Unraveling the Impact of Cysteine-to-Serine Mutations on the Structural and Functional Properties of Cu(I)-Binding Proteins

Abstract

Appropriate maintenance of Cu(I) homeostasis is an essential requirement for proper cell function because its misregulation induces the onset of major human diseases and mortality. For this reason, several research efforts have been devoted to dissecting the inner working mechanism of Cu(I)-binding proteins and transporters. A commonly adopted strategy relies on mutations of cysteine residues, for which Cu(I) has an exquisite complementarity, to serines. Nevertheless, in spite of the similarity between these two amino acids, the structural and functional impact of serine mutations on Cu(I)-binding biomolecules remains unclear. Here, we applied various biochemical and biophysical methods, together with all-atom simulations, to investigate the effect of these mutations on the stability, structure, and aggregation propensity of Cu(I)-binding proteins, as well as their interaction with specific partner proteins. Among Cu(I)-binding biomolecules, we focused on the eukaryotic Atox1-ATP7B system, and the prokaryotic CueR metalloregulator. Our results reveal that proteins containing cysteine-to-serine mutations can still bind Cu(I) ions; however, this alters their stability and aggregation propensity. These results contribute to deciphering the critical biological principles underlying the regulatory mechanism of the in-cell Cu(I) concentration, and provide a basis for interpreting future studies that will take advantage of cysteine-to-serine mutations in Cu(I)-binding systems.

Roles of RcsA, an AhpD Family Protein, in Reactive Chlorine Stress Resistance and Virulence in Pseudomonas aeruginosa

“…Replacement of a cysteine residue at position 60 or 63 with a serine residue caused a migration of the RcsA protein from 14 kDa to 28 kDa due to an inability to form an intramolecular disulfide formation of monomeric RcsA and increased the possibility of forming either intermolecular disulfide linkages or protein aggregations. Recently, an unraveling of the Cys-to-Ser replacement in the CueR protein resulting in increased aggregation and oligomerization of the mutated proteins in vitro was shown (43). This was similar to the Cys-to-Ser replacement in the RcsA protein in this study.…”

An EPR Study on the Interaction between the Cu(I) Metal Binding Domains of ATP7B and the Atox1 Metallochaperone

“…Here, we applied EPR spectroscopy to MBD1-3 and MBD4-6, exploring their interaction with Atox1. Further, we compared these results with our previous ones from MBD3-4 [ 23 , 28 ].…”

Thiol Redox Regulation of Plant β-Carbonic Anhydrase

“…It was surprising that any Cys → Ser exchange inhibited the enzyme. Possibly, each cysteine replacement in the ring collapses the structure and the intramolecular disulfide-dithiol switch fails to activate the enzyme [36,37]. Thiol switches are commonly used throughout the kingdoms of life to adjust process activities in dependence on cellular redox state.…”

“…Mutations of these cysteines should not result in inhibition of the enzyme by redox-regulation, since activity measurements were done after reduction with DTT. Therefore, the mutations themselves likely cause the loss of activity, since Cys-Ser mutations can alter the stability, structure, aggregation, and therefore, the activity of proteins [36,37].…”