Proteins as Play‐Doh: Evolution May Utilize Local Structural Plasticity to Alter Protein Folds

Abstract

Protein fold evolution is a process by which protein structure is altered over time creating the mosaic of diverse protein folds that exists today. A model system for studying protein fold evolution consists of two members of the Cro protein family that have 40% sequence identity but different structures, one all α-helical (Xfaso1) and the other mixed α-helix and β-sheet (Pfl6). Previous work showed that hybrids of these two sequences have combinations of α and β conformations unlike either parent sequence, suggesting that new folds could evolve from preexisting folds in several steps. One such hybrid, XPH1, has the sequence of Xfaso1 with 9 mutations from Pfl6, and has a structure less helical than Xfaso1, but more helical than Pfl6. Here, we introduced subsets of the 9 mutations into Xfaso1 and analyzed the resulting mutant proteins via circular dichroism and NMR to determine which of the 9 mutations were necessary to change the structure of Xfaso1 to that of XPH1. We found that only the E40G and V48Y mutations together were necessary to change the all-α structure of Xfaso1 to that of XPH1. The structural plasticity of helical Cro proteins implies that α-helical folds could have evolved into α+β folds through multiple structural intermediates.