Protein Evolution across Fold Classes: A 3-α-Helix Bundle can Switch to β, α/β, and α+β Folds by Stepwise Mutation

Abstract

The mechanism for the evolution of new protein folds remains obscure. Recent examples of proteins with high levels of sequence identity but different folds have given new impetus to the hypothesis that proteins can evolve via stepwise mutation. Few examples of these metamorphic proteins are available, however, concealing the number of conformational alternatives accessible to a given fold. Here we show that stepwise mutation can switch one fold, a 3-α-helix bundle, to three radically different conformations: β-sheet, α/β/α-sandwich, and α/β-grasp. Specifically, we engineered three variants of the human serum albumin binding domain of protein G (GA, 3-α-helix bundle) to have >50% sequence identity to three other proteins: a subdomain within outer surface protein A (OspA, β-sheet), a subdomain within maltose binding protein (MBP, α/β/α-sandwich), and the IgG-binding domain of protein G (GB, α/β-grasp). Additional modifications to the sequences of these alternative folds yielded three fold pairs with high levels of sequence identity: 77% (GA-OspA), 80% (GA-MBP), and 98% (GA-GB). This network of switchable folds links all four major fold families, mainly α (3-α-helix bundle), mainly β (β-sheet), α/β (α/β/α-sandwich), and α+β (α/β-grasp), demonstrating that stepwise mutation can switch one protein fold to multiple disparate conformations. Furthermore, cross-pair sequence identity levels are consistent with identity cut-offs determined from the PDB: pairwise sequence alignments of all GA variants are >40% identical, while pairwise sequence alignments of the three alternative folds are <30% identical. Together, these results demonstrate that stepwise mutation can induce one parent fold to spawn multiple new folds in a way that is consistent with observations of experimentally determined protein structures.