Epistasis, where the effect of a mutation depends on other mutations, has been observed in virtually every quantitative biochemical feature examined. Despite its ubiquity, epistasis is often difficult to explain mechanistically. Its interpretation is thus often limited to phenomenological descriptions of synergy or antagonism. Here we argue that epistasis quantifies the mismatch between experimental observations and a model for how mutations should sum together. Put differently: it is a fit residual. And like any fit residual, the presence of epistasis is a call to action, not an interesting observation in itself. It signals that the model is inadequate, not (necessarily) that the mutations physically interact. We propose that biochemists and biophysicists should pivot away from analyzing epistasis and into fitting mechanistic models that connect genotype to phenotype. Such models describe the effects of mutations as perturbations to underlying biochemical parameters—stability, binding affinity, catalytic rate—and combine those perturbations on mechanistically appropriate scales. Compared to a standard epistasis analysis, this approach reduces the number of free parameters, improves extrapolative power, and yields interpretable insights into structure-function relationships. We illustrate how this framework works in practice with studies covering a broad range of molecular systems and biochemically relevant scales. By reframing epistasis as a starting point rather than as a final observation, biochemists are uniquely positioned to build predictive models of mutational effects grounded in the physical principles governing biomolecular function.

Previously, we showed Enterobacteriaceae use chemotaxis and the chemoreceptor Tsr for attraction to blood serum (Glenn et al., 2024). Here, we investigated the complementary role of Tsr in mediating chemorepulsion, a behaviour by which motile bacteria avoid deleterious compounds to locate permissive niches. In the gut, indole is a bacteriostatic compound produced by microbiota and thought to act as a chemorepellent for invading pathogens, thereby protecting the host against infection. The principal reservoir of intestinal indole is fecal matter, a complex biological material that contains both attractant and repellent stimuli. Whether indole in its natural context is sufficient for pathogen chemorepulsion or host protection has remained unknown. Using an intestinal explant system, we show that pure indole suppresses an infection advantage mediated through chemotaxis for the enteric pathogen Salmonella Typhimurium, but this effect is abolished in the presence of other fecal chemoeffectors, including the chemoattractant L-Serine (L-Ser), dependent on the chemoreceptor Tsr. Live imaging reveals that although S. Typhimurium is repelled by pure indole, the pathogen is actually strongly attracted to human fecal matter despite its high indole content, and this response is mediated by Tsr, which simultaneously senses both stimuli. Fecal attraction is conserved across diverse Enterobacteriaceae species that harbor Tsr orthologues, including Escherichia coli, Citrobacter koseri, Enterobacter cloacae, and clinical isolates of non-typhoidal Salmonella. In a defined system of fecal chemoeffectors, L-Ser and other fecal chemoattractants override indole chemorepulsion, but the magnitude of bacterial chemoattraction is controlled by indole levels. Together, these findings suggest chemorepulsion elicited by indole is not protective against enteric infection and actually benefits pathogens by helping them locate niches with lower competitor density. Our study highlights the limitations of applying single-effector studies in predicting bacterial behavior in natural environments, where chemotaxis is shaped by the integration of multiple, often opposing, chemical signals.

Abstract Chemotaxis controls motility and colonization in many enteric pathogens, yet most studies have examined bacterial responses to single effectors in isolation. Previously, we reported that Salmonella Typhimurium uses the chemoreceptor Tsr to detect L-serine (L-Ser) in human blood serum, promoting invasion of damaged vasculature (Glenn et al., eLife 2024 1). Tsr also mediates sensing of indole, a microbiota-derived chemorepellent and bactericide proposed to protect against enteric infection by deterring pathogen colonization. The major biological reservoir of indole in the gut is feces, where it accumulates to millimolar levels. Here, we tested whether indole-rich human fecal material is protective against infection and found that exposure to feces instead enhances intestinal invasion in an explant model. Surprisingly, diverse non-typhoidal Salmonella serovars were strongly attracted to feces despite its high indole content. We found that while pure indole is a strong repellent sensed through Tsr, its effects are overridden in the presence of nutrient attractants, including L-Ser. Moreover, indole only minimally impairs growth in the presence of sufficient nutrients. Using video microscopy, we observed that Tsr integrates L-Ser and indole signals in real time, biasing bacterial movement based on the relative concentrations of attractant and repellent. We propose that this chemotactic compromise optimizes pathogen fitness by guiding bacteria to niches with a favorable L-Ser-to-indole ratio, balancing nutrient acquisition and avoidance of high microbial competitor density. These findings highlight the limitations of single-effector studies in predicting bacterial navigation in complex environments, where chemotaxis is shaped by the integration of multiple, often opposing, chemical cues.

Motile bacteria sense and avoid deleterious stimuli in their environment through chemorepulsion, a behavior that helps them locate permissive ecological niches. In the gut, indole is a bacteriostatic compound produced by the microbiota and is thought to act as a chemorepellent for invading pathogens, thereby protecting the host against infection. The principal reservoir of intestinal indole is fecal matter, a complex biological material that contains both attractant and repellent stimuli. Whether indole in its natural context is sufficient for pathogen chemorepulsion or host protection has remained unknown. Using an intestinal explant system, we show that while pure indole indeed suppresses an infection advantage mediated through chemotaxis for the enteric pathogen Salmonella enterica serovar Typhimurium, this effect is abolished in the presence of other chemoeffectors present in feces, including the chemoattractant L-Serine (L-Ser), in a manner dependent on the chemoreceptor Tsr. Live imaging reveals that although S. Typhimurium is repelled by pure indole, the pathogen is actually strongly attracted to human fecal matter despite its high indole content, and that this response is mediated by Tsr, which simultaneously senses both indole and L-Ser. Fecal attraction is conserved across diverse Enterobacteriaceae species that harbor Tsr orthologues, including Escherichia coli, Citrobacter koseri, Enterobacter cloacae, and clinical isolates of non-typhoidal Salmonella. In a defined system of fecal chemoeffectors, we find that L-Ser and other fecal chemoattractants override indole chemorepulsion, but the magnitude of bacterial chemoattraction is controlled by indole levels. Together, these findings suggest that indole in its native context is not protective against enteric infection and that indole taxis actually benefits pathogens during infection by locating niches with low competitor density. Our study highlights the limitations of applying single-effector studies in predicting bacterial behavior in natural environments, where chemotaxis is shaped by the integration of multiple, often opposing, chemical signals.

Previously, we showed Enterobacteriaceae use chemotaxis and the chemoreceptor Tsr for attraction to blood serum (Glenn et al., 2024). Here, we investigated the complementary role of Tsr in mediating chemorepulsion, a behaviour by which motile bacteria avoid deleterious compounds to locate permissive niches. In the gut, indole is a bacteriostatic compound produced by microbiota and thought to act as a chemorepellent for invading pathogens, thereby protecting the host against infection. The principal reservoir of intestinal indole is fecal matter, a complex biological material that contains both attractant and repellent stimuli. Whether indole in its natural context is sufficient for pathogen chemorepulsion or host protection has remained unknown. Using an intestinal explant system, we show that pure indole suppresses an infection advantage mediated through chemotaxis for the enteric pathogen Salmonella Typhimurium, but this effect is abolished in the presence of other fecal chemoeffectors, including the chemoattractant L-Serine (L-Ser), dependent on the chemoreceptor Tsr. Live imaging reveals that although S. Typhimurium is repelled by pure indole, the pathogen is actually strongly attracted to human fecal matter despite its high indole content, and this response is mediated by Tsr, which simultaneously senses both stimuli. Fecal attraction is conserved across diverse Enterobacteriaceae species that harbor Tsr orthologues, including Escherichia coli, Citrobacter koseri, Enterobacter cloacae, and clinical isolates of non-typhoidal Salmonella. In a defined system of fecal chemoeffectors, L-Ser and other fecal chemoattractants override indole chemorepulsion, but the magnitude of bacterial chemoattraction is controlled by indole levels. Together, these findings suggest chemorepulsion elicited by indole is not protective against enteric infection and actually benefits pathogens by helping them locate niches with lower competitor density. Our study highlights the limitations of applying single-effector studies in predicting bacterial behavior in natural environments, where chemotaxis is shaped by the integration of multiple, often opposing, chemical signals.

The protein heterodimer calprotectin and its component proteins, S100A8 and S100A9, play important antibacterial and pro-inflammatory roles in the mammalian innate immune response. Gaining mechanistic insights into the regulation and biological function of calprotectin will help facilitate patient diagnostics and therapy for inflammation and further our understanding of the host-microbe interface. Recent literature has identified zebrafish s100a10b as zebrafish calprotectin based on sequence similarity, genomic context, and transcriptional upregulation during the immune response to bacterial infections. The field would benefit from expanding the breadth of calprotectin studies into a zebrafish innate immunity model. Here, we carefully evaluated the possibility that zebrafish possess a calprotectin ortholog or a paralog that convergently evolved similar function. Using careful bioinformatics approaches, we found that zebrafish do not have an ortholog of either mammalian S100A8 or S100A9. To look for paralogs with convergent function, we identified four zebrafish s100 proteins-including s100a10b-that are expressed in immune cells and upregulated during the immune response. We recombinantly expressed and purified these proteins and measured their antimicrobial activity. None of the zebrafish proteins exhibited activity comparable to mammalian calprotectin. We also generated structural models of all homodimers and heterodimers of all annotated zebrafish s100 genes. None of these complexes were predicted to have an antimicrobial transition metal binding site equivalent to calprotectin. Finally, we measured the ability of our four purified zebrafish s100 proteins to activate inflammation via Toll-like receptor 4, a key feature of human S100A9; none of the proteins activated the receptor. Our work demonstrates conclusively that zebrafish have no ortholog of calprotectin and suggests that similar proteins have not convergently evolved analogous functions.

Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) play coordinated roles in angiogenesis. However, current biomaterial delivery vehicles for these proteins have a limited ability to precisely control the kinetics of protein release, preventing systematic exploration of their temporal effects. Here, we combined yeast surface display and computational protein design to engineer eight VEGF-specific and PDGF-specific protein binders called affibodies with a broad range of affinities for controlled protein release. Soluble affibodies modulated protein bioactivity as evidenced by changes in VEGF-induced endothelial cell proliferation and luminescent output of a PDGF-responsive cell line. Affibody-conjugated hydrogels enabled tunable protein release over 7 days. VEGF and PDGF released from affibody-conjugated hydrogels exhibited higher bioactivity than proteins released from hydrogels without affibodies, suggesting that these engineered affinity interactions could prolong protein bioactivity. This work underscores the power of computational protein design to enhance biomaterial functionality, creating a platform for tunable protein delivery.

S100A9 is a Damage Associated Molecular Pattern (DAMP) that activates inflammatory pathways via Toll-like receptor 4 (TLR4). This activity plays important homeostatic roles in tissue repair, but can also contribute to inflammatory diseases. The mechanism of activation is unknown. Here, we follow up on a previous observation that the protein CD14 is an important co-receptor that enables S100A9 to activate TLR4. Using cell-based functional assays and a combination of mutations and pharmocological perturbations, we found that CD14 must be membrane bound to potentiate TLR4 activation by S100A9. Additionally, S100A9 is sensitive to inhibitors of pathways downstream of TLR4 internalization. Together, this suggests that S100A9 induces activity via CD14-dependent internalization of TLR4. We then used mutagenesis, structural modeling, and in vitro binding experiments to establish that S100A9 binds to CD14's N-terminus in a region that overlaps with, but is not identical to, the region where CD14 binds its canonical ligand, lipopolysaccharide (LPS). In molecular dynamics simulations, this region of the protein is dynamic, allowing it to reorganize to recognize both S100A9 (a soluble protein) and LPS (a small hydrophobic molecule). Our work is the first attempt at a molecular characterization of the S100A9/CD14 interaction, bringing us one step closer to unraveling the full mechanism by which S100A9 activates TLR4/MD-2.

Cucurbit[n]urils (CB[n]s) are cyclic macrocycles with rich host-guest chemistry and can bind a variety of guests, often with high association constants. In many cases, guest binding in CB[n]s results in structural deformations in the host to accommodate guests of different sizes and shapes. Unfortunately, measuring such deformations has remained a major challenge, with only a handful of manual estimations reported in the literature. To address this challenge and to allow for the simple measurement and analysis of CB[n] deformations both in the presence and absence of guests, we have developed the public program ElliptiCB[n], which is available on GitHub, that provides a robust and automated method for measuring the elliptical deformations in CB[n] hosts. We outline the development and validation of this approach, apply ElliptiCB[n] to measure to the ellipticity of the 1113 available CB[n] structures from the Cambridge Structural Database (CSD), and directly investigate the structural deformations of CB[5], CB[6], CB[7], CB[8], and CB[10]. We also report the general landscape of accessible CB[n] elliptical deformations and compare ellipticity distributions across CB[n] hosts and host-guest complexes. We found that in almost all cases guest binding significant impacts the distribution of host ellipticity distributions and that ellipticity distributions are dissimilar across host-guest complexes of differently sized CB[n]s. We anticipate that this work will provide not only be a useful approach for understanding of the flexibility of CB[n] hosts but will also enable future measurement and standardization of ellipticity measurements of CB[n] X-ray and/or computational data.

The protein heterodimer calprotectin and its component proteins, S100A8 and S100A9, play important antibacterial and proinflammatory roles in the mammalian innate immune response. Gaining mechanistic insights into the regulation and biological function of calprotectin will help facilitate patient diagnostics and therapy and further our understanding of the host-microbe interface. Recent literature has identified zebrafish s100a10b as zebrafish calprotectin based on sequence similarity, genomic context, and transcriptional upregulation during the immune response to bacterial infections. The field would benefit from expanding the breadth of calprotectin studies into a zebrafish innate immunity model. Here, we carefully evaluated the possibility that zebrafish possess a calprotectin. We found that zebrafish do not possess an ortholog of mammalian S100A8 or S100A9. We then identified four zebrafish s100 proteins- including s100a10b-that are expressed in immune cells and upregulated during the immune response. We recombinantly expressed and purified these proteins and measured the antimicrobial and proinflammatory characteristics. We found that none of the zebrafish proteins exhibited activity comparable to mammalian calprotectin. Our work demonstrates conclusively that zebrafish have no ortholog of calprotectin, and the most plausible candidate proteins have not convergently evolved similar functions.

Bacteria of the family Enterobacteriaceae are associated with gastrointestinal (GI) bleeding and bacteremia and are a leading cause of death, from sepsis, for individuals with inflammatory bowel diseases. The bacterial behaviors and mechanisms underlying why these bacteria are prone to bloodstream entry remain poorly understood. Herein, we report that clinical isolates of non-typhoidal Salmonella enterica serovars, Escherichia coli , and Citrobacter koseri are rapidly attracted toward sources of human serum. To simulate GI bleeding, we utilized an injection-based microfluidics device and found that femtoliter volumes of human serum are sufficient to induce bacterial attraction to the serum source. This response is orchestrated through chemotaxis and the chemoattractant L-serine, an amino acid abundant in serum that is recognized through direct binding by the chemoreceptor Tsr. We report the first crystal structures of Salmonella Typhimurium Tsr in complex with L-serine and identify a conserved amino acid recognition motif for L-serine shared among Tsr orthologues. We find Tsr to be widely conserved among Enterobacteriaceae and numerous World Health Organization priority pathogens associated with bloodstream infections. Lastly, we find that Enterobacteriaceae use human serum as a source of nutrients for growth and that chemotaxis and the chemoreceptor Tsr provide a competitive advantage for migration into enterohemorrhagic lesions. We define this bacterial behavior of taxis toward serum, colonization of hemorrhagic lesions, and the consumption of serum nutrients as ‘bacterial vampirism’, which may relate to the proclivity of Enterobacteriaceae for bloodstream infections.

Evolutionarily conserved structural folds can give rise to diverse biological functions, yet predicting atomic-scale interactions that contribute to the emergence of novel activities within such folds remains challenging. Pancreatic-type ribonucleases illustrate this complexity, sharing a core structure that has evolved to accommodate varied functions. In this study, we used ancestral sequence reconstruction to probe evolutionary and molecular determinants that distinguish biological activities within eosinophil members of the RNase 2/ 3 subfamily. Our investigation unveils functional, structural, and dynamical behaviors that differentiate the evolved ancestral ribonuclease (AncRNase) from its contemporary eosinophil RNase orthologs. Leveraging the potential of ancestral reconstruction for protein engineering, we used AncRNase predictions to design a minimal 4-residue variant that transforms human RNase 2 into a chimeric enzyme endowed with the antimicrobial and cytotoxic activities of RNase 3 members. This work provides unique insights into mutational and evolutionary pathways governing structure, function, and conformational states within the eosinophil RNase subfamily, offering potential for targeted modulation of RNase-associated functions.

A protein's sequence determines its conformational energy landscape. This, in turn, determines the protein's function. Understanding the evolution of new protein functions therefore requires understanding how mutations alter the protein energy landscape. Ancestral sequence reconstruction (ASR) has proven a valuable tool for tackling this problem. In ASR, one phylogenetically infers the sequences of ancient proteins, allowing characterization of their properties. When coupled to biophysical, biochemical, and functional characterization, ASR can reveal how historical mutations altered the energy landscape of ancient proteins, allowing the evolution of enzyme activity, altered conformations, binding specificity, oligomerization, and many other protein features. In this article, we review how ASR studies have been used to dissect the evolution of energy landscapes. We also discuss ASR studies that reveal how energy landscapes have shaped protein evolution. Finally, we propose that thinking about evolution from the perspective of an energy landscape can improve how we approach and interpret ASR studies.

S100A9 is a Damage Associated Molecular Pattern (DAMP) that activates the innate immune system via Toll-like receptor 4 (TLR4). Despite many years of study, the mechanism of activation remains unknown. To date, much of the biochemical characterization of S100A9 has been performed using recombinant S100A9 expressed in E. coli (S100A9ec). TLR4 is the canonical receptor for LPS, a molecule found in the outer membrane of E. coli, raising the possibility of artifacts due to LPS contamination. Here we report characterization of LPS-free recombinant S100A9 expressed in insect cells (S100A9in). We show that S100A9in does not activate TLR4. This difference does not appear to be due to LPS contamination, protein misfolding, purification artifacts, or differences in phosphorylation. We show instead that S100A9in adopts an altered oligomeric state compared to S100A9ec. Disrupting oligomer formation with the E. coli disaggregase SlyD restores activity to S100A9in. Our results also indicate that the oligomeric state of S100A9 is a major factor in its ability to activate TLR4 and that this can be altered in unexpected ways by the recombinant expression system used to produce the protein.

A protein sequence encodes its energy landscape-all the accessible conformations, energetics, and dynamics. The evolutionary relationship between sequence and landscape can be probed phylogenetically by compiling a multiple sequence alignment of homologous sequences and generating common ancestors via Ancestral Sequence Reconstruction or a consensus protein containing the most common amino acid at each position. Both ancestral and consensus proteins are often more stable than their extant homologs-questioning the differences between them and suggesting that both approaches serve as general methods to engineer thermostability. We used the Ribonuclease H family to compare these approaches and evaluate how the evolutionary relationship of the input sequences affects the properties of the resulting consensus protein. While the consensus protein derived from our full Ribonuclease H sequence alignment is structured and active, it neither shows properties of a well-folded protein nor has enhanced stability. In contrast, the consensus protein derived from a phylogenetically-restricted set of sequences is significantly more stable and cooperatively folded, suggesting that cooperativity may be encoded by different mechanisms in separate clades and lost when too many diverse clades are combined to generate a consensus protein. To explore this, we compared pairwise covariance scores using a Potts formalism as well as higher-order sequence correlations using singular value decomposition (SVD). We find the SVD coordinates of a stable consensus sequence are close to coordinates of the analogous ancestor sequence and its descendants, whereas the unstable consensus sequences are outliers in SVD space.

The organization of chromatin - including the positions of nucleosomes and the binding of other proteins to DNA - helps define transcriptional profiles in eukaryotic organisms. While techniques like ChIP-Seq and MNase-Seq can map protein-DNA and nucleosome localization separately, assays designed to simultaneously capture nucleosome positions and protein-DNA interactions can produce a detailed picture of the chromatin landscape. Most assays that monitor chromatin organization and protein binding rely on antibodies, which often exhibit nonspecific binding, and/or the addition of bulky adducts to the DNA-binding protein being studied, which can affect their expression and activity. Here, we describe SpyCatcher Linked Targeting of Chromatin Endogenous Cleavage (SpLiT-ChEC), where a 13-amino acid SpyTag peptide, appended to a protein of interest, serves as a highly-specific targeting moiety for in situ enzymatic digestion. The SpyTag/SpyCatcher system forms a covalent bond, linking the target protein and a co-expressed MNase-SpyCatcher fusion construct. SpyTagged proteins are expressed from endogenous loci, whereas MNase-SpyCatcher expression is induced immediately before harvesting cultures. MNase is activated with high concentrations of calcium, which primarily digests DNA near target protein binding sites. By sequencing the DNA fragments released by targeted MNase digestion, we found that this method recovers information on protein binding and proximal nucleosome positioning. SpLiT-ChEC provides precise temporal control that we anticipate can be used to monitor chromatin under various conditions and at distinct points in the cell cycle.

Ancestral sequence reconstruction (ASR) is a powerful tool to study the evolution of proteins and thus gain deep insight into the relationships among protein sequence, structure, and function. A major barrier to its broad use is the complexity of the task: it requires multiple software packages, complex file manipulations, and expert phylogenetic knowledge. Here we introduce topiary, a software pipeline that aims to overcome this barrier. To use topiary, users prepare a spreadsheet with a handful of sequences. Topiary then: (1) Infers the taxonomic scope for the ASR study and finds relevant sequences by BLAST; (2) Does taxonomically informed sequence quality control and redundancy reduction; (3) Constructs a multiple sequence alignment; (4) Generates a maximum-likelihood gene tree; (5) Reconciles the gene tree to the species tree; (6) Reconstructs ancestral amino acid sequences; and (7) Determines branch supports. The pipeline returns annotated evolutionary trees, spreadsheets with sequences, and graphical summaries of ancestor quality. This is achieved by integrating modern phylogenetics software (Muscle5, RAxML-NG, GeneRax, and PastML) with online databases (NCBI and the Open Tree of Life). In this paper, we introduce non-expert readers to the steps required for ASR, describe the specific design choices made in topiary, provide a detailed protocol for users, and then validate the pipeline using datasets from a broad collection of protein families. Topiary is freely available for download: https://github.com/harmslab/topiary.

Mutations introduced into macromolecules often exhibit epistasis, where the effect of one mutation alters the effect of another. Knowing the mechanisms that lead to epistasis is important for understanding how macromolecules work and evolve, as well as for effective macromolecular engineering. Here, we investigate the interplay between “contact epistasis” (epistasis arising from physical interactions between mutated residues) and “ensemble epistasis” (epistasis that occurs when a mutation redistributes the conformational ensemble of a macromolecule, thus changing the effect of the second mutation). We argue that the two mechanisms can be distinguished in allosteric macromolecules by measuring epistasis at differing allosteric effector concentrations. Contact epistasis manifests as nonadditivity in the microscopic equilibrium constants describing the conformational ensemble. This epistatic effect is independent of allosteric effector concentration. Ensemble epistasis manifests as nonadditivity in thermodynamic observables—such as ligand binding—that are determined by the distribution of ensemble conformations. This epistatic effect strongly depends on allosteric effector concentration. Using this framework, we experimentally investigated the origins of epistasis in three pairwise mutant cycles introduced into the adenine riboswitch aptamer domain by measuring ligand binding as a function of allosteric effector concentration. We found evidence for both contact and ensemble epistasis in all cycles. Furthermore, we found that the two mechanisms of epistasis could interact with each other. For example, in one mutant cycle we observed 6 kcal/mol of contact epistasis in a microscopic equilibrium constant. In that same cycle, the maximum epistasis in ligand binding was only 1.5 kcal/mol: shifts in the ensemble masked the contribution of contact epistasis. Finally, our work yields simple heuristics for identifying contact and ensemble epistasis based on measurements of a biochemical observable as a function of allosteric effector concentration.