Structure of the saxiphilin:saxitoxin (STX) complex reveals a convergent molecular recognition strategy for paralytic toxins

Abstract

Dinoflagelates and cyanobacteria produce saxitoxin (STX), a lethal bis-guanidinium neurotoxin causing paralytic shellfish poisoning. A number of metazoans have soluble STX-binding proteins that may prevent STX intoxication. However, their STX molecular recognition mechanisms remain unknown. Here, we present structures of saxiphilin (Sxph), a bullfrog high-affinity STX-binding protein, alone and bound to STX. The structures reveal a novel high-affinity STX-binding site built from a "proto-pocket" on a transferrin scaffold that also bears thyroglobulin domain protease inhibitor repeats. Comparison of Sxph and voltage-gated sodium channel STX-binding sites reveals a convergent toxin recognition strategy comprising a largely rigid binding site where acidic side chains and a cation-π interaction engage STX. These studies reveal molecular rules for STX recognition, outline how a toxin-binding site can be built on a naïve scaffold, and open a path to developing protein sensors for environmental STX monitoring and new biologics for STX intoxication mitigation.

Fig. 1. Sxph structure.

(A) R. catesbeiana Sxph:STX: complex ribbon diagram. Domains are indicated and are colored as follows: N1 (smudge), N2 (limon), thyroglobulin (Thy; bright orange), C1 (marine), and C2 (cyan). STX (red) is shown as space filling. (B) Superposition of Sxph and rabbit transferrin [Protein Data Bank (PDB): 1JNF] (32). Transferrin N-lobe and C-lobe are colored purple and pink, respectively. Sxph Thy1 repeats are not shown. Insets show transferrin Fe³⁺ ligands and Sxph equivalents as sticks. N domain: transferrin (purple) and Sxph (green); C domain: transferrin (pink) and Sxph (blue). STX (red) is shown as space filling. Right hand inset shows distance between the STX center and transferrin Fe³⁺. (C) Cartoon diagram showing unique Sxph disulfide bonds in space filling representation: SS3 (Cys²⁷ to Cys⁴¹⁷) SS4 (Cys⁹¹ to Cys¹¹¹), SS5 (Cys¹²² to Cys¹²⁹), SS6 (Cys¹³¹ to Cys¹⁵³), SS7 (Cys¹⁶¹ to Cys¹⁸³), SS8 (Cys²⁰³ to Cys²²⁵), and SS9 (Cys²³⁴ to Cys⁸²⁵). Colors and labels are the same as in (A).

Fig. 2. Comparison of Sxph Thy1-1 and Thy1-2 with p41 Ii.

(A) Sequence comparison. Thy1-1 and Thy1-2 secondary structure elements and disulfide bonds are indicated. Cysteines and conserved residues are highlighted yellow and blue, respectively. (B) Cartoon diagram superposition of Thy1-1 (light orange), Thy1-2 (marine), and p41 Ii (magenta) (PDB: 1ICF) (38). Disulfide bonds (italics) and select residues are labeled. Thy1-1 and Thy1-2 have root mean square deviation of Cα position (RMSD_Cα) = 0.61 and 0.64 Å over 43 and 42 residues, respectively. p41 Ii has RMSD_Cα = 0.57 Å over 53 residues of Sxph Thy1-1 and Thy1-2. (C and D) Superposition of Sxph on the p41 Ii:cathespin L complex (PDB: 1ICF) (38) using the (C) Thy1-1 and (D) Thy1-2 domains. In (D), red oval indicates cathepsin L and Sxph C1 clash. Sxph colors are the same as in Fig. 1A. (E) Superposition of Sxph Thy1-1 (light orange), Thy1-2 (marine), and p41 Ii (magenta) in the context of the p41 Ii:cathepsin L interface.

Fig. 3. Sxph STX-binding site.

(A) Apo-Sxph (olive) and STX-bound Sxph (slate) superposition cartoon diagram. STX-interacting residues are shown as sticks. Key secondary structure elements are labeled. Black and gray dashed lines indicate hydrogen bond networks and the cation-π interaction, respectively. STX is shown as red sticks. Gdm-5, Gdm-6, and HK indicate the five- and six-membered guanidinium rings and hemiketal, respectively. (B) STX-binding site highlighting the cation-π interaction (gray) and Asp⁷⁸⁵ movement. (C) LIGPLOT diagram of the STX-binding site. α6C1 is shown for orientation.

Fig. 4. Sxph STX-binding site and transferrin proto-pocket.

(A and B) Superposition of the Sxph (marine) STX-binding site with (A) Fe³⁺-bound rabbit serum transferrin (PDB: 1JNF) (41) (pink) and (B) apo-human serum transferrin (PDB: 2HAU) (32) (gray). STX (red) is shown as sticks. Select residues are shown. Blue labels indicate Sxph residues. Orange arrows indicate changes between transferrin and Sxph. (C to E) Transferrin proto-pocket and Sxph STX-binding pocket comparisons. (C) to (E) show apo-transferrin (pink), transferrin (gray), and Sxph (marine) surfaces, respectively. In (C) and (D), labels indicate Sxph residues that break through the transferrin surface. Red circle highlights the STX-binding site. STX is shown as space filling. Sxph surface is colored by atom type, where red and blue denote oxygen and nitrogen, respectively.

Fig. 5. Sxph and Na_Vs share STX recognition strategies.

(A) Na_VPaS:STX (PDB: 6a91) (29) and Sxph:STX STX-binding site superposition. Na_VPaS is shown as a cartoon viewed from the central channel cavity. Pore domains are colored as follows: DI, green; DII, orange; DIII, yellow; and DIV, pink. STX coordinating and selectivity filter “DEKA” motif (white) residues are shown as sticks. Sxph STX-binding site side chains are blue. STX from Sxph:STX (red) and Na_VPaS:STX (cyan) are superposed. (B) Closeup view of the STX-binding sites from (A). (C) Diagram of the Na_VPaS:STX interactions. (D) Diagram of the Sxph:STX interactions. (E) Comparison of common STX interactions for Sxph (blue), Na_VPaS (green), and Na_V1.7 (magenta). STX from the Sxph:STX complex (red), Na_VPaS:STX complex (cyan), and Na_V1.7:STX complex (violet) are indicated. (C) and (D) were generated using LIGPLOT (67) and a 3.35-Å cutoff. Hydrogen bonding networks (black dashed lines) and cation-π interactions (gray dashed lines) are indicated. (D) is the same as Fig. 3C.