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Alkaloids are important bioactive molecules throughout the natural world, and in many animals they serve as a source of chemical defense against predation. Dendrobatid poison frogs bioaccumulate alkaloids from their diet to make themselves toxic or unpalatable to predators. Despite the proposed roles of plasma proteins as mediators of alkaloid trafficking and bioavailability, the responsible proteins have not been identified. We use chemical approaches to show that a ~50 kDa plasma protein is the principal alkaloid binding molecule in blood from poison frogs. Proteomic and biochemical studies establish this plasma protein to be liver-derived alkaloid-binding globulin (ABG) that is a member of the serine-protease inhibitor (serpin) family. In addition to alkaloid binding activity, ABG sequesters and regulates the bioavailability of “free” plasma alkaloidsin vitro. Unexpectedly, ABG is not related to saxiphilin, albumin, or other known vitamin carriers, but instead exhibits sequence and structural homology to mammalian hormone carriers and amphibian biliverdin binding proteins. Alkaloid-binding globulin (ABG) represents a new small molecule binding functionality in serpin proteins, a novel mechanism of plasma alkaloid transport in poison frogs, and more broadly points towards serpins acting as tunable scaffolds for small molecule binding and transport across different organisms.

American bullfrog ( Rana castesbeiana ) saxiphilin ( Rc Sxph) is a high-affinity “toxin sponge” protein thought to prevent intoxication by saxitoxin (STX), a lethal bis-guanidinium neurotoxin that causes paralytic shellfish poisoning (PSP) by blocking voltage-gated sodium channels (Na V s). How specific Rc Sxph interactions contribute to STX binding has not been defined and whether other organisms have similar proteins is unclear. Here, we use mutagenesis, ligand binding, and structural studies to define the energetic basis of Sxph:STX recognition. The resultant STX “recognition code” enabled engineering of Rc Sxph to improve its ability to rescue Na V s from STX and facilitated discovery of 10 new frog and toad Sxphs. Definition of the STX binding code and Sxph family expansion among diverse anurans separated by ∼140 My of evolution provides a molecular basis for understanding the roles of toxin sponge proteins in toxin resistance and for developing novel proteins to sense or neutralize STX and related PSP toxins.

The ability to use small molecule alkaloids as defensive chemicals has evolved in many organisms, often via trophic interactions due to dietary specialization. Animals with diet-derived defenses must balance food choices to maintain their defense reservoirs along with other physiological needs. However, environmental availability of prey and dietary preferences of vertebrate animals with acquired defenses remains largely unexplored. Here, we test the hypothesis that poison frogs that sequester alkaloids from their arthropod prey display prey preference. We collected alkaloid-defended Diablito frogs (Oophaga sylvatica) and leaf litter samples in five localities in northwestern Ecuador. Additionally, we collected the undefended Chimbo rocket frogs (Hyloxalus infraguttatus) from one locality in which it is living in sympatry with O. sylvatica. We found that both diets and skin alkaloids of O. sylvatica frogs were distinct among localities, and that undefended and sympatric H. infraguttatus ate fewer ants and mites. Unexpectedly, across O. sylvatica populations, we found a negative correlation between their summed skin alkaloid content and number of ants and mites they consumed. Finally, we compared leaf litter ants to frog stomach contents, and found that frogs eat a small subset of the available ant genera found in surrounding leaf litter, as only 13% of all the ants recovered from leaf litter samples belong to the 16 ant genera consumed by the frogs. Our results suggest that the consumption of diet-acquired defenses depends on preference-informed food choices in addition to environmental availability. This impacts our understanding of chemical defenses as a whole, as behavioral reinforcement may be an understudied, yet important selection factor in the evolution of acquired defenses.ResumenLa capacidad de usar moléculas pequeñas alcaloides como productos químicos defensivos ha evolucionado en muchos organismos, a menudo a través de interacciones tróficas debido a la especialización dietética. Los animales con defensas derivadas de la dieta deben equilibrar la selección de los alimentos para mantener sus reservorios de defensa junto con otras necesidades fisiológicas. Sin embargo, la disponibilidad ambiental de presas y las preferencias dietéticas de los animales vertebrados con defensas adquiridas son aspectos en gran parte desconocidos. Aquí, probamos la hipótesis de que las ranas venenosas que obtienen alcaloides de sus presas de artrópodos muestran preferencia por las presas. Recolectamos especímenes de ranas diablito (Oophaga sylvatica), con alcaloides defensivos, y muestras de hojarasca en cinco localidades del noroeste de Ecuador. Adicionalmente, recolectamos la rana cohete de Chimbo (Hyloxalus infraguttatus), sin alcaloides defensivos, de una localidad en la que vive en simpatría con O. sylvatica. Encontramos que tanto las dietas como los alcaloides de la piel de las ranas O. sylvatica eran distintas entre localidades, y que la simpátrica y no venenosa H. infraguttatus comía menos hormigas y ácaros. Inesperadamente, entre poblaciones de O. sylvatica, encontramos una correlación negativa entre la sumatoria del contenido de alcaloides cutáneos y el número de hormigas y ácaros que consumieron. Finalmente, comparamos las hormigas de hojarasca con el contenido del estómago de las ranas y descubrimos que las ranas comen un pequeño subconjunto de los géneros de hormigas disponibles que se encuentran en la hojarasca circundante, ya que solo el 13% de todas las hormigas obtenidas de las muestras de hojarasca pertenecen a los 16 géneros de hormigas consumidos por las ranas. Nuestros resultados sugieren que el consumo de defensas adquiridas a través de la dieta depende de la selección preferencial de alimentos, además de la disponibilidad ambiental. Esto influye en nuestra comprensión de las defensas químicas en su conjunto, ya que el refuerzo conductual puede ser un factor de selección poco estudiado, pero importante, en la evolución de las defensas adquiridas.

Poison frogs bioaccumulate alkaloids for chemical defense from their arthropod diet. Although many alkaloids are accumulated without modification, some poison frog species can metabolize pumiliotoxin (PTX 251D) into the more potent allopumiliotoxin (aPTX 267A). Despite extensive research characterizing the chemical arsenal of poison frogs, the physiological mechanisms involved in the sequestration and metabolism of individual alkaloids remain unclear. We first performed a feeding experiment with the Dyeing poison frog (Dendrobates tinctorius) to ask if this species can metabolize PTX 251D into aPTX 267A and what gene expression changes are associated with PTX 251D exposure in the intestines, liver, and skin. We found that D. tinctorius can metabolize PTX 251D into aPTX 267A, and that PTX 251D exposure changed the expression level of genes involved in immune system function and small molecule metabolism and transport. To better understand the functional significance of these changes in gene expression, we then conducted a series of high-throughput screens to determine the molecular targets of PTX 251D and identify potential proteins responsible for metabolism of PTX 251D into aPTX 267A. Although screens of PTX 251D binding human voltage-gated ion channels and G-protein coupled receptors were inconclusive, we identified human CYP2D6 as a rapid metabolizer of PTX 251D in a cytochrome P450 screen. Furthermore, a CYP2D6-like gene had increased expression in the intestines of animals fed PTX, suggesting this protein may be involved in PTX metabolism. These results show that individual alkaloids can modify gene expression across tissues, including genes involved in alkaloid metabolism. More broadly, this work suggests that specific alkaloid classes in wild diets may induce physiological changes for targeted accumulation and metabolism.

Despite RNA's diverse secondary and tertiary structures and its complex conformational changes, nature utilizes a limited set of structural "motifs"-helices, junctions, and tertiary contact modules-to build diverse functional RNAs. Thus, in-depth descriptions of a relatively small universe of RNA motifs may lead to predictive models of RNA tertiary conformational landscapes. Motifs may have different properties depending on sequence and secondary structure, giving rise to subclasses that expand the universe of RNA building blocks. Yet we know very little about motif subclasses, given the challenges in mapping conformational properties in high throughput. Previously, we used "RNA on a massively parallel array" (RNA-MaP), a quantitative, high-throughput technique, to study thousands of helices and two-way junctions. Here, we adapt RNA-MaP to study the thermodynamic and conformational properties of tetraloop/tetraloop receptor (TL/TLR) tertiary contact motifs, analyzing 1,493 TLR sequences from different classes. Clustering analyses revealed variability in TL specificity, stability, and conformational behavior. Nevertheless, natural GAAA/11ntR TL/TLRs, while varying in tertiary stability by ∼2.5 kcal/mol, exhibited conserved TL specificity and conformational properties. Thus, RNAs may tune stability without altering the overall structure of these TL/TLRs. Furthermore, their stability correlated with natural frequency, suggesting thermodynamics as the dominant selection pressure. In contrast, other TL/TLRs displayed heterogenous conformational behavior and appear to not be under strong thermodynamic selection. Our results build toward a generalizable model of RNA-folding thermodynamics based on the properties of isolated motifs, and our characterized TL/TLR library can be used to engineer RNAs with predictable thermodynamic and conformational behavior.

Gene duplication is a common and powerful mechanism by which cells create new signalling pathways1,2, but recently duplicated proteins typically must become insulated from each other and from other paralogues to prevent unwanted crosstalk3. A similar challenge arises when new sensors or synthetic signalling pathways are engineered within cells or transferred between genomes. How easily new pathways can be introduced into cells depends on the density and distribution of paralogous pathways in the sequence space that is defined by their specificity-determining residues4,5. Here we directly investigate how crowded this sequence space is, by generating novel two-component signalling proteins in Escherichia coli using cell sorting coupled to deep sequencing to analyse large libraries designed on the basis of coevolutionary patterns. We produce 58 insulated pathways comprising functional kinase–substrate pairs that have different specificities than their parent proteins, and demonstrate that several of these new pairs are orthogonal to all 27 paralogous pathways in E. coli. Additionally, from the kinase–substrate pairs generated, we identify sets consisting of six pairs that are mutually orthogonal to each other, which considerably increases the two-component signalling capacity of E. coli. These results indicate that sequence space is not densely occupied. The relative sparsity of paralogues in sequence space suggests that new insulated pathways can arise easily during evolution, or be designed de novo. We demonstrate the latter by engineering a signalling pathway in E. coli that responds to a plant cytokinin, without crosstalk to extant pathways. Our work also demonstrates how coevolution-guided mutagenesis and the mapping of sequence space can be used to design large sets of orthogonal protein–protein interactions. Engineered two-component signalling proteins in Escherichia coli have residue specificities different to their parent proteins and are orthogonal to all extant paralogues, demonstrating that sequence space is not densely occupied.