Side-chain pairing preferences in the parallel coiled-coil dimer motif: insight on ion pairing between core and flanking sites

Abstract

A new strategy for rapid evaluation of sequence-stability relationships in the parallel coiled-coil motif is described. The experimental design relies upon thiol-thioester exchange equilibria, an approach that is particularly well suited to examination of heterodimeric systems. Our model system has been benchmarked by demonstrating that it can quantitatively reproduce previously reported trends in interhelical a-a' side-chain pairing preferences at the coiled-coil interface. This new tool has been used to explore the role of Coulombic interactions between a core position on one helix and a flanking position on the other helix (a-g'). This type of interhelical contact has received relatively little attention to date. Our results indicate that such interactions can influence coiled-coil partner preferences.

Figure 1

(a) Primary sequence of C_T-C' (X = Ψ =Ile). (b) Helical wheel diagram showing the proposed helical regions of C_T-C'. Guest sites are highlighted in dashed boxes (c) Cartoon illustrating the thiol-thioester exchange process.

Figure 2

(a) Thermodynamic data (ΔG_CC) values determined from thioester exchange of C_T-C' mutants. Assays were conducted in 50 mM sodium phosphate buffer (pH 7) with 2 mM TCEP at 25 °C. (b) Correlation diagram comparing Δ(ΔG) data from the Vinson laboratory, and our work using thioester exchange. Δ(ΔG) values are normalized to the Ala-Ala homotypic pairing.

Figure 3

Partial helical net diagrams and ΔG_CC values depicting for different a–g' or a'–g ion pairs.