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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 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 + β-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. Protein secondary structures–α-helices and β-sheets–are generally assumed to be fixed over evolutionary history. By leveraging sequence information and sensitive statistical techniques, this work proposes that secondary structures in naturally occurring DNA-binding proteins switched in response to stepwise mutation.

Protein families might evolve paralogous functions on their common tertiary scaffold in two ways. First, the locations of functionally-important sites might be \"hard-wired\" into the structure, with novel functions evolved by altering the amino acid (e.g. Ala vs Ser) at these positions. Alternatively, the tertiary scaffold might be adaptable, accommodating a unique set of functionally important sites for each paralogous function. To discriminate between these possibilities, we compared the set of functionally important sites in the six largest paralogous subfamilies of the LacI/GalR transcription repressor family. LacI/GalR paralogs share a common tertiary structure, but have low sequence identity (≤ 30%), and regulate a variety of metabolic processes. Functionally important positions were identified by conservation and co-evolutionary sequence analyses. Results showed that conserved positions use a mixture of the \"hard-wired\" and \"accommodating\" scaffold frameworks, but that the co-evolution networks were highly dissimilar between any pair of subfamilies. Therefore, the tertiary structure can accommodate multiple networks of functionally important positions. This possibility should be included when designing and interpreting sequence analyses of other protein families. Software implementing conservation and co-evolution analyses is available at https://sourceforge.net/projects/coevolutils/.

The millions of protein sequences generated by genomics are expected to transform protein engineering and personalized medicine. To achieve these goals, tools for predicting outcomes of amino acid changes must be improved. Currently, advances are hampered by insufficient experimental data about nonconserved amino acid positions. Since the property \"nonconserved\" is identified using a sequence alignment, we designed experiments to recapitulate that context: Mutagenesis and functional characterization was carried out in 15 LacI/GalR homologs (rows) at 12 nonconserved positions (columns). Multiple substitutions were made at each position, to reveal how various amino acids of a nonconserved column were tolerated in each protein row. Results showed that amino acid preferences of nonconserved positions were highly context-dependent, had few correlations with physico-chemical similarities, and were not predictable from their occurrence in natural LacI/GalR sequences. Further, unlike the \"toggle switch\" behaviors of conserved positions, substitutions at nonconserved positions could be rank-ordered to show a \"rheostatic\", progressive effect on function that spanned several orders of magnitude. Comparisons to various sequence analyses suggested that conserved and strongly co-evolving positions act as functional toggles, whereas other important, nonconserved positions serve as rheostats for modifying protein function. Both the presence of rheostat positions and the sequence analysis strategy appear to be generalizable to other protein families and should be considered when engineering protein modifications or predicting the impact of protein polymorphisms.

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.

For protein mutagenesis, a common expectation is that important positions will behave like on/off “toggle” switches ( i.e ., a few substitutions act like wildtype, most abolish function). However, there exists another class of important positions that manifests a wide range of functional outcomes upon substitution: “rheostat” positions. Previously, we evaluated rheostat positions located near the allosteric binding sites for inhibitor alanine (Ala) and activator fructose-1,6-bisphosphate (Fru-1,6-BP) in human liver pyruvate kinase. When substituted with multiple amino acids, many positions demonstrated moderate rheostatic effects on allosteric coupling between effector binding and phosphoenolpyruvate (PEP) binding in the active site. Nonetheless, the combined outcomes of all positions sampled the full range of possible allosteric coupling (full tunability). However, that study only evaluated allosteric tunability of “local” positions, i.e ., positions were located near the binding sites of the allosteric ligand being assessed. Here, we evaluated tunability of allosteric coupling when mutated sites were distant from the allosterically-coupled binding sites. Positions near the Ala binding site had rheostatic outcomes on allosteric coupling between Fru-1,6-BP and PEP binding. In contrast, positions in the Fru-1,6-BP site exhibited modest effects on coupling between Ala and PEP binding. Analyzed in aggregate, both PEP/Ala and PEP/Fru-1,6-BP coupling were again fully tunable by amino acid substitutions at this limited set of distant positions. Furthermore, some positions exhibited rheostatic control over multiple parameters and others exhibited rheostatic effects on one parameter and toggle control over a second. These findings highlight challenges in efforts to both predict/interpret mutational outcomes and engineer functions into proteins.

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.

Successfully predicting the effects of amino acid substitutions on protein function and stability remains challenging. Recent efforts to improve computational models have included training and validation on high-throughput experimental datasets, such as those generated by deep mutational scanning (DMS) approaches. However, DMS signals typically conflate a substitution’s effects on protein function with those on in vivo protein abundance; this limits the resolution of mechanistic insights that can be gleaned from DMS data. Distinguishing functional changes from abundance-related effects is particularly important for substitutions that exhibit intermediate outcomes (e.g., partial loss-of-function), which are difficult to predict. Here, we explored changes in in vivo abundance for substitutions at representative positions in the SARS-CoV-2 Main Protease (Mpro). For this study, we used previously published DMS results to identify “rheostat” positions, which are defined by having substitutions that sample a broad range of intermediate outcomes. We generated 10 substitutions at each of six positions and separately measured effects on function and abundance. Results revealed an ∼45-fold range of change for abundance, demonstrating that it can make significant contributions to DMS outcomes. Moreover, the six tested positions showed diverse substitution sensitivities for function and abundance. Some positions influenced only one parameter. Others exhibited rheostatic effects on both parameters, which to our knowledge, provides the first example of such behavior. Since effects on function and abundance may arise through different biophysical bases, these results underscore the need for datasets that independently measure these parameters in order to build predictors with enhanced mechanistic insights. Changing one amino acid in a protein can affect its function, its abundance, or both. Understanding these separate effects will help scientists predict how protein changes alter biology, which is important for understanding pathogen evolution, improving personalized medicine, and bioengineering. This work reports a study to experimentally separate these effects for the main protease of SARS-CoV-2 and suggests strategies for building better prediction models for emerging variants of this key viral protein.

High resolution structures of the Tet and Lac repressors bound to their respective operator DNA sites provide endpoints for the functional allosteric pathways in these distinct regulatory systems.

In , 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 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 Δ strain and complementation show that FruK has a broader role in metabolism than fructose catabolism. The Δ strain also alters biofilm formation. Since itself is repressed by Cra, these newly-reported events add layers to the dynamic regulation of 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.

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.Competing Interest StatementThe authors have declared no competing interest.Footnotes* https://github.com/DevlinaC/FixJ_KdpE