Evolutionary selection of proteins with two folds

Although most globular proteins fold into a single stable structure[1][1], an increasing number have been shown to remodel their secondary and tertiary structures in response to cellular stimuli[2][2]. State-of-the-art algorithms[3][3]-[5][4] predict that these fold-switching proteins assume only one stable structure[6][5],[7][6], 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 over-represented single-fold sequences may be masking these signatures, we developed an approach to search both highly diverse protein superfamilies–composed of single-fold and fold-switching variants–and protein subfamilies with more fold-switching variants. This approach successfully revealed coevolution of amino acid pairs uniquely corresponding to both conformations of 56/58 fold-switching proteins from distinct families. Then, using a set of coevolved amino acid pairs predicted by our approach, we successfully biased AlphaFold2[5][4] to predict two experimentally consistent conformations of a candidate protein with unsolved structure. 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. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-5 [5]: #ref-6 [6]: #ref-7