Cooperativity of complex salt bridges

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Abstract
The energetic contribution of complex salt bridges, in which one charged residue (anchor residue) forms salt bridges with two or more residues simultaneously, has been suggested to have importance for protein stability. Detailed analysis of the net energetics of complex salt bridge formation using double‐ and triple‐mutant cycle analysis revealed conflicting results. In two cases, it was shown that complex salt bridge formation is cooperative, i.e., the net strength of the complex salt bridge is more than the sum of the energies of individual pairs. In one case, it was reported that complex salt bridge formation is anti‐cooperative. To resolve these different findings, we performed analysis of the geometries of salt bridges in a representative set of structures from the PDB and found that over 87% of all complex salt bridges anchored by Arg/Lys have a geometry such that the angle formed by their Cα atoms, Θ, is <90°. This preferred geometry is observed in the two reported instances when the energetics of complex salt bridge formation is cooperative, while in the reported anti‐cooperative complex salt bridge, Θ is close to 160°. Based on these observations, we hypothesized that complex salt bridges are cooperative for Θ < 90° and anti‐cooperative for 90° < Θ < 180°. To provide a further experimental test for this hypothesis, we engineered a complex salt bridge with Θ = 150° into a model protein, the activation domain of human procarboxypeptidase A2 (ADA2h). Experimentally derived stabilities of the ADA2h variants allowed us to show that the complex salt bridge in ADA2h is anti‐cooperative.