which coordinate the interaction between EAA and hGSTP1-1 were primarily conserved within CLIC1, whereas the residues from the buffer binding site (the proposed ligandin binding site (L-site)) of hGSTP1-1 were conserved within EcSspA. As a result, this provided insight as to why CLIC1 and EcSspA could not interact with IAA-94 and EAA, respectively. In fact, the crystal structure of hGSTP1-1 in complex with IAA-94 suggested that IAA-94 did bind at the buffer binding site, but due to crystal contacts, only the acetate moiety of IAA-94 could be solved. These findings therefore supported previous studies which suggested that the buffer binding site is also the L-site of hGSTP1-1. As a result, considering that the EAA and IAA-94 have distinct binding sites within hGSTP1-1 and that the EAA binding site is structurally conserved within CLIC1, the binding of EAA to the active site of CLIC1 was significant considering that it would inhibit the enzyme function of CLIC1 by masking the thiol group of Cys24. In fact, Cys24 was shown to be the main reactive cysteine residue in CLIC1 and in turn it was also shown to play a key role in maintaining the structural stability of CLIC1. Furthermore, Cys24 was shown to be a target site for S-nitrosylation and similar to the C24A mutation, it appeared that this PTM could influence the structural stability of CLIC1. Therefore, although CLIC1 did not bind to IAA-94, its ability to bind to EAA provides insight into the rational drug design of novel inhibitors that could effectively bind and stabilise the soluble conformation of CLIC1 in order to attenuate its structural transition into to a membrane-bound conformation