Evolutionary selection of proteins with two folds*

Although most globular proteins fold into a single stable structure, an increasing number have been shown to remodel their secondary and tertiary structures in response to cellular stimuli. State-of-the-art algorithms predict that these fold-switching proteins adopt only one stable structure, missing their functionally critical alternative folds. Why these algorithms predict a single fold is unclear, but all of them infer protein structure from coevolved amino acid pairs. Here, we hypothesize that coevolutionary signatures are being missed. Suspecting that single-fold variants could be masking these signatures, we developed an approach, called Alternative Contact Enhancement (ACE), to search both highly diverse protein superfamilies–composed of single-fold and fold-switching variants–and protein subfamilies with more fold-switching variants. ACE successfully revealed coevolution of amino acid pairs uniquely corresponding to both conformations of 56/56 fold-switching proteins from distinct families. Then, we used ACE-derived contacts to predict two experimentally consistent conformations of a candidate protein with unsolved structure and develop a blind prediction pipeline for fold-switching proteins. The discovery of widespread dual-fold coevolution indicates that fold-switching sequences have been preserved by natural selection, implying that their functionalities provide evolutionary advantage and paving the way for predictions of diverse protein structures from single sequences.