Rheostats, toggles, and neutrals, Oh my! A new framework for understanding how amino acid changes modulate protein function.

Advances in personalized medicine and protein engineering require accurately predicting outcomes of amino acid substitutions. Many algorithms correctly predict that evolutionarily-conserved positions show "toggle" substitution phenotypes, which is defined when a few substitutions at that position retain function. In contrast, predictions often fail for substitutions at the less-studied "rheostat" positions, which are defined when different amino acid substitutions at a position sample at least half of the possible functional range. This review describes efforts to understand the impact and significance of rheostat positions: (1) They have been observed in globular soluble, integral membrane, and intrinsically disordered proteins; within single proteins, their prevalence can be up to 40%. (2) Substitutions at rheostat positions can have biological consequences and ∼10% of substitutions gain function. (3) Although both rheostat and "neutral" (defined when all substitutions exhibit wild-type function) positions are nonconserved, the two classes have different evolutionary signatures. (4) Some rheostat positions have pleiotropic effects on function, simultaneously modulating multiple parameters (e.g., altering both affinity and allosteric coupling). (5) In structural studies, substitutions at rheostat positions appear to cause only local perturbations; the overall conformations appear unchanged. (6) Measured functional changes show promising correlations with predicted changes in protein dynamics; the emergent properties of predicted, dynamically coupled amino acid networks might explain some of the complex functional outcomes observed when substituting rheostat positions. Overall, rheostat positions provide unique opportunities for using single substitutions to tune protein function. Future studies of these positions will yield important insights into the protein sequence/function relationship.

Fructose-1-kinase has pleiotropic roles in Escherichia coli.

In Escherichia coli, the master transcription regulator catabolite repressor activator (Cra) regulates >100 genes in central metabolism. Cra binding to DNA is allosterically regulated by binding to fructose-1-phosphate (F-1-P), but the only documented source of F-1-P is from the concurrent import and phosphorylation of exogenous fructose. Thus, many have proposed that fructose-1,6-bisphosphate (F-1,6-BP) is also a physiological regulatory ligand. However, the role of F-1,6-BP has been widely debated. Here, we report that the E. coli enzyme fructose-1-kinase (FruK) can carry out its "reverse" reaction under physiological substrate concentrations to generate F-1-P from F-1,6-BP. We further show that FruK directly binds Cra with nanomolar affinity and forms higher order, heterocomplexes. Growth assays with a ΔfruK strain and fruK complementation show that FruK has a broader role in metabolism than fructose catabolism. Since fruK itself is repressed by Cra, these newly-reported events add layers to the dynamic regulation of E. coli's central metabolism that occur in response to changing nutrients. These findings might have wide-spread relevance to other γ-proteobacteria, which conserve both Cra and FruK.

The 2.4 Å structure of Zymomonas mobilis pyruvate kinase: Implications for stability and regulation.

Human liver pyruvate kinase (hlPYK) catalyzes the final step in glycolysis, the formation of pyruvate (PYR) and ATP from phosphoenolpyruvate (PEP) and ADP. Fructose 1,6-bisphosphate (FBP), a pathway intermediate of glycolysis, serves as an allosteric activator of hlPYK. Zymomonas mobilis pyruvate kinase (ZmPYK) performs the final step of the Entner-Doudoroff pathway, which is similar to glycolysis in that energy is harvested from glucose and pyruvate is generated. The Entner-Doudoroff pathway does not have FBP as a pathway intermediate, and ZmPYK is not allosterically activated. In this work, we solved the 2.4 Å X-ray crystallographic structure of ZmPYK. The protein is dimeric in solution as determined by gel filtration chromatography, but crystallizes as a tetramer. The buried surface area of the ZmPYK tetramerization interface is significantly smaller than that of hlPYK, and yet tetramerization using the standard interfaces from higher organisms provides an accessible low energy crystallization pathway. Interestingly, the ZmPYK structure showed a phosphate ion in the analogous location to the 6-phosphate binding site of FBP in hlPYK. Circular Dichroism (CD) was used to measure melting temperatures of hlPYK and ZmPYK in the absence and presence of substrates and effectors. The only significant difference was an additional phase of small amplitude for the ZmPYK melting curves. We conclude that the phosphate ion plays neither a structural or allosteric role in ZmPYK under the conditions tested. We hypothesize that ZmPYK does not have sufficient protein stability for activity to be tuned by allosteric effectors as described for rheostat positions in the allosteric homologues.

A clinically relevant polymorphism in the Na+/taurocholate cotransporting polypeptide (NTCP) occurs at a rheostat position.

Conventionally, most amino acid substitutions at "important" protein positions are expected to abolish function. However, in several soluble-globular proteins, we identified a class of nonconserved positions for which various substitutions produced progressive functional changes; we consider these evolutionary "rheostats". Here, we report a strong rheostat position in the integral membrane protein, Na+/taurocholate (TCA) cotransporting polypeptide, at the site of a pharmacologically relevant polymorphism (S267F). Functional studies were performed for all 20 substitutions (S267X) with three substrates (TCA, estrone-3-sulfate, and rosuvastatin). The S267X set showed strong rheostatic effects on overall transport, and individual substitutions showed varied effects on transport kinetics (Km and Vmax) and substrate specificity. To assess protein stability, we measured surface expression and used the Rosetta software (https://www.rosettacommons.org) suite to model structure and stability changes of S267X. Although buried near the substrate-binding site, S267X substitutions were easily accommodated in the Na+/TCA cotransporting polypeptide structure model. Across the modest range of changes, calculated stabilities correlated with surface-expression differences, but neither parameter correlated with altered transport. Thus, substitutions at rheostat position 267 had wide-ranging effects on the phenotype of this integral membrane protein. We further propose that polymorphic positions in other proteins might be locations of rheostat positions.

Allosteric regulation within the highly interconnected structural scaffold of AraC/XylS homologs tolerates a wide range of amino acid changes.

To create bacterial transcription "circuits" for biotechnology, one approach is to recombine natural transcription factors, promoters, and operators. Additional novel functions can be engineered from existing transcription factors such as the E. coli AraC transcriptional activator, for which binding to DNA is modulated by binding L-arabinose. Here, we engineered chimeric AraC/XylS transcription activators that recognized ara DNA binding sites and responded to varied effector ligands. The first step, identifying domain boundaries in the natural homologs, was challenging because (i) no full-length, dimeric structures were available and (ii) extremely low sequence identities (≤10%) among homologs precluded traditional assemblies of sequence alignments. Thus, to identify domains, we built and aligned structural models of the natural proteins. The designed chimeric activators were assessed for function, which was then further improved by random mutagenesis. Several mutational variants were identified for an XylS•AraC chimera that responded to benzoate; two enhanced activation to near that of wild-type AraC. For an RhaR•AraC chimera, a variant with five additional substitutions enabled transcriptional activation in response to rhamnose. These five changes were dispersed across the protein structure, and combinatorial experiments testing subsets of substitutions showed significant non-additivity. Combined, the structure modeling and epistasis suggest that the common AraC/XylS structural scaffold is highly interconnected, with complex intra-protein and inter-domain communication pathways enabling allosteric regulation. At the same time, the observed epistasis and the low sequence identities of the natural homologs suggest that the structural scaffold and function of transcriptional regulation are nevertheless highly accommodating of amino acid changes.

Substitutions at Nonconserved Rheostat Positions Modulate Function by Rewiring Long-Range, Dynamic Interactions.

Amino acid substitutions at nonconserved protein positions can have noncanonical and "long-distance" outcomes on protein function. Such outcomes might arise from changes in the internal protein communication network, which is often accompanied by changes in structural flexibility. To test this, we calculated flexibilities and dynamic coupling for positions in the linker region of the lactose repressor protein. This region contains nonconserved positions for which substitutions alter DNA-binding affinity. We first chose to study 11 substitutions at position 52. In computations, substitutions showed long-range effects on flexibilities of DNA-binding positions, and the degree of flexibility change correlated with experimentally measured changes in DNA binding. Substitutions also altered dynamic coupling to DNA-binding positions in a manner that captured other experimentally determined functional changes. Next, we broadened calculations to consider the dynamic coupling between 17 linker positions and the DNA-binding domain. Experimentally, these linker positions exhibited a wide range of substitution outcomes: Four conserved positions tolerated hardly any substitutions ("toggle"), ten nonconserved positions showed progressive changes from a range of substitutions ("rheostat"), and three nonconserved positions tolerated almost all substitutions ("neutral"). In computations with wild-type lactose repressor protein, the dynamic couplings between the DNA-binding domain and these linker positions showed varied degrees of asymmetry that correlated with the observed toggle/rheostat/neutral substitution outcomes. Thus, we propose that long-range and noncanonical substitutions outcomes at nonconserved positions arise from rewiring long-range communication among functionally important positions. Such calculations might enable predictions for substitution outcomes at a range of nonconserved positions.

Structural Plasticity Is a Feature of Rheostat Positions in the Human Na+/Taurocholate Cotransporting Polypeptide (NTCP).

In the Na+/taurocholate cotransporting polypeptide (NTCP), the clinically relevant S267F polymorphism occurs at a "rheostat position". That is, amino acid substitutions at this position ("S267X") lead to a wide range of functional outcomes. This result was particularly striking because molecular models predicted the S267X side chains are buried, and thus, usually expected to be less tolerant of substitutions. To assess whether structural tolerance to buried substitutions is widespread in NTCP, here we used Rosetta to model all 19 potential substitutions at another 13 buried positions. Again, only subtle changes in the calculated stabilities and structures were predicted. Calculations were experimentally validated for 19 variants at codon 271 ("N271X"). Results showed near wildtype expression and rheostatic modulation of substrate transport, implicating N271 as a rheostat position. Notably, each N271X substitution showed a similar effect on the transport of three different substrates and thus did not alter substrate specificity. This differs from S267X, which altered both transport kinetics and specificity. As both transport and specificity may change during protein evolution, the recognition of such rheostat positions may be important for evolutionary studies. We further propose that the presence of rheostat positions is facilitated by local plasticity within the protein structure. Finally, we note that identifying rheostat positions may advance efforts to predict new biomedically relevant missense variants in NTCP and other membrane transport proteins.

Spectroscopic evidence of tetanus toxin translocation domain bilayer-induced refolding and insertion.

Tetanus neurotoxin (TeNT) is an A-B toxin with three functional domains: endopeptidase, translocation (HCT), and receptor binding. Endosomal acidification triggers HCT to interact with and insert into the membrane, translocating the endopeptidase across the bilayer. Although the function of HCT is well defined, the mechanism by which it accomplishes this task is unknown. To gain insight into the HCT membrane interaction on both local and global scales, we utilized an isolated, beltless HCT variant (bHCT), which retained the ability to release potassium ions from vesicles. To examine which bHCT residues interact with the membrane, we widely sampled the surface of bHCT using 47 single-cysteine variants labeled with the environmentally sensitive fluorophore NBD. At neutral pH, no interaction was observed for any variant. In contrast, all NBD-labeled positions reported environmental change in the presence of acidic pH and membranes containing anionic lipids. We then examined the conformation of inserted bHCT using circular dichroism and intrinsic fluorescence. Upon entering the membrane, bHCT retained predominantly α-helical secondary structure, whereas the tertiary structure exhibited substantial refolding. The use of lipid-attached quenchers revealed that at least one of the three tryptophan residues penetrated deep into the hydrocarbon core of the membrane, suggesting formation of a bHCT transmembrane conformation. The possible conformational topology was further explored with the hydropathy analysis webtool MPEx, which identified a large, potential α-helical transmembrane region. Altogether, the spectroscopic evidence supports a model in which, upon acidification, the majority of TeNT bHCT entered the membrane with a concurrent change in tertiary structure.

Identification of biochemically neutral positions in liver pyruvate kinase.

Understanding how each residue position contributes to protein function has been a long-standing goal in protein science. Substitution studies have historically focused on conserved protein positions. However, substitutions of nonconserved positions can also modify function. Indeed, we recently identified nonconserved positions that have large substitution effects in human liver pyruvate kinase (hLPYK), including altered allosteric coupling. To facilitate a comparison of which characteristics determine when a nonconserved position does vs does not contribute to function, the goal of the current work was to identify neutral positions in hLPYK. However, existing hLPYK data showed that three features commonly associated with neutral positions-high sequence entropy, high surface exposure, and alanine scanning-lacked the sensitivity needed to guide experimental studies. We used multiple evolutionary patterns identified in a sequence alignment of the PYK family to identify which positions were least patterned, reasoning that these were most likely to be neutral. Nine positions were tested with a total of 117 amino acid substitutions. Although exploring all potential functions is not feasible for any protein, five parameters associated with substrate/effector affinities and allosteric coupling were measured for hLPYK variants. For each position, the aggregate functional outcomes of all variants were used to quantify a "neutrality" score. Three positions showed perfect neutral scores for all five parameters. Furthermore, the nine positions showed larger neutral scores than 17 positions located near allosteric binding sites. Thus, our strategy successfully enriched the dataset for positions with neutral and modest substitutions.

Rheostat positions: A new classification of protein positions relevant to pharmacogenomics.

To achieve the full potential of pharmacogenomics, one must accurately predict the functional out comes that arise from amino acid substitutions in proteins. Classically, researchers have focused on understanding the consequences of individual substitutions. However, literature surveys have shown that most substitutions were created at evolutionarily conserved positions. Awareness of this bias leads to a shift in perspective, from considering the outcomes of individual substitutions to understanding the roles of individual protein positions. Conserved positions tend to act as "toggle" switches, with most substitutions abolishing function. However, nonconserved positions have been found equally capable of affecting protein function. Indeed, many nonconserved positions act like functional dimmer switches ("rheostat" positions): This is revealed when multiple substitutions are made at a single position. Each substitution has a different functional outcome; the set of substitutions spans arange of outcomes. Finally, some nonconserved positions appear neutral, capable of accommodating all amino acid types without modifying function. This manuscript reviews the currently-known properties of rheost at positions, with examples shown for pyruvate kinase, organic anion transporting polypeptide 1B1, the beta-lactamase inhibitory protein, and angiotensin-converting enzyme 2. Outcomes observed for rheostat positions have implications for the rational design of drug analogs and allosteric drugs. Furthermore, this new framework - comprising three types of protein positions - provides a new approach to interpreting disease and population-based databases of amino acid changes. In conclusion, although a full understanding of substitution out comes at rheostat positions poses a challenge, utilization of this new frame of reference will further advance the application of pharmacogenomics.

RheoScale: A tool to aggregate and quantify experimentally determined substitution outcomes for multiple variants at individual protein positions.

Human mutations often cause amino acid changes (variants) that can alter protein function or stability. Some variants fall at protein positions that experimentally exhibit "rheostatic" mutation outcomes (different amino acid substitutions lead to a range of functional outcomes). In ongoing studies of rheostat positions, we encountered the need to aggregate experimental results from multiple variants, to describe the overall roles of individual positions. Here, we present "RheoScale" which generates quantitative scores to discriminate rheostat positions from those with "toggle" (most substitutions abolish function) or "neutral" (most substitutions have wild-type function) outcomes. RheoScale scores facilitate correlations of experimental data (such as binding affinity or stability) with structural and bioinformatic analyses. The RheoScale calculator is encoded into a Microsoft Excel workbook and an R script. Example analyses are shown for three model protein systems, including one assessed via deep mutational scanning. The RheoScale calculator quickly and efficiently provided quantitative descriptions that were in good agreement with prior qualitative observations. As an example application, scores were compared to the example proteins' structures; strong rheostat positions tended to occur in dynamic locations. In the future, RheoScale scores can be easily integrated into computational studies to facilitate improved algorithms for predicting outcomes of human variants.

Using Evolution to Guide Protein Engineering: The Devil IS in the Details.

For decades, protein engineers have endeavored to reengineer existing proteins for novel applications. Overall, protein folds and gross functions can be readily transferred from one protein to another by transplanting large blocks of sequence (i.e., domain recombination). However, predictably fine-tuning function (e.g., by adjusting ligand affinity, specificity, catalysis, and/or allosteric regulation) remains a challenge. One approach has been to use the sequences of protein families to identify amino acid positions that change during the evolution of functional variation. The rationale is that these nonconserved positions could be mutated to predictably fine-tune function. Evolutionary approaches to protein design have had some success, but the engineered proteins seldom replicate the functional performances of natural proteins. This Biophysical Perspective reviews several complexities that have been revealed by evolutionary and experimental studies of protein function. These include 1) challenges in defining computational and biological thresholds that define important amino acids; 2) the co-occurrence of many different patterns of amino acid changes in evolutionary data; 3) difficulties in mapping the patterns of amino acid changes to discrete functional parameters; 4) the nonconventional mutational outcomes that occur for a particular group of functionally important, nonconserved positions; 5) epistasis (nonadditivity) among multiple mutations; and 6) the fact that a large fraction of a protein's amino acids contribute to its overall function. To overcome these challenges, new goals are identified for future studies.

Flexibility and Disorder in Gene Regulation: LacI/GalR and Hox Proteins.

To modulate transcription, a variety of input signals must be sensed by genetic regulatory proteins. In these proteins, flexibility and disorder are emerging as common themes. Prokaryotic regulators generally have short, flexible segments, whereas eukaryotic regulators have extended regions that lack predicted secondary structure (intrinsic disorder). Two examples illustrate the impact of flexibility and disorder on gene regulation: the prokaryotic LacI/GalR family, with detailed information from studies on LacI, and the eukaryotic family of Hox proteins, with specific insights from investigations of Ultrabithorax (Ubx). The widespread importance of structural disorder in gene regulatory proteins may derive from the need for flexibility in signal response and, particularly in eukaryotes, in protein partner selection.

Amino acid positions subject to multiple coevolutionary constraints can be robustly identified by their eigenvector network centrality scores.

As proteins evolve, amino acid positions key to protein structure or function are subject to mutational constraints. These positions can be detected by analyzing sequence families for amino acid conservation or for coevolution between pairs of positions. Coevolutionary scores are usually rank-ordered and thresholded to reveal the top pairwise scores, but they also can be treated as weighted networks. Here, we used network analyses to bypass a major complication of coevolution studies: For a given sequence alignment, alternative algorithms usually identify different, top pairwise scores. We reconciled results from five commonly-used, mathematically divergent algorithms (ELSC, McBASC, OMES, SCA, and ZNMI), using the LacI/GalR and 1,6-bisphosphate aldolase protein families as models. Calculations used unthresholded coevolution scores from which column-specific properties such as sequence entropy and random noise were subtracted; "central" positions were identified by calculating various network centrality scores. When compared among algorithms, network centrality methods, particularly eigenvector centrality, showed markedly better agreement than comparisons of the top pairwise scores. Positions with large centrality scores occurred at key structural locations and/or were functionally sensitive to mutations. Further, the top central positions often differed from those with top pairwise coevolution scores: instead of a few strong scores, central positions often had multiple, moderate scores. We conclude that eigenvector centrality calculations reveal a robust evolutionary pattern of constraints-detectable by divergent algorithms--that occur at key protein locations. Finally, we discuss the fact that multiple patterns coexist in evolutionary data that, together, give rise to emergent protein functions.

Novel insights from hybrid LacI/GalR proteins: family-wide functional attributes and biologically significant variation in transcription repression.

LacI/GalR transcription regulators have extensive, non-conserved interfaces between their regulatory domains and the 18 amino acids that serve as 'linkers' to their DNA-binding domains. These non-conserved interfaces might contribute to functional differences between paralogs. Previously, two chimeras created by domain recombination displayed novel functional properties. Here, we present a synthetic protein family, which was created by joining the LacI DNA-binding domain/linker to seven additional regulatory domains. Despite 'mismatched' interfaces, chimeras maintained allosteric response to their cognate effectors. Therefore, allostery in many LacI/GalR proteins does not require interfaces with precisely matched interactions. Nevertheless, the chimeric interfaces were not silent to mutagenesis, and preliminary comparisons suggest that the chimeras provide an ideal context for systematically exploring functional contributions of non-conserved positions. DNA looping experiments revealed higher order (dimer-dimer) oligomerization in several chimeras, which might be possible for the natural paralogs. Finally, the biological significance of repression differences was determined by measuring bacterial growth rates on lactose minimal media. Unexpectedly, moderate and strong repressors showed an apparent induction phase, even though inducers were not provided; therefore, an unknown mechanism might contribute to regulation of the lac operon. Nevertheless, altered growth correlated with altered repression, which indicates that observed functional modifications are significant.

Rheostatic contributions to protein stability can obscure a position's functional role.

Rheostat positions, which can be substituted with various amino acids to tune protein function across a range of outcomes, are a developing area for advancing personalized medicine and bioengineering. Current methods cannot accurately predict which proteins contain rheostat positions or their substitution outcomes. To compare the prevalence of rheostat positions in homologs, we previously investigated their occurrence in two pyruvate kinase (PYK) isozymes. Human liver PYK contained numerous rheostat positions that tuned the apparent affinity for the substrate phosphoenolpyruvate (Kapp-PEP) across a wide range. In contrast, no functional rheostat positions were identified in Zymomonas mobilis PYK (ZmPYK). Further, the set of ZmPYK substitutions included an unusually large number that lacked measurable activity. We hypothesized that the inactive substitution variants had reduced protein stability, precluding detection of Kapp-PEP tuning. Using modified buffers, robust enzymatic activity was obtained for 19 previously-inactive ZmPYK substitution variants at three positions. Surprisingly, both previously-inactive and previously-active substitution variants all had Kapp-PEP values close to wild-type. Thus, none of the three positions were functional rheostat positions, and, unlike human liver PYK, ZmPYK's Kapp-PEP remained poorly tunable by single substitutions. To directly assess effects on stability, we performed thermal denaturation experiments for all ZmPYK substitution variants. Many diminished stability, two enhanced stability, and the three positions showed different thermal sensitivity to substitution, with one position acting as a "stability rheostat." The differences between the two PYK homologs raises interesting questions about the underlying mechanism(s) that permit functional tuning by single substitutions in some proteins but not in others.

Simulated pressure changes in LacI suggest a link between hydration and functional conformational changes.

The functions of many proteins are associated with interconversions among conformational substates. However, these substates can be difficult to measure experimentally, and determining contributions from hydration changes can be especially difficult. Here, we assessed the use of pressure perturbations to sample the substates accessible to the Escherichia coli lactose repressor protein (LacI) in various liganded forms. In the presence of DNA, the regulatory domain of LacI adopts an Open conformation that, in the absence of DNA, changes to a Closed conformation. Increasing the simulation pressure prevented the transition from an Open to a Closed conformation, in a similar manner to the binding of DNA and anti-inducer, ONPF. The results suggest the hydration of specific residues play a significant role in determining the population of different LacI substates and that simulating pressure perturbation could be useful for assessing the role of hydration changes that accompany functionally-relevant amino acid substitutions.

The intrinsically disordered transcriptional activation domain of CIITA is functionally tuneable by single substitutions: An exception or a new paradigm?

During protein evolution, some amino acid substitutions modulate protein function ("tuneability"). In most proteins, the tuneable range is wide and can be sampled by a set of protein variants that each contains multiple amino acid substitutions. In other proteins, the full tuneable range can be accessed by a set of variants that each contains a single substitution. Indeed, in some globular proteins, the full tuneable range can be accessed by the set of site-saturating substitutions at an individual "rheostat" position. However, in proteins with intrinsically disordered regions (IDRs), most functional studies-which would also detect tuneability-used multiple substitutions or small deletions. In disordered transcriptional activation domains (ADs), studies with multiple substitutions led to the "acidic exposure" model, which does not anticipate the existence of rheostat positions. In the few studies that did assess effects of single substitutions on AD function, results were mixed: the ADs of two full-length transcription factors did not show tuneability, whereas a fragment of a third AD was tuneable by single substitutions. In this study, we tested tuneability in the AD of full-length human class II transactivator (CIITA). Sequence analyses and experiments showed that CIITA's AD is an IDR. Functional assays of singly-substituted AD variants showed that CIITA's function was highly tuneable, with outcomes not predicted by the acidic exposure model. Four tested positions showed rheostat behavior for transcriptional activation. Thus, tuneability of different IDRs can vary widely. Future studies are needed to illuminate the biophysical features that govern whether an IDR is tuneable by single substitutions.

Enzymatic reaction sequences as coupled multiple traces on a multidimensional landscape.

We propose that an enzyme-catalyzed reaction is best described by a large, three-dimensional potential energy surface, defined by the number of enzyme conformers in one dimension, the number of reaction steps as the second and Gibbs free energy as the third. Aside from accommodating experimental observations that do not fit current mechanistic paradigms, such a surface enables multiple intersecting reaction pathways, pathway funneling, ligand binding energy transduction and kinetic coupling between alternative reaction pathways. The landscape also confers flexibility, enabling an enzyme to seek out an optimal pathway for any reaction conditions that might occur. Thus, coupled pathways enable relatively minor differences in experimental conditions to result in abrupt phenomenological changes in the observed behavior of the reaction.

Identification of a covert evolutionary pathway between two protein folds.

Although homologous protein sequences are expected to adopt similar structures, some amino acid substitutions can interconvert α-helices and ß-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 overcome these barriers by applying multiple statistical methods to a family of ~600,000 bacterial response regulator proteins. We find that their homologous DNA-binding subunits assume divergent structures: helix-turn-helix versus α-helix + ß-sheet (winged helix). Phylogenetic analyses, ancestral sequence reconstruction, and AlphaFold2 models indicate 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 a methodology to identify secondary structure switching in other protein families.