Evolutionary selection of proteins with two folds

Abstract

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 assume 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 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 variant--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 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.

AlphaFold2 has more to learn about protein energy landscapes

“…As mentioned previously, both SPEACH_AF and AF-cluster postulate that AF2 can predict alternative protein conformations when sufficient coevolutionary information is provided. A recent computational approach called Alternative Contact Enhancement (ACE) identified coevolutionary information unique to both folds of 56 fold-switching proteins, confirming that MSAs often contain structural information unique to both conformations (11). Nevertheless, after combining all correctly predicted fold switch pairs from 282,000 predicted structures ( Figure 1B ), AlphaFold2 misses this information in 43/56 cases.…”

“…Our results suggest a way to potentially improve AF2-based predictions of fold-switching proteins. Previous work from our lab shows that coevolutionary signals for both folds of fold-switching proteins are often present in MSAs (11). Deep MSAs show strong signal for a dominant conformation, while shallower subfamily-specific MSAs show increased signal for the alternative.…”

“…Here, we systematically assess AF2’s ability to sample and score both experimentally determined conformations of 93 fold-switching proteins (10). This newly emerging class of proteins has been evolutionarily selected to assume two distinctly folded states (11), presumably for functionally important reasons (12). Previously, we showed that AF2.2.0 is systematically biased to predict one conformation of fold switchers while missing the other (13).…”

SSDraw: Software for generating comparative protein secondary structure diagrams

“…That is, the secondary structure of Protein A at position X can be compared directly to the secondary structure of its homolog, Protein B, at position X if their secondary structures are both registered to the same MSA (Figure 1). Comparative secondary structure diagrams also simplify the visualization of fold‐switching proteins, single sequences evolutionarily selected to remodel their secondary and tertiary structures in response to cellular stimuli (Murzin, 2008; Porter & Looger, 2018; Schafer & Porter, 2023). In short, as increasing evidence indicates that highly similar or identical protein sequences can assume folds with drastically different secondary structures (Porter, 2023), the need to graphically depict structural differences among homologous proteins and relate them to other protein properties increases.…”

SSDraw: software for generating comparative protein secondary structure diagrams

“…That is, the secondary structure of Protein A at position X can be compared directly to the secondary structure of its homolog, Protein B, at position X if their secondary structures are both registered to the same MSA (Figure 1). Comparative secondary structure diagrams also simplify the visualization of fold-switching proteins, single sequences evolutionarily selected to remodel their secondary and tertiary structures in response to cellular stimuli [18][19][20] . In short, as increasing evidence indicates that highly similar or identical protein sequences can assume folds with drastically different secondary structures 21 , the need to graphically depict structural differences among homologous proteins and relate them to other protein properties increases.…”