Definition of a saxitoxin (STX) binding code enables discovery and characterization of the anuran saxiphilin family

Significance Saxitoxin (STX) is a lethal neurotoxin made by cyanobacteria and dinoflagellates associated with oceanic red tides. Its accumulation in seafood causes paralytic shellfish poisoning (PSP), a public health hazard that is increasing due to climate change. Bullfrog saxiphilin (RcSxph) is a soluble STX binding protein that acts as a ‘toxin sponge’ involved in toxin resistance. Studying RcSxph:STX interaction energetics defines a STX ‘recognition code’ that enabled discovery of ten Sxphs in frogs and toads separated by ∼140 million years of evolution. These findings provide a molecular basis for understanding the roles of toxin sponge proteins in toxin resistance and should enable STX binding site identification in natural proteins and design of proteins to sense or neutralize STX and related toxins.

American bullfrog (Rana castesbeiana) saxiphilin (RcSxph) is a high-affinity "toxin sponge" protein thought to prevent intoxication by saxitoxin (STX), a lethal bisguanidinium neurotoxin that causes paralytic shellfish poisoning (PSP) by blocking voltage-gated sodium channels (Na V s). How specific RcSxph 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 RcSxph 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.
saxitoxin j saxiphilin j toxin resistance j anuran Saxitoxin (STX), one of the most potent nonpeptidyl neurotoxins, blocks the bioelectrical signals in nerve and muscle required for life by inhibiting select voltage-gated sodium channel (Na V ) isoforms (1)(2)(3). Cyanobacteria and dinoflagellate species associated with oceanic red tides produce this bis-guanidinium small molecule and its congeners, whose accumulation in seafood can cause paralytic shellfish poisoning (PSP), a commercial fishing and public health hazard of growing importance due to climate change (1,(3)(4)(5). Its lethality has also earned STX the unusual distinction of being the only marine toxin declared a chemical weapon (1,3). Select vertebrates, particularly frogs, resist STX poisoning (6)(7)(8)(9), a property that is thought to rely on the ability of the soluble "toxin sponge" protein saxiphilin (Sxph) to sequester STX (8,9). Recent structural studies (10) defined the molecular architecture of the American bullfrog [Rana (Lithobates) castesbeiana] Sxph (RcSxph) (8,(11)(12)(13)(14) showing that this 91-kDa soluble, transferrin-related protein from frog heart and plasma has a single, highaffinity STX binding site on its C lobe. Remarkably, even though RcSxph and Na V s are unrelated, both engage STX through similar types of interactions (10). This structural convergence raises the possibility that determination of the factors that underlie the high-affinity Sxph:STX interaction could provide a generalizable molecular recognition code for STX that would enable the identification or engineering of STX binding sites in natural and designed proteins.
To characterize RcSxph:STX interactions in detail, we developed a suite of assays comprising thermofluor (TF) measurements of ligand-induced changes in RcSxph stability, fluorescence polarization (FP) binding to a fluorescein-labeled STX, and isothermal titration calorimetry (ITC). We paired these assays with a scanning mutagenesis strategy (15,16) to dissect the energetic contributions of RcSxph STX binding pocket residues. These studies show that the core RcSxph STX recognition code comprises two "hot spot" triads. One engages the STX tricyclic bis-guanidinium core through a pair of carboxylate groups and a cation-π interaction (17) in a manner that underscores the convergent STX recognition strategies shared by RcSxph and Na V s (17)(18)(19)(20)(21)(22). The second triad largely interacts with the C13 carbamate group of STX and is the site of interactions that can enhance STX binding affinity and the ability of RcSxph to act as a "toxin sponge" that can reverse the effects of STX inhibition of Na V s (8,9).
Although Sxph-like STX binding activity has been reported in extracts from diverse organisms including arthropods (13), amphibians (11,13,23), fish (13), and reptiles (13), the molecular origins of this activity have remained obscure. Definition of the RcSxph STX recognition code enabled identification of 10 new Sxphs from diverse frogs and toads. This substantial enlargement of the Sxph family beyond RcSxph and the previously identified High Himalaya frog (Nanorana parkeri) Sxph (NpSxph) (10) reveals a varied STX binding pocket that surrounds a conserved core of "hot spot" Significance Saxitoxin (STX) is a lethal neurotoxin made by cyanobacteria and dinoflagellates associated with oceanic red tides. Its accumulation in seafood causes paralytic shellfish poisoning (PSP), a public health hazard that is increasing due to climate change. Bullfrog saxiphilin (RcSxph) is a soluble STX binding protein that acts as a 'toxin sponge' involved in toxin resistance. Studying RcSxph:STX interaction energetics defines a STX 'recognition code' that enabled discovery of ten Sxphs in frogs and toads separated by ∼140 million years of evolution. These findings provide a molecular basis for understanding the roles of toxin sponge proteins in toxin resistance and should enable STX binding site identification in natural proteins and design of proteins to sense or neutralize STX and related toxins.
positions. Comparison of the new Sxph family members further identifies dramatic differences in the number of thyroglobulin (Thy1) domains inserted into the modified transferrin fold upon which the Sxph family is built. Biochemical characterization of NpSxph, Oophaga sylvatica Sxph (OsSxph) (24), Mantella aurantiaca Sxph (MaSxph), and Ranitomeya imitator Sxph (RiSxph), together with structural determination of NpSxph, alone and as STX complexes, shows that the different Sxphs share the capacity to form high-affinity STX complexes and that binding site preorganization (10) is a critical factor for tight STX association. Together, these studies establish an STX molecular recognition code that provides a template for understanding how diverse STX binding proteins engage the toxin and its congeners and uncover that Sxph family members are abundantly found in the most varied and widespread group of amphibians, the anurans. This knowledge and suite of diverse Sxphs, conserved among anuran families separated by at least 140 My of evolution (25), provide a starting point for defining the physiological roles of Sxph in toxin resistance (9,24,26), should facilitate identification or design of other STX binding proteins, and may enable the development of new biologics to detect or neutralize STX and related PSPs.

Results
Establishment of a Suite of Assays to Probe RcSxph Toxin-Binding Properties. To investigate the molecular details of the high-affinity RcSxph:STX interaction, we developed three assays to assess the effects of STX binding site mutations. A key criterion was to create assays that could be performed in parallel on many RcSxph mutants using minimal amounts of purified protein and toxin. To this end, we first tested whether we could detect STX binding using a TF assay (27,28) in which STX binding would manifest as concentration-dependent change in the apparent RcSxph melting temperature (Tm). Addition of STX, but not the related guanidinium toxin tetrodotoxin (TTX), over a 0-to-20 μM range to samples containing 1.1 μM RcSxph caused concentration-dependent shifts in the RcSxph melting curve (Fig. 1A) (ΔTm = 3.6°C ± 0.2 versus 0.3°C ± 0.4 for STX and TTX, respectively). These differential effects of STX and TTX are in line with the ability of RcSxph to bind STX (8,13,14) but not TTX (8,9) and indicate that ΔTm is a consequence of the RcSxph:STX interaction.
To investigate the contributions of residues that comprise the STX binding site, we coupled the TF assay with alanine scanning (15), as well as deeper mutagenesis studies, targeting the eight residues that directly contact STX (Glu540, Phe561, Thr563, Tyr558, Pro727, Phe784, Asp785, and Asp794) (10) and four second-shell sites that support these residues (Tyr795, Ile782, Gln787, and Lys789) (Figs. 1 A and B and SI Appendix, Fig.  S1A). Measurement of the STX-induced ΔTm changes for the purified RcSxph mutants revealed ΔTm changes spread over a ∼4°C range that included ΔTm increases relative to wild type (e.g., I782A and D785N) as well as those that caused complete loss of the thermal shift (e.g., E540A and D794A). All mutations had minimal effects on protein stability (SI Appendix, Fig. S1B) and there was no evident correlation between Tm and ΔTm (SI Appendix, Fig. S1C). Hence, the varied ΔTms indicate that each of the 12 positions contributes differently to STX binding.
Because ΔTm interpretation can be complex, especially in the case of a multidomain protein such as RcSxph, and may not necessarily indicate changes in ligand affinity (27,28), we developed a second assay to measure the effects of mutations on RcSxph binding affinity. We synthesized a fluorescein-labeled STX derivative (F-STX) by functionalization of the pendant carbamate group with a six-carbon linker and fluorescein (29, 30) ( Fig. 1C and SI Appendix, Fig. S2) and established an FP assay (31,32) to measure toxin binding. FP measurements revealed a high-affinity interaction between F-STX and RcSxph (dissociation constant [K d ] = 7.4 nM ± 2.6) that closely agrees with prior radioligand assay measurements of RcSxph affinity for STX (∼1 nM) (14). The similarity between the F-STX and STX K d values is consistent with the expectation from the RcSxph:STX structure that STX carbamate derivatization should have a minimal effect on binding, as this element resides on the solvent-exposed side of the STX binding pocket (10). To investigate the F-STX interaction further, we soaked RcSxph crystals with F-STX and determined the structure of the RcSxph:F-STX complex at 2.65-Å resolution by X-ray crystallography (SI Appendix, Fig. S3A and Table S1). Inspection of the STX binding pocket revealed clear electron density for the F-STX bis-guanidinium core as well as weaker density that we could assign to the fluorescein heterocycle (SI Appendix, Fig. S3A), although the high B-factors of the linker and fluorescein indicate that these moieties are highly mobile (SI Appendix, Fig. S3B). Structural comparison with the RcSxph: STX complex (10) showed no changes in the core STX binding pose or STX binding pocket residues (RMSD Cα = 0.279 Å) (SI Appendix, Fig. S3C). Together, these data demonstrate that both F-STX and STX bind to Sxph in the same manner and indicate that there are no substantial interactions with the fluorescein label.
FP measurement of the RcSxph alanine scan mutants uncovered binding affinity changes spanning a ∼13,000-fold range that correspond to free energy perturbations (ΔΔG) of up to ∼5.60 kcalÁmol À1 (Fig. 1D, SI Appendix, Fig. S4, and Table 1). The effects were diverse, encompassing enhanced affinity changes (Y558A K d = 1.2 nM ± 0.3) and large disruptions (E540A K d = 15.3 μM ± 4.1). As indicated by the TF data, each STX binding pocket residue contributes differently to STX recognition energetics. Comparison of the TF ΔTm and FP ΔΔG values shows a strong correlation between the two measurements ( Fig. 1E). This concordance between ΔTm and ΔΔG indicates that the changes in unfolding free energies caused by protein mutation and changes in STX binding affinity do not incur large heat capacity or entropy changes relative to the wild-type protein (33,34). Hence, ΔTm values provide an accurate estimate of the STX binding affinity differences.
To investigate the STX affinity changes further, we used ITC ( Fig. 1F and SI Appendix, Table S2), a label-free method that reports directly on ligand association energetics (35), to examine the interaction of STX with RcSxph and six mutants having varied effects on binding (E540D, Y558I, Y558A, F561A, P727A, and D794E) (Figs. 1F and 2A and SI Appendix, Fig. S5 A-C and Table S2). Experiments with RcSxph confirm the 1:1 stoichiometry and high affinity of the RcSxph:STX interaction (K d ∼nanomolar) reported previously (8,10,14) and reveal a large, favorable binding enthalpy (ΔH À16.1 ± 0.2 kcalÁmol À1 ) in line with previous radioligand binding studies (14). In almost all mutants, binding affinity loss correlated with a reduction in enthalpy, consistent with a loss of interactions (SI Appendix, Table S2). The one exception to this trend is E540D for which STX association yielded a binding enthalpy (ΔH = À16.3 ± 1.7 kcalÁmol À1 ) very similar to wild-type RcSxph that was offset by an approximately twofold unfavorable change in binding entropy. The ITC measurements were unable to measure the affinity enhancement for Y558A and Y558I accurately due to the fact that these mutants, as well as RcSxph, have K d s at the detection limit of direct titration methods (∼1 nM) (35). Nevertheless,  Table 1.
ΔG ITC from mutants having STX K d s within the ITC dynamic range (K d s ∼30 to 300 nM) showed an excellent agreement with ΔG FP measurements made with F-STX (Fig. 1G). These data further validate the TF and FP assay trends and support the conclusion that RcSpxh:F-STX binding interactions are very similar to the RcSpxh:STX interactions. Together, these three assays (Fig. 1 E and G) provide a robust and versatile suite of options for characterizing STX:Sxph interactions.
Sxph STX binding code is focused on two sets of "hot spot" residues. To understand the structural code underlying STX binding, we classified the effects of the alanine mutations into six groups based on ΔΔG values ( Fig. 2A and Table 1) and mapped these onto the RcSxph structure (Fig. 2B). This analysis identified a binding "hot spot" comprising three residues that directly contact the STX bis-guanidinium core (Glu540, Phe784, and Asp794) (10) and an additional site near the carbamate (Pro727) where alanine mutations caused substantial STX binding losses (ΔΔG ≥1 kcalÁmol À1 ). Conversely, we also identified a site (Tyr558) where alanine caused a notable enhancement of STX binding (ΔΔG ≤À1 kcalÁmol À1 ) ( Fig. 2B and Table 1).
To examine the physicochemical nature of key residues critical for STX binding further, we made mutations at select positions guided by the alanine scan. Mutations at Glu540 and Asp794 (10), residues involved in charge pair interactions with the STX guanidinium rings, that neutralized the side chain while preserving shape and volume ( Fig. 2A and Table 1) disrupted binding strongly, similar to their alanine counterparts (ΔΔG = 4.30 and 3.60 kcalÁmol À1 for E540Q and D794N, respectively) ( Fig. 2A, SI Appendix, Fig. S4, and Table 1). Altering side-chain length while preserving the negative charge at these sites also greatly diminished STX affinity but was notably less problematic at Glu540 (ΔΔG = 1.54 and 2.94 kcalÁmol À1 for E540D and D794E, respectively). To probe contacts with Phe784, which makes a cation-π interaction (17) with the STX five-membered guanidinium ring (10), we tested changes that preserved this interface (F784Y), maintained side-chain volume and hydrophobicity (F784L), and that mimicked substitutions (F784C and F784S) found in the analogous residue in STX-resistant Na V s (Na V 1.5, Na V 1.8, and Na V 1.9) (10, 17, 19-22) ( Fig. 2A and Table 1). Preserving the cation-π interaction with F784Y caused a modest binding reduction (ΔΔG = 0.37 kcalÁmol À1 ), whereas F784L was disruptive (ΔΔG = 1.11 kcalÁmol À1 ) and F784C and F784S were even more destabilizing than F784A (ΔΔG = 3.15, 3.60, and 2.71 kcalÁmol À1 , respectively).
We also examined two other positions that form part of the Sxph binding pocket near the five-membered STX guanidinium ring. Asp785 undergoes the most dramatic conformational change of any residue associated with STX binding, moving from an external-facing conformation to one that engages this STX element (10). Surprisingly, D785A and D785N mutations caused only relatively modest binding changes ( Fig. 2A and Table 1) (ΔΔG = 0.57 and À0.30 kcalÁmol À1 for D785A and D785N, respectively). Because of the proximity of the secondshell residue Gln787 to Asp785 and Asp794 (Fig. 2B), two residues that coordinate the five-membered STX guanidinium ring (10), we also asked whether adding additional negative charge to this part of the STX binding pocket would enhance toxin binding affinity. However, Q787E had essentially no effect on binding (ΔΔG = À0.09 kcalÁmol À1 ).
Two residues, Tyr558 and Ile782, stood out as sites where alanine substitutions enhanced STX affinity (Fig. 2 A and B and Table 1). Tyr558 interacts with both the STX five-membered guanidinium ring and carbamate and moves away from the STX binding pocket upon toxin binding (10), whereas Ile782 is a   (40). Residues that are energetically important for the STX interaction are shown in space filling. Na V 1.7 selectivity filter "DEKA" ring residues are shown (white). Italics indicate corresponding residue numbers for rat Na V 1.4 (18).
second-shell site that buttresses Tyr558. Hence, we hypothesized that affinity enhancements observed in the Tyr558 and Ile782 mutants resulted from the reduction of Tyr558-STX clashes. In accord with this idea, Y558F had little effect on STX binding (ΔΔG = À0.10 kcalÁmol À1 ), whereas shortening the side chain but preserving its hydrophobic character, Y558I, enhanced binding as much as Y558A (ΔΔG = À1.07 and À1.00 kcalÁmol À1 , respectively). Conversely, increasing the side-chain volume at the buttressing position, I782F, a change expected to make it more difficult for Tyr558 to move out of the binding pocket, reduced STX binding affinity (ΔΔG = 0.46 kcalÁmol À1 ). Combining the two affinity enhancing mutants, Y558A/I782A, yielded only a marginal increase in affinity in comparison to Y558A (ΔΔG = À1.07 and À1.00 kcalÁmol À1 , respectively) but was better than I782A alone (ΔΔG = À0.53 kcalÁmol À1 ). This nonadditivity in binding energetics (36) is in line with the physical interaction of the two sites and the direct contacts of Tyr558 with the toxin. Together, these data support the idea that the Tyr558 clash with STX is a key factor affecting STX affinity and suggest that it should be possible to engineer Sxph variants with enhanced binding properties by altering this site.
Taken together, these studies of the energetic map of the RcSxph STX binding pocket highlight the importance of two amino acid triads. One (Glu540, Phe784, and Asp794) engages the STX bis-guanidinium core of the toxin. The second (Tyr558, Phe561, and Pro727) forms the surface surrounding the carbamate unit (Fig. 2B). The central role of the Glu540/ Phe784/Asp794 triad in the energetics of binding the bisguanidinium STX core underscores the toxin receptor site similarities between RcSxph and Na V s (10) (Fig. 2 C and D). In both, STX binding relies on two acidic residues that coordinate the fiveand six-membered STX rings [Sxph Asp794 and Glu540 and rat Na V 1.4 Glu403 and Glu758 (18)] and a cation-π interaction [Sxph Phe784 and rat Na V 1.4 Tyr401 (18) and its equivalents in other Na V s (19)(20)(21)(22)]. Hence, both the basic architecture and binding energetics appear to be conserved even though the overall protein structures presenting these elements are dramatically different. Structures of enhanced-affinity RcSxph mutants. To investigate the structural underpinnings of the affinity enhancement caused by mutations at the Tyr558 site, we determined crystal structures of RcSxph-Y558A and RcSxph-Y558I alone (2.60-Å and 2.70-Å resolution, respectively) and as cocrystallized STX complexes (2.60 Å and 2.15 Å, respectively) (SI Appendix, Fig. S6 A-D and Table S1). Comparison of the apo-and STX-bound structures reveals little movement in the STX binding pocket upon ligand binding (RMSD Cα = 0.209 Å and 0.308 Å comparing apo-and STX-bound RcSxph-Y558A and RcSxph-Y558I, respectively) ( Fig. 3 A and B and Movies S1 and S2). In both, the largest conformational change is the rotation of Asp785 into the binding pocket to interact with the five-membered guanidinium ring of STX, as seen for RcSxph (Fig. 3C) (10). By contrast, unlike in RcSxph, there is minimal movement of residue 558 and its supporting loop, indicating that both Y558A and Y558I eliminate the clash incurred by the Tyr558 side chain. Comparison with the RcSxph:STX complex also shows that the STX carbamate in both structures has moved into a pocket formed by the mutation at Tyr558 (RMSD Cα = 0.279 Å and 0.327 Å comparing RcSxph:STX with RcSxph-Y558A:STX and RcSxph-Y558I:STX, respectively) (Fig. 3C). This structural change involves a repositioning of the carbamate carbon by 2 Å in the RcSxph-Y558I:STX complex relative to the RcSxph:STX complex. These findings are in line with the nearly equivalent toxin-binding affinities of Y558A and Y558I, as well as with the idea that changes at the Tyr558 buttressing residue, Ile782, relieve the steric clash with STX. They also demonstrate that one strategy for increasing STX affinity is to engineer a highly organized binding pocket that requires minimal conformational changes to bind STX.
Sxph STX binding-affinity changes alter Na v rescue from STX block. RcSxph acts as a "toxin sponge" that can reverse STX inhibition of Na V s (9). To test the extent to which this property is linked to the intrinsic affinity of RcSxph for STX, we evaluated how STX-affinity-altering mutations affected RcSxph rescue of channels blocked by STX. As shown previously, titration of different RcSxph:STX ratios against Phyllobates terribilis Na V 1.4 (PtNa V 1.4), a Na V having a concentration that inhibits response by 50% for STX (IC 50 ) of 12.6 nM (9), completely reverses the effects of STX at ratios of 2:1 RcSxph:STX or greater ( Fig. 4 A and F). Incorporation of mutations that affect STX affinity altered the ability of RcSxph to rescue Na V s and followed the binding assay trends. Mutants that increased STX affinity, Y558I and I782A, improved the ability of RcSxph to rescue PtNa V 1.4 (Effective Rescue Ratio 50 [ERR 50 ] = 0.81 ± 0.01, 0.87 ± 0.02, and 1.07 ± 0.02 for Y558I, I782A, and RcSxph, respectively), whereas mutations that compromised STX binding reduced (P727A, ERR 50 >4) or eliminated (E540A) the ability of RcSxph to reverse the STX inhibition ( Fig. 4 B-F). This strong correlation indicates that the "toxin sponge" property of Sxph (9) depends on the capacity of Sxph to sequester STX and adds further support to the idea that Sxph has a role in toxin resistance mechanisms (8,9). Expansion of the Sxph family. STX binding activity has been reported in the plasma, hemolymph, and tissues of diverse arthropods, amphibians, fish, and reptiles (11,13), suggesting that many organisms harbor Sxph-like proteins. Besides RcSpxh, similar Sxphs have been identified in only two other frogs, the High Himalaya frog N. parkeri (10) and the little devil poison frog O. sylvatica (24). As a number of poison frogs exhibit resistance to STX poisoning (9), we asked whether the STX binding site "recognition code" could enable identification of Sxph homologs in other amphibians. To this end, we determined the sequences of 10 new Sxphs (Fig. 5 A and B  show that all of the new Sxphs share the transferrin fold found in RcSxph comprising N and C lobes each having two subdomains (N1, N2 and C1, C2, respectively) (10, 12) and the signature "EFDD" motif (10) or a close variant in the core of the C-lobe STX binding site (Fig. 5A). Similar to RcSxph, the new Sxphs also have amino acid differences relative to transferrin that should eliminate Fe 3+ binding (10,12,37), as well as a number of protease inhibitor thyroglobulin domains (Thy1) inserted between the N1 and N2 N-lobe subdomains (10, 38) ( Fig. 5A and SI Appendix, Fig. S7-S9). These Thy1 domain insertions range from two in RcSxph, NpSxph, and MaSxph to three in the dendrobatid poison frog and cane toad Sxphs to 16 and 15 in toad BbSxph and BgSxph, respectively ( Fig. 5A and SI Appendix, Figs. S7-S9).
We used the STX recognition code defined by our studies as a template for investigating cross-species variation in the residues that contribute to STX binding (Fig. 5B). This analysis shows a conservation of residues that interact with the STX bis-guanidinium core (Glu540, Phe784, Asp785, Asp794, and Tyr795) and carbamate (Phe561). Surprisingly, five of the Sxphs (D. tinctorius, R. imitator, A. femoralis, B. bufo, and B. gargarizans) have an aspartate instead of a glutamate at the Glu540 position in RcSxph that contributes the most binding energy (Fig. 2A). The equivalent change in RcSxph, E540D, reduced STX affinity by ∼100-fold (Table 1) and uniquely alters enthalpy and entropy binding parameters compared to other affinity-lowering mutations (SI Appendix, Table S2). Additionally, we identified variations at two sites for which mutations increase RcSxph STX binding, Tyr558 and Ile782 (Figs. 2A and 4 C and E and Table 1). NpSxph and MaSxph have an Ile at the Tyr558 site, whereas eight of the new Sxphs have hydrophobic substitutions at the Ile782 position (Fig.  5B). The striking conservation of the Sxph scaffold and STX binding site indicate that this class of "toxin sponge" proteins is widespread among diverse anurans, while the amino acid variations in key positions (Glu540, Tyr558, and Ile782) raise the possibility that the different Sxph homologs have varied STX affinity or selectivity for STX congeners.

Diverse Sxph family members have conserved STX binding
properties. To explore the STX binding properties of this new set of Sxphs and to begin to understand whether changes in the binding site composition affect toxin affinity, we expressed and purified four representative variants. These included two Sxphs having STX binding-site sequences similar to RcSxph (NpSxph and MaSxph) and two Sxphs bearing more diverse amino acid differences (RiSxph and OsSxph), including one displaying the E540D substitution (RiSxph). This set also represents Sxphs  having either two Thy1 domains similar to RcSxph (NpSxph and MaSxph) or three Thy1 domains (OsSxph and RiSxph) (Fig. 5A). TF experiments showed STX-dependent ΔTms for all four Sxphs. By contrast, equivalent concentrations of TTX had no effect (Fig. 5C), indicating that, similar to RcSpxh (Fig. 1A) (8,9), all four Sxphs bind STX but not TTX. Unlike the other Sxphs, the RiSxph melting curve showed two thermal transitions; however, only the first transition was sensitive to STX concentration (Fig. 5C). FP binding assays showed that all four Sxphs bound F-STX and revealed affinities stronger than RcSxph (Fig. 5D and Table 2). The enhanced affinity of NpSxph and MaSxph for STX relative to RcSxph is consistent with the presence of the Y558I variant (Fig. 5B). Importantly, the observation that RiSxph has a higher affinity for STX than RcSxph despite the presence of the E540D difference suggests that the other sequence variations in the RiSxph STX binding pocket compensate for this Glu!Asp change at Glu540. Because NpSxph has a higher affinity for STX than RcSxph (Figs. 1D and 5D and Table 2) and has an isoleucine at the Tyr558 site (Fig. 5B), we asked whether the NpSxph I559Y mutant that converts the NpSxph binding site to match RcSxph would lower STX affinity. TF measurements showed that NpSxph I559Y had a ∼1°C smaller ΔTm than NpSxph (ΔTm = 3.2°C ± 0.3 versus 2.5°C ± 0.2 for NpSxph and NpSxph I559Y, respectively), indicative of a decreased binding affinity (Fig. 5 C and E). This result was validated by FP (ΔΔG = À1.56 kcalÁmol À1 ), yielding a result of similar magnitude to the RcSxph Y558I differences (Fig. 5E and Tables 1  and 2). ITC confirmed the high affinity of the interaction (SI Appendix, Fig. S5 D, E and F), but could not yield an explicit K d given its low nanomolar value (SI Appendix, Fig. S5F). Nevertheless, these experiments validate the 1:1 stoichiometry of the NpSxph:STX interaction (SI Appendix, Table S2) and show that the I559Y change reduced the binding enthalpy, consistent with perturbation of NpSxph:STX interactions (ΔH = À18.7 ± 0.2 vs. À16.8 ± 0.2 kcalÁmol À1 , NpSxph and NpSxph I559Y, respectively) (SI Appendix, Table S2). Taken together, these experiments establish the conserved nature of the STX binding pocket among diverse Sxph homologs and show that the STX recognition code derived from RcSxph studies (Fig. 5B) can identify key changes that influence toxin binding.
Structures of apo-and STX-bound NpSxph reveal a preorganized STX binding site. To compare STX binding modes among Sxph family members, we crystallized and determined the structure of NpSxph, alone and cocrystallized with STX. NpSxph and STX:NpSxph crystals diffracted X-rays to resolutions of 2.2 Å and 2.0 Å, respectively, and were solved by molecular replacement ( Fig. 6A and SI Appendix, Fig. S10 A and B). As expected from the similarity to RcSxph, NpSxph is built on a transferrin fold (Fig. 6A) and has the same 21 disulfides found in RcSxph, as well as an additional 22nd disulfide in the Type 1A thyroglobulin domain of NpSxph Thy1-2. However, structural comparison of NpSxph and RcSxph reveals a number of unexpected large-scale domain rearrangements.
The NpSxph N lobe is displaced along the plane of the molecule by ∼30°and rotated around the central axis by a similar amount (SI Appendix, Fig. S10C). NpSxph N lobe and C lobe lack Fe 3+ binding sites (Fig. 5A), and despite the N-lobe displacement relative to RcSxph adopt closed and open conformations, respectively, as in RcSxph (10) (SI Appendix, Fig. S10 D and E) (RMSD Cα = 1.160 Å and 1.373 Å for NpSxph and RcSxph N and C lobes, respectively). Surprisingly, the two NpSxph Thy1 domains are in different positions than in RcSxph and appear to move as a unit by ∼90°with respect to the central transferrin scaffold (SI Appendix, Fig. S10F and Movie S3) and a translation of ∼30 Å of Thy1-2 (SI Appendix, Fig. S10G). Thy1-1 is displaced from a site over the N lobe in RcSxph to one in which it interacts with the NpSxph C-lobe C2 subdomain and Thy1-2 moves from between the N and C lobes in RcSxph where it interacts with the C1 subdomain, to a position in NpSxph where it interacts with both N-lobe subdomains. Consequently, the interaction between the C-lobe β-strand β7C1 and Thy1-2 β5 observed in RcSxph is absent in NpSxph. Despite these domain-scale differences, Thy1-1 and Thy1-2 are structurally similar to each other (RMSD Cα = 1.056 Å) and to their RcSxph counterparts (SI Appendix, Fig.  S10H) (RMSD Cα = 1.107 Å and 0.837, respectively). Further, none of these large-scale changes impact the STX binding site, which is found on the C1 domain as in RcSxph (Fig. 5A).
Comparison of the apo-and STX-bound NpSxph structures shows that there are essentially no STX binding site conformational changes upon STX engagement, apart from the movement of Asp786 to interact with the STX five-membered guanidinium ring (Fig. 6B and Movie S4). This conformational change is shared with RcSxph (10) and appears to be a common element of Sxph binding to STX. The movements of Tyr558 and its loop away from the STX binding site observed in RcSxph (10) are largely absent in NpSxph for the Tyr558 equivalent position, Ile559, and its supporting loop. Hence, the NpSxph STX binding site is better-organized to accommodate STX (Fig. 6B), similar to RcSxph Y558I (Fig. 3B). We also noted an electron density in the apo-NpSxph STX binding site that we assigned as a PEG400 molecule from the crystallization solution (SI Appendix, Fig. S10A). This density occupies a site different from STX and is not present in the STX-bound complex (SI Appendix, Fig. S10B). Its presence suggests that other molecules may be able to bind the STX binding pocket. Similar to RcSxph, the NpSxph STX binding site is very electronegative (SI Appendix, Fig. S11), consistent with the strong conservation of the E540 and D794 positions (Fig. 5 A and B) that are the most energetically important and coordinate the positively charged STX bis-guanidinium core (Fig. 2 A and B and Table 1).
We also determined the structure of an NpSxph:F-STX complex at 2.2-Å resolution (SI Appendix, Table S1). This structure shows no density for the fluorescein moiety and has an identical STX pose to the NpSxph:STX complex (SI Appendix, Fig. S12). These data provide further evidence that fluorescein does not interact with Sxph (cf. SI Appendix, Fig. S3) even though it is tethered to the STX binding pocket and support the idea that the FP assay faithfully reports on Sxph:STX interactions. Comparison of the NpSxph and RcSpxh STX poses shows essentially identical interactions with the tricyclic bis-guanidinium core and reveals that the carbamate is able to occupy the pocket opened by the Y!I variant (Fig. 6C), as observed in RcSxph Y558I (Fig. 3B). This change, together with the more rigid nature of the NpSxph STX binding pocket, likely contributes to the higher affinity of NpSxph for STX relative to RcSxph (Table 1). Taken together, the various structures of Sxph:STX complexes show how subtle changes, particularly at the Tyr558 position, can influence STX binding and underscore that knowledge of the STX binding code can be used to tune the STX binding properties of different Sxphs.

Discussion
Our biochemical and structural characterization of a set of RcSxph mutants and Sxphs from diverse anurans reveals a conserved STX recognition code centered around six amino acid residues comprising two triads. One triad engages the STX bis-guanidinium core using carboxylate groups that coordinate each ring (RcSxph Glu540 and Asp794) and an aromatic residue that makes a cation-π interaction (RcSxph Phe784) with the STX concave face. This recognition motif is shared with Na V s, the primary target of STX in PSP (18,39,40) (Fig. 2 C and D), and showcases a remarkably convergent STX recognition strategy for the tricyclic, bis-guandinium STX core. The second amino acid triad (RcSxph residues Tyr558, Phe561, and Pro727) largely interacts with the carbamate moiety and contains a site, Tyr558, and its supporting residue, Ile782, where amino acid changes, including those found in some anuran Sxphs (Fig. 5), enhance STX binding. Structural studies of RcSxph mutants and the High Himalaya frog NpSxph show that STX-affinity-enhancing substitutions in this area of the binding pocket act by reducing the degree of conformational change associated with STX binding (Figs. 3 and 6 C and D). These findings reveal one strategy for creating high-affinity STX binding sites. Importantly, enhancing the affinity of Sxph for STX through changes at either residue increases the capacity of RcSxph to rescue Na V s from STX block (Fig. 4), demonstrating that an understanding of the STX recognition code enables rational modification of Sxph binding properties. Thus, exploiting the information in the STX recognition code defined here should enable design of Sxphs as STX sensors or agents for treating STX poisoning. Although STX binding activity has been reported in a variety of diverse invertebrates (13) and vertebrates (13,23), only two types of STX binding proteins have been identified and validated, frog Sxphs (8,9) and the pufferfish STX and TTX binding proteins (41,42). Our discovery of a set of 10 new Sxphs that bind STX with high affinity (Fig. 5 and SI Appendix, Figs. S7 and S8) that share a conserved STX binding site represents a substantial expansion of the Sxph family and reveals natural variation in the residues that are important for STX binding (Fig. 5B). Most notably, E540D, a change that reduces STX binding in RcSxph by ∼14-fold, occurs in five of the newly identified Sxphs. Nevertheless, functional studies show that RiSxph, which bears an Asp at this site, binds STX more strongly than RcSxph (Table 2). Hence, the natural variations at other STX binding pocket residues must provide compensatory interactions to maintain a high STX binding affinity. Understanding how such variations impact STX engagement or influence the capacity of these proteins to discriminate among STX congeners (13) remain important unanswered questions. The striking abundance of Sxphs in diverse amphibians, representing lineages separated by ∼140 My (25) and that are not known to carry STX, raises intriguing questions regarding the selective pressures that have caused these disparate amphibians to maintain this STX binding protein and its capacity to sequester this lethal toxin.
Besides the conserved STX binding site, all of the amphibian Sxphs possess a set of Thy1 domains similar to those in RcSxph that have been shown to act as protease inhibitors (38). Comparison of anuran Sxphs shows that these domains are a common feature of the Sxph family and occur in strikingly varied numbers, comprising two or three in most Sxphs but having a remarkable expansion to 15 to 16 in some toad Sxphs (Fig. 5A and SI Appendix, Fig. S7-S9). Structural comparisons between RcSxph and NpSxph, representing the class that has two Thy1 domains (Fig. 5A), show that these domains can adopt different positions with respect to the shared, modified transferrin core (SI Appendix, Fig. S10F). Whether the Sxph Thy1 domains and their varied numbers are important for Sxph-mediated toxin resistance mechanisms (8,9) or serve some other function remains unknown. Our definition of the Sxph STX binding code, which provides a guide for deciphering variation in the Sxph STX binding site (Fig. 5B), and discovery of high variability in Thy1 repeats among anuran Sxphs constitutes a template for identifying other Sxphs within this widespread and diverse family of amphibians.
STX interacts with a variety of target proteins including select Na V isoforms (43), other channels (44,45), diverse soluble STX binding proteins (8,10,41,42,46,47), and some enzymes (3,48,49). The identification of the Sxph STX recognition code together with the substantial expansion of the Sxph family provides a foundation for developing a deeper understanding of the factors that enable proteins to bind STX. Exploration of such factors should be facilitated by the assays established here that allow Sxph:STX interactions to be probed using a range of sample quantities (TF: 25 μg Sxph, 600 ng STX; FP: 3 μg Sxph, 1 ng F-STX; ITC: 300 μg Sxph, 5 μg STX) and that should be adaptable to other types of STX targets. In cases of limited samples, such as difficult to obtain STX congeners, the excellent agreement among the assays should provide a reliable basis for interpretation of Sxph binding properties. Our delineation of the Sxph STX binding code and discovery of numerous Sxph family homologs among diverse amphibians set a broad framework for understanding the lethal effects of this potent neurotoxin and "toxin sponge" STX resistance mechanisms (8,9). This knowledge may enable the design of novel PSP toxin sensors and agents that could mitigate STX intoxication.

Materials and Methods
R. catesbeiana Sxph (RcSxph), N. parkeri Sxph (NpSxph), M. aurantiaca Sxph (MaSxph), O. sylvatica Sxph (OsSxph), and R. imitator Sxph (RiSxph), and mutants were expressed and purified using a previously described RcSxph baculovirus expression system (10). TF assays for STX and TTX binding were developed as outlined in ref. 27. FP assays were performed as described (50). Structure determination was done following previously described methods for RcSxph (10). Two-electrode voltage clamp experiments evaluating RcSxph rescue of Na V s from STX block were done as previously described (9). Details on cloning, expression, purification, binding assays, electrophysiology, structure determination, and F-STX synthesis can be found in SI Appendix.