Identification of a covert evolutionary pathway between two protein folds

Abstract

Although homologous protein sequences are expected to adopt similar structures, some amino acid substitutions can interconvert α-helices and β-sheets. Such fold switching may have occurred over evolutionary history, but supporting evidence has been limited by the: (1) abundance and diversity of sequenced genes, (2) quantity of experimentally determined protein structures, and (3) assumptions underlying the statistical methods used to infer homology. Here, we overcame these barriers by applying multiple statistical methods to a family of ~600,000 bacterial response regulator proteins. We found that their homologous DNA-binding subunits assume divergent structures: helix-turn-helix versus α-helix+β-sheet (winged helix). Phylogenetic analyses, ancestral sequence reconstruction, and AlphaFold2 models indicated that amino acid substitutions facilitated a switch from helix-turn-helix into winged helix. This structural transformation likely expanded DNA-binding specificity. Our approach uncovers an evolutionary pathway between two protein folds and provides methodology to identify secondary structure switching in other protein families.

Distinguishing features of fold‐switching proteins

“…A recent preprint reports that AlphaFold2 was able to accurately predict the alternative conformations of 3/6‐fold switchers when shallow, subfamily‐specific MSAs were inputted (Wayment‐Steele et al, 2022). Furthermore, AlphaFold2 predicted α‐helix ↔ β‐sheet switching in reconstructed ancestors of bacterial response regulator proteins with high pairwise sequence identity (Chakravarty et al, 2022).…”

Fluid protein fold space and its implications

“…Three lines of evidence suggest that protein fold space is fluid: (1) experimentally characterized amino acid sequences that interconvert between two stable folds with distinct secondary structures (fold-switching, [29] or metamorphic [27] proteins), (2) discoveries of short mutational pathways interconnecting proteins with distinct folds, [34,39,40] and (3) evolutionary analyses coupled with experiments demonstrating that fold switching is conserved among diverse homologs. [36,37] Demonstrating the first line of evidence, Figure 1B shows two proteins that switch between distinct stable folds: XCL1 (green), a human chemokine, and the C-terminal domain (CTD) of RfaH (yellow) a bacterial transcription factor.…”

“…Both HTH 4 and wH proteins bind DNA, but the HTH 4 tends to contact the major groove only, while wH contacts both the major and minor grooves. [34] Thus, this likely evolved fold switching event may have increased binding specificity.…”

“…Using a massive number of homologous sequences, we reported a natural evolutionary pathway between two protein folds – helix‐turn‐helix (HTH 4 ) and winged helix (wH). [ 34 ] Phylogenetic analysis, ancestral sequence reconstruction, and AF2 models suggest that the C‐terminal β‐sheet “wing” of the wH evolved from the C‐terminal helix of the HTH 4 . Both HTH 4 and wH proteins bind DNA, but the HTH 4 tends to contact the major groove only, while wH contacts both the major and minor grooves.…”

“…Repeating the procedure used to generate Figure 1A with fold‐switching proteins included yields Figure 1B (full methodological details in Supplementary Text), a graphical representation of fluid fold space. Fluid fold space is supported by three recent observations: (1) some amino acid sequences interconvert between folds with distinct secondary structures, [ 34,35 ] (2) some naturally occurring sequences have switched folds by stepwise mutation, [ 36 ] and (3) fold switching is evolutionarily selected and likely confers advantage. [ 14,37,38 ] Most work on fold‐switching proteins has been published in the past 15 years, and readers are directed toward reviews covering earlier studies.…”