Identification and prediction of fold-switching proteins.

Abstract

Folded proteins are assumed to be built upon fixed scaffolds of secondary structure, α-helices and β-sheets. Experimentally determined structures of >58,000 non-redundant proteins support this assumption, though it has recently been challenged by ∼100 fold-switching proteins. Though ostensibly rare, these proteins raise the question of how many uncharacterized proteins have shapeshifting, rather than fixed, secondary structures. We used a comparative sequence-based approach to predict fold switching in the universally conserved NusG transcription factor family, one member of which has a 50-residue regulatory subunit experimentally shown to switch between α-helical and β-sheet folds. Our approach predicts that 24% of sequences in this family undergo similar α-helix ⇌ β-sheet transitions. While these predictions cannot be reproduced by other state-of-the-art computational methods, they are confirmed by circular dichroism and nuclear magnetic resonance spectroscopy for 10 out of 10 sequence-diverse variants. Extending principles underlying our predictive approach, we identified coevolution of amino acid pairs uniquely corresponding to both conformations of 58 fold-switching proteins from distinct families. Using a set of these coevolved amino acid pairs, we successfully biased AlphaFold2 to predict two experimentally consistent conformations of a candidate protein with unsolved structure. These results indicate that fold-switching sequences have been preserved by natural selection, suggesting that their functionalities provide evolutionary advantage and paving the way for predictions of multiple distinct protein structures from single sequences.