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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., Competing Interests: Conflict of interests During the course of this work, Dr Bose served on the Scientific Advisory Board and was a consultant for Azitra, Inc and Merck & Co, Inc. These activities did not financially support and are unrelated to the current manuscript. The other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2023. Published by Elsevier B.V.)

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., (© 2021 Wiley Periodicals LLC.)

When amino acids vary during evolution, the outcome can be functionally neutral or biologically-important. We previously found that substituting a subset of nonconserved positions, "rheostat" positions, can have surprising effects on protein function. Since changes at rheostat positions can facilitate functional evolution or cause disease, more examples are needed to understand their unique biophysical characteristics. Here, we explored whether "phylogenetic" patterns of change in multiple sequence alignments (such as positions with subfamily specific conservation) predict the locations of functional rheostat positions. To that end, we experimentally tested eight phylogenetic positions in human liver pyruvate kinase (hLPYK), using 10-15 substitutions per position and biochemical assays that yielded five functional parameters. Five positions were strongly rheostatic and three were non-neutral. To test the corollary that positions with low phylogenetic scores were not rheostat positions, we combined these phylogenetic positions with previously-identified hLPYK rheostat, "toggle" (most substitution abolished function), and "neutral" (all substitutions were like wild-type) positions. Despite representing 428 variants, this set of 33 positions was poorly statistically powered. Thus, we turned to the in vivo phenotypic dataset for E. coli lactose repressor protein (LacI), which comprised 12-13 substitutions at 329 positions and could be used to identify rheostat, toggle, and neutral positions. Combined hLPYK and LacI results show that positions with strong phylogenetic patterns of change are more likely to exhibit rheostat substitution outcomes than neutral or toggle outcomes. Furthermore, phylogenetic patterns were more successful at identifying rheostat positions than were co-evolutionary or eigenvector centrality measures of evolutionary change., (© 2021 The Protein Society.)

To bridge biological and biochemical disciplines, the relationship between in vitro protein biochemical function and in vivo activity must be established. Such studies can (a) help determine whether properties measured in simple, dilute solutions extrapolate to the complex in vivo conditions and (b) illuminate cryptic biological factors that are new avenues for study. We have explored the in vivo-in vitro relationship for chimeras built from LacI/GalR transcription regulators. In prior studies of individual chimeras, amino acid changes that altered in vitro DNA binding affinity exhibited correlated changes in in vivo transcription repression. However, discrepancies arose when the two datasets were compared to each other: Although their DNA binding domains were identical and their in vitro binding affinities spanned the same range, their in vivo repression ranges differed by >50-fold. Here, we determined that the presence of endogenous ligand for one chimera further exacerbated the offset, but that different abilities to simultaneously bind and "loop" two DNA operators resolves the discrepancy. Indeed, results suggest that the lac operon can be looped by even weakly interacting repressor dimers. For looping-competent repressors, we measured in vitro binding to the secondary operator. Surprisingly, this was largely insensitive to amino acid changes in the repressor protein, which reflects altered specificity; this supports the emerging view that the locations of specificity determining positions can be unique to each protein homolog. In aggregate, this work illustrates how a comparative approach can enrich understanding of the in vivo-in vitro relationship and suggest unexpected avenues for future study., (© 2019 The Protein Society.)

In prokaryotes, the construction of synthetic, multi-input promoters is constrained by the number of transcription factors that can simultaneously regulate a single promoter. This fundamental engineering constraint is an obstacle to synthetic biologists because it limits the computational capacity of engineered gene circuits. Here, we demonstrate that complex multi-input transcriptional logic gating can be achieved through the use of ligand-inducible chimeric transcription factors assembled from the LacI/GalR family. These modular chimeras each contain a ligand-binding domain and a DNA-binding domain, both of which are chosen from a library of possibilities. When two or more chimeras have the same DNA-binding domain, they independently and simultaneously regulate any promoter containing the appropriate operator site. In this manner, simple transcriptional AND gating is possible through the combination of two chimeras, and multiple-input AND gating is possible with the simultaneous use of three or even four chimeras. Furthermore, we demonstrate that orthogonal DNA-binding domains and their cognate operators allow the coexpression of multiple, orthogonal AND gates. Altogether, this work provides synthetic biologists with novel, ligand-inducible logic gates and greatly expands the possibilities for engineering complex synthetic gene circuits.

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/.

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.

One emphasis of the Gibbs Conference on Biothermodynamics is the value of thermodynamic measurements for understanding behaviors of biological systems. In this study, the correlation between thermodynamic measurements of in vitro DNA binding affinity with in vivo transcription repression was investigated for two transcription repressors. In the first system, which comprised an engineered LacI/GalR homolog, mutational changes altered the equilibrium constant for binding DNA. Changes correlated with altered repression, but estimates of in vivo repressor concentration suggest a ≥25-fold discrepancy with in vitro conditions. In the second system, changes in ligand binding to BirA altered dimerization and subsequent DNA occupancy. Again, these changes correlate with altered in vivo repression, but comparison with in vitro measurements reveals a ~10-fold discrepancy. Further analysis of each system suggests that the observed discrepancies between in vitro and in vivo results reflect the contributions of additional equilibria to the transcription repression process., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Concomitant with the genomic era, many bioinformatics programs have been developed to identify functionally important positions from sequence alignments of protein families. To evaluate these analyses, many have used the LacI/GalR family and determined whether positions predicted to be "important" are validated by published experiments. However, we previously noted that predictions do not identify all of the experimentally important positions present in the linker regions of these homologs. In an attempt to reconcile these differences, we corrected and expanded the LacI/GalR sequence set commonly used in sequence/function analyses. Next, a variety of analyses were carried out (1) for the entire LacI/GalR sequence set and (2) for a subset of homologs with functionally-important "YxPxxxAxxL" motifs in their linkers. This strategy was devised to determine whether predictions could be improved by knowledge-based sequence sorting and-for some analyses-did increase the number of linker positions identified. However, two functionally important linker positions were not reliably identified by any analysis. Finally, we compared the new predictions to all known experimental data for E. coli LacI and three homologous linkers. From these, we estimate that >50% of positions are important to the functions of the LacI/GalR homologs. In corollary, neutral positions might occur less frequently and might be easier to detect in sequence analyses. Although analyses have successfully guided mutations that partially exchange protein functions, a better experimental understanding of the sequence/function relationships in protein families would be helpful for uncovering the remaining rules used by nature to evolve new protein functions., (Copyright © 2011 Wiley-Liss, Inc.)

The lactose repressor protein (LacI) was among the very first genetic regulatory proteins discovered, and more than 1000 members of the bacterial LacI/GalR family are now identified. LacI has been the prototype for understanding how transcription is controlled using small metabolites to modulate protein association with specific DNA sites. This understanding has been greatly expanded by the study of other LacI/GalR homologues. A general picture emerges in which the conserved fold provides a scaffold for multiple types of interactions - including oligomerization, small molecule binding, and protein-protein binding - that in turn influence target DNA binding and thereby regulate mRNA production. Although many different functions have evolved from this basic scaffold, each homologue retains functional flexibility: For the same protein, different small molecules can have disparate impact on DNA binding and hence transcriptional outcome. In turn, binding to alternative DNA sequences may impact the degree of allosteric response. Thus, this family exhibits a symphony of variations by which transcriptional control is achieved.

In protein families, conserved residues often contribute to a common general function, such as DNA-binding. However, unique attributes for each homolog (e.g. recognition of alternative DNA sequences) must arise from variation in other functionally-important positions. The locations of these "specificity determinant" positions are obscured amongst the background of varied residues that do not make significant contributions to either structure or function. To isolate specificity determinants, a number of bioinformatics algorithms have been developed. When applied to the LacI/GalR family of transcription regulators, several specificity determinants are predicted in the 18 amino acids that link the DNA-binding and regulatory domains. However, results from alternative algorithms are only in partial agreement with each other. Here, we experimentally evaluate these predictions using an engineered repressor comprising the LacI DNA-binding domain, the LacI linker, and the GalR regulatory domain (LLhG). "Wild-type" LLhG has altered DNA specificity and weaker lacO(1) repression compared to LacI or a similar LacI:PurR chimera. Next, predictions of linker specificity determinants were tested, using amino acid substitution and in vivo repression assays to assess functional change. In LLhG, all predicted sites are specificity determinants, as well as three sites not predicted by any algorithm. Strategies are suggested for diminishing the number of false negative predictions. Finally, individual substitutions at LLhG specificity determinants exhibited a broad range of functional changes that are not predicted by bioinformatics algorithms. Results suggest that some variants have altered affinity for DNA, some have altered allosteric response, and some appear to have changed specificity for alternative DNA ligands.

Substitutions within the interdomain linkers of the AraC/XylS family proteins RhaS and RhaR were tested to determine whether side chain identity or linker structure was required for function. Neither was found crucial, suggesting that the linkers do not play a direct role in activation, but rather simply connect the two domains.

LacI and PurR are highly homologous proteins. Their functional units are homodimers, with an N-terminal DNA binding domain that comprises the helix-turn-helix (HTH), N-linker, and hinge regions from both monomers. Hinge structural changes are known to occur upon DNA dissociation but are difficult to monitor experimentally. The initial steps of hinge unfolding were therefore examined using molecular dynamics simulations, utilizing a truncated, chimeric protein comprising the LacI HTH/N-linker and PurR hinge. A terminal Gly-Cys-Gly was added to allow "dimerization" through disulfide bond formation. Simulations indicate that differences in LacI and PurR hinge primary sequence affect the quaternary structure of the hinge x hinge' interface. However, these alternate hinge orientations would be sterically restricted by the core domain. These results prompted detailed comparison of recently available DNA-bound structures for LacI and truncated LacI(1-62) with the PurR structure. Examination revealed that different N-linker and hinge contacts to the core domain of the partner monomer (which binds effector molecule) affect the juxtapositions of the HTH, N-linker, and hinge regions in the DNA binding domain. In addition, the two full-length repressors exhibit significant differences in the interactions between the core and the C-linker connection to the DNA binding domain. Both linkers and the hinge have been implicated in the allosteric response of these repressors. Intriguingly, one functional difference between these two proteins is that they exhibit opposite allosteric response to effector. Simulations and observed structural distinctions are correlated with mutational analysis and sequence information from the LacI/GalR family to formulate a mechanism for fine-tuning individual repressor function.