Comparative study of His- and Non-His-tagged CLIC proteins, reveals changes in their enzymatic activity

Daniel R Turkewitz,Saba Moghaddasi,Amani Alghalayini,Claudia D'Amario,Hala M Ali,Michael Wallach,Stella M Valenzuela

doi:10.1016/j.bbrep.2021.101015

Daniel R Turkewitz, Saba Moghaddasi + Show 5 more

Open Access

https://doi.org/10.1016/j.bbrep.2021.101015

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Abstract

The chloride intracellular ion channel protein (CLIC) family are a unique set of ion channels that can exist as soluble and integral membrane proteins. New evidence has emerged that demonstrates CLICs' possess oxidoreductase enzymatic activity and may function as either membrane-spanning ion channels or as globular enzymes. To further characterize the enzymatic profile of members of the CLIC family and to expand our understanding of their functions, we expressed and purified recombinant CLIC1, CLIC3, and a non-functional CLIC1-Cys24A mutant using a Histidine tag, bacterial protein expression system. We demonstrate that the presence of the six-polyhistidine tag at the amino terminus of the proteins led to a decrease in their oxidoreductase enzymatic activity compared to their non-His-tagged counterparts, when assessed using 2-hydroxyethyl disulfide as a substrate. These results strongly suggest the six-polyhistidine tag alters CLIC's structure at the N-terminus, which also contains the enzyme active site. It also raises the need for caution in use of His-tagged proteins when assessing oxidoreductase protein enzymatic function.

Highlights

Enzymes as biological macromolecules have been extensively stud ied and are considered vital for the survival of all living organisms
As an initial comparison to analyze the effects of the two different buffers on CLIC1’s enzymatic activity, puri fied recombinant CLIC1 proteins were run in the hydrox yethyl disulphide (HEDS) enzyme assay
The His-tagged CLIC1 stored in the Imidazole elution buffer, revealed an almost two-fold higher activity (− 0.299 = ±0.57) compared to its column sizing buffer counterpart (P < 0.001) and showed a significant in crease in activity compared to the non-His-tagged CLIC1 control in column sizing buffer, an unexpected result (P < 0.01)

Summary

Introduction

Enzymes as biological macromolecules have been extensively stud ied and are considered vital for the survival of all living organisms. The chloride intracellular ion channel protein family, CLICs, have been shown to have enzymatic activity [1,2] and are unusual as they exist in both monomeric soluble and integral membrane-bound states. CLICs possess structural similarities to the Glutathione S-Transferase (GST) superfamily, the GST-Omega class [7,8] along with a strong homology to the Glutaredoxin (Grx) enzyme family which contain an active site dithiol motif (Cys-X-X-Cys) located in their N-terminal domain. Structural studies have shown that the soluble form of the CLIC proteins display either a conserved monothiol (CLICs 1, 4, 5, and 6) or dithiol (CLICs 2 and 3) active G-site motif [1,8]. HEDS assays were employed to demonstrate CLIC1-C24A and CLIC1-C24S had a loss of enzyme activity [1], with only a decrease in the activity for CLIC3-C22A [1,2], while the double mutant CLIC3- C22A&C25A showed almost a complete loss of enzymatic activity (Supplementary Fig. S4)

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