David Ginsburg

Animal venoms vary greatly in compositional complexity, where complex venoms are hypothesized to be maintained by greater dietary breadth. Beyond explaining venom composition, the dietary breadth hypothesis predicts that these more complex venoms should show greater functional breadth in terms of overall toxicity and by disrupting multiple prey physiological processes. We evaluate these predictions with six distinct physiological assays of cardiac and blood clotting functions and compared the effects of venoms from snake species with a range of taxonomic dietary diversity levels. We compared the taxonomic dietary generalists Agkistrodon piscivorus and Sistrurus miliarius to taxa with varying taxonomic specialization, namely Agkistrodon contortrix and Crotalus adamanteus, and Azemiops feae. Comparing fish thrombocyte and mammal platelet aggregation and fibrin clot formation, only species with broader diets disrupted both fish and mammal hemostatic function. Venom of A. piscivorus, a uniquely fish-eating species, was the most disruptive of zebrafish heart rate, thrombocyte activation, vascular permeability, and clotting after injury. S. miliarius was also highly toxic, whereas mammal-specialists’ venoms scarcely altered zebrafish physiology. Our results support the hypothesis that dietary breadth selects functionally-complex venoms. Understanding venom gene evolution, snakebite symptoms, and searching for therapeutics in venom should be guided by evolutionary ecology.

Group A Streptococcus (GAS) expresses streptokinase (SK), a critical virulence factor that non-enzymatically activates the host’s plasminogen (PLG), to an active form, PLG _SK , resulting in the degradation of fibrin clots and subsequent bacterial dissemination. PLG _SK formation contrasts with the physiologic activation of PLG to the serine protease plasmin via a proteolytic mechanism. As a potent thrombolytic, SK has been used as a therapeutic to treat heart attacks and strokes. GAS SK is highly specific for human PLG, and sequence variation between SK from different GAS strains has been linked to differences in PLG binding and disease severity. We now report the application of deep mutational scanning (DMS) to map the effects of ∼71% of single amino acid substitutions within SK from Group C Streptococcus, which shares high sequence homology with GAS on its ability to bind human PLG. We first demonstrate that SK expressed as a fusion protein to the p3 coat protein of M13 filamentous phage retains its capacity to bind human PLG. Our subsequent DMS analysis using this phage system identifies regions of SK in which amino acid substitutions are likely to increase or decrease its affinity for PLG. Our findings suggest a complex protein-protein interaction in which long-range protein dynamics influence the conformational activation of PLG to PLG _SK . These data lay the foundation for linking SK variation between GAS strains to differences in virulence, mapping the determinants of GAS SK’s human specificity, and potentially contributing to the development of improved therapeutics for heart attack and stroke.

Tandem duplication of genes can play a critical role in the evolution of functional novelty, yet our understanding is limited concerning the role of gene duplication in coevolution between species. Much is known about the evolution and function of tandemly duplicated snake venom genes, however, the potential of gene duplication to fuel venom resistance within prey species is poorly understood. The SERPINA subfamily of genes produces globular serine protease inhibitors and carrier molecules, and SERPINA1 has previously been shown to inhibit snake venom serine proteases. In this study, we characterize patterns of duplication within the tandem array of SERPINA, documenting trends in copy number evolution between species. We find the hallmarks of rapid birth-death evolution of SERPINA1-like and SERPINA3-like genes within and between rodent lineages, and evidence for diversifying selection acting on rodent genes. To explore the functional significance of copy number evolution, we recombinantly expressed the full set of 12 paralogous duplicates of SERPINA3 found in the genome of the Big-eared woodrat (Neotoma macrotis), a species known for resistance to protease-rich rattlesnake venoms. Two SERPINA3 paralogs inhibited venom serine proteases, indicating that these proteins may serve as resistance factors. In addition, functional variation is apparent among paralogs, including neofunctionalization to inhibit both chymotrypsin-like and trypsin-like proteases simultaneously for one venom-inhibiting paralog. Our results provide further evidence that the rapid evolution of SERPINA1 and SERPINA3 gene copy number across rodents has adaptive potential by producing functionally diverse venom inhibitors.

Plasminogen activator inhibitor-1 (PAI-1), like other serine protease inhibitors (SERPINs), exists in a functionally active metastable conformation. Unlike other SERPINs, PAI-1 undergoes a relatively rapid rearrangement to a lower energy latent conformation. We employ deep mutational scanning (DMS) to simultaneously probe the effects of nearly all single amino acid substitutions (92%) in PAI-1 on its latency transition as well as its ability to inhibit its canonical protease target, urokinase-type plasminogen activator (uPA). The DMS results are interpreted in the context of variant effect predictors (EVE, AlphaMissense, and CPT) and protein stability predictors (EvoEF and FoldX), as well as the evolutionary conservation of the PAI-1 sequence space across extant vertebrate species. We demonstrate that while variant effect predictors can generally partition PAI-1 as functional or non-functional with respect to uPA inhibition, they perform poorly when attempting to discriminate the effects of PAI-1 variants on its latency transition. However, by employing protein stability predictors, we demonstrate that the PAI-1 latency transition is most likely driven by changes in the energy barrier to the latency transition. Finally, we show that PAI-1’s ability to inhibit uPA, as well as its thermodynamic instability in its active conformation are both under purifying selection that limits the sequence space available to PAI-1. These findings suggest that DMS of collateral fitness effects, including PAI-1’s latency transition, may be better interpreted in contexts other than variant effect predictors, including protein thermodynamics and evolution.

While members of large paralogous protein families share structural features, their functional niches often diverge significantly. Serine protease inhibitors (SERPINs), whose members typically function as covalent inhibitors of serine proteases, are one such family. Plasminogen activator inhibitor‐1 (PAI‐1) is a prototypic SERPIN, which canonically inhibits tissue‐ and urokinase‐type plasminogen activators (tPA and uPA) to regulate fibrinolysis. PAI‐1 has been shown to also inhibit other serine proteases, including coagulation factor XIIa (FXIIa) and transmembrane serine protease 2 (TMPRSS2). The structural determinants of PAI‐1 inhibitory function toward these non‐canonical protease targets, and the biological significance of these functions, are unknown. We applied deep mutational scanning (DMS) to assess the effects of ~80% of all possible single‐amino acid substitutions in PAI‐1 on its ability to inhibit three putative serine protease targets (uPA, FXIIa, and TMPRSS2). Selection with each target protease generated a unique PAI‐1 mutational landscape, with the determinants of protease specificity distributed throughout PAI‐1's primary sequence. Next, we conducted a comparative analysis of extant orthologous sequences, demonstrating that key residues modulating PAI‐1 inhibition of uPA and FXIIa, but not TMPRSS2, are maintained by purifying selection (also referred to as “negative selection”). PAI‐1's activity toward FXIIa may reflect how protease evolutionary relationships predict SERPIN functional divergence, which we support via a cophylogenetic analysis of secreted SERPINs and their cognate serine proteases. This work provides insight into the functional diversification of SERPINs and lays the framework for extending these studies to other proteases and their regulators.

Interactions between predators and prey are often characterized by strong selection pressures that shape extreme physiological adaptations. Venom resistance in large-bodied South American opossums (Clade Didelphini) is a striking example, as these marsupials prey on venomous snakes and exhibit remarkable resistance to their venom. While resistance is well documented in Didelphini, relatively little is known about venom resistance in the smaller, more diverse members of Didelphidae, which inhabit the same regions and encounter the same predators. Here, we investigate venom resistance in the small-bodied opossum, Monodelphis domestica , through multi-level physiological assays, examining responses to purified venom components and whole venom from sympatric and allopatric vipers. Our results show M. domestica resists venom-induced disruptions to blood coagulation, retains platelet function in the presence of platelet-disrupting venoms, and inhibits snake venom metalloproteinases. Unexpectedly, we find that M. domestica von Willebrand Factor (VWF) requires increased shear force to elongate, a previously unknown aspect of opossum blood physiology that may contribute to venom resistance and may have relevance to human coagulopathies. These findings expand the extent of venom resistance beyond large-bodied Didelphini, suggesting it is a widespread trait in South American marsupials and providing new insights into venom-mammal coevolution. Abstract Figure: