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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.

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.

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.

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.

During protein evolution, some amino acid substitutions modulate protein function (tuneability). In most proteins, the accessible tuneable range is wide and can be sampled by a set of protein variants that each contain multiple amino acid substitutions. In other proteins, the full range can be accessed by a set of variants that each contains a single substitution. Indeed, site-saturating substitutions at individual rheostat positions can sample the full range for some folded globular proteins. In proteins with intrinsically disordered regions (IDRs), many functional studies, which would also detect tuneability, used multiple substitutions or deleted small regions. These results have led to proposed mechanisms such as the acidic exposure model for transcriptional activation domains (AD). Only a few IDRs have been assessed with single substitutions. Results have been mixed: (i) The disordered ADs of two full-length transcription factors did not show tuneability, yet (ii) a fragment of another AD was tuneable by single substitutions. Here, we tested tuneability in the AD of a full-length, non-DNA-binding transcription factor, human class II transactivator (CIITA). Sequence analyses and experiments showed that AD of CIITA is an IDR. Functional assays of singly-substituted variants showed that this IDR was highly tuneable, and some outcomes were not predicted by the acidic exposure model. Instead, four tested positions showed rheostat behaviour, with a wide range of effects on transcriptional activation. Thus, tuneability of different IDRs can vary widely; future studies are needed to illuminate the biophysical features that govern whether an IDR can be tuned by single substitutions.Competing Interest StatementThe authors have declared no competing interest.Footnotes* All sections in the main text have been revised to clarify concepts and improve readability; Supplementary files updated.