Targeting a helix-in-groove interaction between E1 and E2 blocks ubiquitin transfer.

The ubiquitin-proteasome system (UPS) is a highly regulated protein disposal process critical to cell survival. Inhibiting the pathway induces proteotoxic stress and can be an effective cancer treatment. The therapeutic window observed upon proteasomal blockade has motivated multiple UPS-targeting strategies, including preventing ubiquitination altogether. E1 initiates the cascade by transferring ubiquitin to E2 enzymes. A small molecule that engages the E1 ATP-binding site and derivatizes ubiquitin disrupts enzymatic activity and kills cancer cells. However, binding-site mutations cause resistance, motivating alternative approaches to block this promising target. We identified an interaction between the E2 N-terminal alpha-1 helix and a pocket within the E1 ubiquitin-fold domain as a potentially druggable site. Stapled peptides modeled after the E2 alpha-1 helix bound to the E1 groove, induced a consequential conformational change and inhibited E1 ubiquitin thiotransfer, disrupting E2 ubiquitin charging and ubiquitination of cellular proteins. Thus, we provide a blueprint for a distinct E1-targeting strategy to treat cancer.

Homogeneous Oligomers of Pro-apoptotic BAX Reveal Structural Determinants of Mitochondrial Membrane Permeabilization.

BAX is a pro-apoptotic protein that transforms from a cytosolic monomer into a toxic oligomer that permeabilizes the mitochondrial outer membrane. How BAX monomers assemble into a higher-order conformation, and the structural determinants essential to membrane permeabilization, remain a mechanistic mystery. A key hurdle has been the inability to generate a homogeneous BAX oligomer (BAXO) for analysis. Here, we report the production and characterization of a full-length BAXO that recapitulates physiologic BAX activation. Multidisciplinary studies revealed striking conformational consequences of oligomerization and insight into the macromolecular structure of oligomeric BAX. Importantly, BAXO enabled the assignment of specific roles to particular residues and α helices that mediate individual steps of the BAX activation pathway, including unexpected functionalities of BAX α6 and α9 in driving membrane disruption. Our results provide the first glimpse of a full-length and functional BAXO, revealing structural requirements for the elusive execution phase of mitochondrial apoptosis.

Identification of a Structural Determinant for Selective Targeting of HDMX.

p53 is a critical tumor-suppressor protein that guards the human genome against mutations by inducing cell-cycle arrest or apoptosis. Cancer cells subvert p53 by deletion, mutation, or overexpression of the negative regulators HDM2 and HDMX. For tumors that retain wild-type p53, its reactivation by pharmacologic targeting of HDM2 and/or HDMX represents a promising strategy, with a series of selective small-molecule HDM2 inhibitors and a dual HDM2/HDMX stapled-peptide inhibitor being evaluated in clinical trials. Because selective HDM2 targeting can cause hematologic toxicity, selective HDMX inhibitors could provide an alternative p53-reactivation strategy, but clinical candidates remain elusive. Here, we applied a mutation-scanning approach to uncover p53-based stapled peptides that are selective for HDMX. Crystal structures of stapled-peptide/HDMX complexes revealed a molecular mechanism for the observed specificity, which was validated by HDMX mutagenesis. Thus, we provide a blueprint for the development of HDMX-selective inhibitors to dissect and target the p53/HDMX interaction.

Identification of a Covalent Molecular Inhibitor of Anti-apoptotic BFL-1 by Disulfide Tethering.

The BCL-2 family is composed of anti- and pro-apoptotic members that respectively protect or disrupt mitochondrial integrity. Anti-apoptotic overexpression can promote oncogenesis by trapping the BCL-2 homology 3 (BH3) "killer domains" of pro-apoptotic proteins in a surface groove, blocking apoptosis. Groove inhibitors, such as the relatively large BCL-2 drug venetoclax (868 Da), have emerged as cancer therapies. BFL-1 remains an undrugged oncogenic protein and can cause venetoclax resistance. Having identified a unique C55 residue in the BFL-1 groove, we performed a disulfide tethering screen to determine if C55 reactivity could enable smaller molecules to block BFL-1's BH3-binding functionality. We found that a disulfide-bearing N-acetyltryptophan analog (304 Da adduct) effectively targeted BFL-1 C55 and reversed BFL-1-mediated suppression of mitochondrial apoptosis. Structural analyses implicated the conserved leucine-binding pocket of BFL-1 as the interaction site, resulting in conformational remodeling. Thus, therapeutic targeting of BFL-1 may be achievable through the design of small, cysteine-reactive drugs.

Interactions of a Bacterial RND Transporter with a Transmembrane Small Protein in a Lipid Environment.

The small protein AcrZ in Escherichia coli interacts with the transmembrane portion of the multidrug efflux pump AcrB and increases resistance of the bacterium to a subset of the antibiotic substrates of that transporter. It is not clear how the physical association of the two proteins selectively changes activity of the pump for defined substrates. Here, we report cryo-EM structures of AcrB and the AcrBZ complex in lipid environments, and comparisons suggest that conformational changes occur in the drug-binding pocket as a result of AcrZ binding. Simulations indicate that cardiolipin preferentially interacts with the AcrBZ complex, due to increased contact surface, and we observe that chloramphenicol sensitivity of bacteria lacking AcrZ is exacerbated when combined with cardiolipin deficiency. Taken together, the data suggest that AcrZ and lipid cooperate to allosterically modulate AcrB activity. This mode of regulation by a small protein and lipid may occur for other membrane proteins.

The why, when, and how of computing in biology classrooms.

Many biologists are interested in teaching computing skills or using computing in the classroom, despite not being formally trained in these skills themselves. Thus biologists may find themselves researching how to teach these skills, and therefore many individuals are individually attempting to discover resources and methods to do so. Recent years have seen an expansion of new technologies to assist in delivering course content interactively. Educational research provides insights into how learners absorb and process information during interactive learning. In this review, we discuss the value of teaching foundational computing skills to biologists, and strategies and tools to do so. Additionally, we review the literature on teaching practices to support the development of these skills. We pay special attention to meeting the needs of diverse learners, and consider how different ways of delivering course content can be leveraged to provide a more inclusive classroom experience. Our goal is to enable biologists to teach computational skills and use computing in the classroom successfully.

Phylogenetics of Kingsnakes, Lampropeltis getula Complex (Serpentes: Colubridae), in Eastern North America.

Kingsnakes of the Lampropeltis getula complex range throughout much of temperate and subtropical North America. Studies over the last century have used morphology and color pattern to describe numerous subspecies. More recently, DNA analyses have made invaluable contributions to our understanding of their evolution and taxonomy. We use genetic and ecological methods to test previous hypotheses of distinct evolutionary lineages by examining 66 total snakes and 1) analyzing phylogeographic structure using 2 mtDNA loci and 1 nuclear locus, 2) estimating divergence dates and historical demography among lineages in a Bayesian coalescent framework, and 3) applying ecological niche modeling (ENM). Our molecular data and ENMs illustrate that 3 previously recognized subspecies in the eastern United States comprise well-supported monophyletic lineages that diverged during the Pleistocene. The geographic boundaries of these 3 lineages correspond closely to known biogeographic barriers (Florida peninsula, Appalachian Mountains, and Apalachicola River) previously identified for other plants and animals, indicating shared geographic influences on evolutionary history. We conclude that genetic, ecological, and morphological data support recognition of these 3 lineages as distinct species (Lampropeltis floridana, Lampropeltis getula, and Lampropeltis meansi).

The dynamic evolutionary history of genome size in North American woodland salamanders.

The genus Plethodon is the most species-rich salamander genus in North America, and nearly half of its species face an uncertain future. It is also one of the most diverse families in terms of genome sizes, which range from 1C = 18.2 to 69.3 pg, or 5-20 times larger than the human genome. Large genome size in salamanders results in part from accumulation of transposable elements and is associated with various developmental and physiological traits. However, genome sizes have been reported for only 25% of the species of Plethodon (14 of 55). We collected genome size data for Plethodon serratus to supplement an ongoing phylogeographic study, reconstructed the evolutionary history of genome size in Plethodontidae, and inferred probable genome sizes for the 41 species missing empirical data. Results revealed multiple genome size changes in Plethodon: genomes of western Plethodon increased, whereas genomes of eastern Plethodon decreased, followed by additional decreases or subsequent increases. The estimated genome size of P. serratus was 21 pg. New understanding of variation in genome size evolution, along with genome size inferences for previously unstudied taxa, provide a foundation for future studies on the biology of plethodontid salamanders.

Sequence capture and next-generation sequencing of ultraconserved elements in a large-genome salamander.

Amidst the rapid advancement in next-generation sequencing (NGS) technology over the last few years, salamanders have been left behind. Salamanders have enormous genomes-up to 40 times the size of the human genome-and this poses challenges to generating NGS data sets of quality and quantity similar to those of other vertebrates. However, optimization of laboratory protocols is time-consuming and often cost prohibitive, and continued omission of salamanders from novel phylogeographic research is detrimental to species facing decline. Here, we use a salamander endemic to the southeastern United States, Plethodon serratus, to test the utility of an established protocol for sequence capture of ultraconserved elements (UCEs) in resolving intraspecific phylogeographic relationships and delimiting cryptic species. Without modifying the standard laboratory protocol, we generated a data set consisting of over 600 million reads for 85 P. serratus samples. Species delimitation analyses support recognition of seven species within P. serratus sensu lato, and all phylogenetic relationships among the seven species are fully resolved under a coalescent model. Results also corroborate previous data suggesting nonmonophyly of the Ouachita and Louisiana regions. Our results demonstrate that established UCE protocols can successfully be used in phylogeographic studies of salamander species, providing a powerful tool for future research on evolutionary history of amphibians and other organisms with large genomes.

Thriving in the Cold: Glacial Expansion and Post-Glacial Contraction of a Temperate Terrestrial Salamander (Plethodon serratus).

The dynamic geologic history of the southeastern United States has played a major role in shaping the geographic distributions of amphibians in the region. In the phylogeographic literature, the predominant pattern of distribution shifts through time of temperate species is one of contraction during glacial maxima and persistence in refugia. However, the diverse biology and ecology of amphibian species suggest that a "one-size-fits-all" model may be inappropriate. Nearly 10% of amphibian species in the region have a current distribution comprised of multiple disjunct, restricted areas that resemble the shape of Pleistocene refugia identified for other temperate taxa in the literature. Here, we apply genetics and spatially explicit climate analyses to test the hypothesis that the disjunct regions of these species ranges are climatic refugia for species that were more broadly distributed during glacial maxima. We use the salamander Plethodon serratus as a model, as its range consists of four disjunct regions in the Southeast. Phylogenetic results show that P. serratus is comprised of multiple genetic lineages, and the four regions are not reciprocally monophyletic. The Appalachian salamanders form a clade sister to all other P. serratus. Niche and paleodistribution modeling results suggest that P. serratus expanded from the Appalachians during the cooler Last Glacial Maximum and has since been restricted to its current disjunct distribution by a warming climate. These data reject the universal applicability of the glacial contraction model to temperate taxa and reiterate the importance of considering the natural history of individual species.

Cryptic diversity in metropolis: confirmation of a new leopard frog species (Anura: Ranidae) from New York City and surrounding Atlantic coast regions.

We describe a new cryptic species of leopard frog from the New York City metropolitan area and surrounding coastal regions. This species is morphologically similar to two largely parapatric eastern congeners, Rana sphenocephala and R. pipiens. We primarily use bioacoustic and molecular data to characterize the new species, but also examine other lines of evidence. This discovery is unexpected in one of the largest and most densely populated urban parts of the world. It also demonstrates that new vertebrate species can still be found periodically even in well-studied locales rarely associated with undocumented biodiversity. The new species typically occurs in expansive open-canopied wetlands interspersed with upland patches, but centuries of loss and impact to these habitats give some cause for conservation concern. Other concerns include regional extirpations, fragmented extant populations, and a restricted overall geographic distribution. We assign a type locality within New York City and report a narrow and largely coastal lowland distribution from central Connecticut to northern New Jersey (based on genetic data) and south to North Carolina (based on call data).

Model selection as a tool for phylogeographic inference: an example from the willow Salix melanopsis.

Phylogeographic inference has typically relied on analyses of data from one or a few genes to provide estimates of demography and population histories. While much has been learned from these studies, all phylogeographic analysis is conditioned on the data, and thus, inferences derived from data that represent a small sample of the genome are unavoidably tenuous. Here, we demonstrate one approach for moving beyond classic phylogeographic research. We use sequence capture probes and Illumina sequencing to generate data from >400 loci in order to infer the phylogeographic history of Salix melanopsis, a riparian willow with a disjunct distribution in coastal and the inland Pacific Northwest. We evaluate a priori phylogeographic hypotheses using coalescent models for parameter estimation, and the results support earlier findings that identified post-Pleistocene dispersal as the cause of the disjunction in S. melanopsis. We also conduct a series of model selection exercises using IMa2, Migrate-n and ∂a∂i. The resulting ranking of models indicates that refugial dynamics were complex, with multiple regions in the inland regions serving as the source for postglacial colonization. Our results demonstrate that new sources of data and new approaches to data analysis can rejuvenate phylogeographic research by allowing for the identification of complex models that enable researchers to both identify and estimate the most relevant parameters for a given system.

Misleading phylogenetic inferences based on single-exemplar sampling in the turtle genus Pseudemys.

Reconstructing species trees for clades containing weakly delimited or incorrectly identified taxa is one of the most serious challenges facing systematists because building phylogenetic trees is generally predicated on correctly identifying species membership for the terminals in an analysis. A common practice, particularly in large-scale phylogenetic analyses, is to use single-exemplar sampling under the implicit assumption that the resulting phylogenetic trees will be poorly supported if the sampled taxa are not good species. We examine this fundamental assumption in the North American turtle genus Pseudemys, a group of common, widely distributed freshwater turtles whose species boundaries and phylogenetic relationships have challenged systematists for over half a century. We sequenced 10 nuclear and three mitochondrial genes from the nine currently recognized species and subspecies of Pseudemys using geographically-widespread sampling of each taxon, and analyzed the resulting 86-individual data set using population-genetic and phylogenetic methods. We found little or no evidence supporting the division of Pseudemys into its currently recognized species/subspecies. Rather, our data strongly suggest that the group has been oversplit and contains fewer species than currently recognized. Even so, when we conducted 100 replicated, single-exemplar phylogenetic analyses of these same nine taxa, most Bayesian trees were well resolved, had high posterior probabilities, and yet returned completely conflicting topologies. These analyses suggest that phylogenetic analyses based on single-exemplar sampling may recover trees that depend on the individuals that are sampled, rather than the underlying species tree that systematists assume they are estimating. Our results clearly indicate that final resolution of Pseudemys will require an integrated analysis of morphology and historical biogeographic data coupled with extensive geographic sampling and large amounts of molecular data, and we do not recommend taxonomic changes based on our analyses. If our 100-tree resampling experiments generalize to other taxa, they suggest that single-exemplar phylogenies should be interpreted with caution, particularly for groups where species are shallowly diverged or inadequately delimited.

A new species of leopard frog (Anura: Ranidae) from the urban northeastern US.

Past confusion about leopard frog (genus Rana) species composition in the Tri-State area of the US that includes New York (NY), New Jersey (NJ), and Connecticut (CT) has hindered conservation and management efforts, especially where populations are declining or imperiled. We use nuclear and mitochondrial genetic data to clarify the identification and distribution of leopard frog species in this region. We focus on four problematic frog populations of uncertain species affiliation in northern NJ, southeastern mainland NY, and Staten Island to test the following hypotheses: (1) they are conspecific with Rana sphenocephala or R. pipiens, (2) they are hybrids between R. sphenocephala and R. pipiens, or (3) they represent one or more previously undescribed cryptic taxa. Bayesian phylogenetic and cluster analyses revealed that the four unknown populations collectively form a novel genetic lineage, which represents a previously undescribed cryptic leopard frog species, Rana sp. nov. Statistical support for R. sp. nov. was strong in both the Bayesian (pp=1.0) and maximum-likelihood (bootstrap=99) phylogenetic analyses as well as the Structure cluster analyses. While our data support recognition of R. sp. nov. as a novel species, we recommend further study including fine-scaled sampling and ecological, behavioral, call, and morphological analyses before it is formally described.

Phylogeographic analyses of the southern leopard frog: the impact of geography and climate on the distribution of genetic lineages vs. subspecies.

The southeastern United States is a major phylogeographic break hotspot for amphibians, but the processes underlying this hotspot remain to be explicitly tested. We test the correlation of genetic lineages with subspecies breaks in the southeastern United States and the association of such breaks with climate, using Rana sphenocephala as a case study, and place our results in the broader context of the Alabama-Appalachian suture zone (AL-Appalachian SZ). We use genetic and ecological methods to (i) determine whether genetic lineages are coincident with the AL-Appalachian SZ or the subspecies and (ii) test the correlation of major climatic breaks with genetic structure and morphological variation in R. sphenocephala. Bayesian phylogenetic analyses of the ND1 mtDNA gene and microsatellite cluster analyses revealed two distinct lineages with over 4% sequence divergence. The geographic distributions of the two lineages are concordant with the AL-Appalachian SZ but do not correspond to the ranges of the subspecies based on morphology. Mantel tests revealed that isolation by distance and historical barriers to gene flow, rather than climate, are the major drivers of genetic divergence at neutral loci. Examination of climate breaks across the Southeast revealed a pattern incongruent with suture zone hotspots, suggesting that phylogenetic structure has been driven primarily by historical factors, such as isolation, the Appalachian Mountains and the Apalachicola/Chattahoochee/Flint River Basin. However, climate breaks are consistent with the geographic distribution of the subspecies of R. sphenocephala, suggesting that environmental pressures may be driving divergence in morphological traits that outpaces molecular evolution.