Crystal structure of the complex between venom toxin and serum inhibitor from Viperidae snake

Abstract

Venomous snakes have endogenous proteins that neutralize the toxicity of their venom components. We previously identified five small serum proteins (SSP-1-SSP-5) from a highly venomous snake belonging to the family Viperidae as inhibitors of various toxins from snake venom. The endogenous inhibitors belong to the prostate secretory protein of 94 amino acids (PSP94) family. SSP-2 interacts with triflin, which is a member of the cysteine-rich secretory protein (CRISP) family that blocks smooth muscle contraction. However, the structural basis for the interaction and the biological roles of these inhibitors are largely unknown. Here, we determined the crystal structure of the SSP-2-triflin complex at 2.3 Å resolution. A concave region centrally located in the N-terminal domain of triflin is fully occupied by the terminal β-strands of SSP-2. SSP-2 does not bind tightly to the C-terminal cysteine-rich domain of triflin; this domain is thought to be responsible for its channel-blocker function. Instead, the cysteine-rich domain is tilted 7.7° upon binding to SSP-2, and the inhibitor appears to sterically hinder triflin binding to calcium channels. These results help explain how an endogenous inhibitor prevents the venomous protein from maintaining homeostasis in the host. Furthermore, this interaction also sheds light on the binding interface between the human homologues PSP94 and CRISP-3, which are up-regulated in prostate and ovarian cancers.

Keywords: calcium channel; crystal structure; inhibitor; protein complex; serum; snake venom.

Figure 1.

Crystal structure of the SSP-2–triflin complex. A, cartoon representation of the SSP-2–triflin complex structure (left). SSP-2 is shown in orange; triflin is shown in pale green. The β-strands involved in the interaction are highlighted in red for SSP-2 (β1 and β5) and in dark green for triflin (β4). Disulfide bonds that are conserved among PSP94 family proteins are represented with sticks. The sulfur atoms are indicated in yellow. The disordered regions of SSP-2 (Ser¹⁰–Pro¹⁷) and triflin (Gln¹⁹⁷–Asn²⁰⁴) in the crystal structure are indicated with a dotted line. The surface representation of the SSP-2–triflin complex is shown (top right). The left model is the same view as the cartoon representation on the left, whereas the right model represents a view rotated by 90° around a vertical axis. The enlarged view (bottom right) shows the 2F_o − F_c electron density map of SSP-2 contoured at 2.0 σ (sky blue) at the interface with triflin. The structure of the complex shows that β5 (Leu⁵⁹–Glu⁶¹) of SSP-2 forms a parallel β-sheet structure with β4 of triflin to interact with the toxin. B, sequence alignment of SSP-1 to SSP-5 from P. flavoviridis and the PSP94 family protein human PSP94. Universal Protein Resource (UniProt) accession numbers are as follows: A7VN13 (SSP-1), A7VN14 (SSP-2), A7VN15 (SSP-3), A7VN16 (SSP-4), and A7VN17 (SSP-5) from P. flavoviridis; P08118 (PSP94) from Homo sapiens. Highly conserved residues are shown in white font on a red background, and other conserved residues are shown in red font. Cysteine residues forming disulfide bridges are indicated below the alignment with a light green number. The same number indicates the paired residues for the disulfide bond. The secondary structures of SSP-2 and PSP94 obtained from the SSP-2–triflin complex and the PSP94 crystal structure (22) are shown above and below the alignment, respectively. Black arrow indicates β-strand. T indicates a β-turn. The alignment figure was generated using Esprit (35). The residues whose side chains are involved in the interaction between SSP-2 and triflin are indicated with double circles. The residue numbers used throughout the manuscript are derived from this sequence alignment. Missing indicates a disordered region. The box with dashed lines indicates the β-strands that form an interchain β-sheet with triflin. The residues involved in the interaction with CRISP identified by the NMR experiment (20) are indicated with diamonds below the alignment. Natural variants of PSP94 are also indicated with a yellow box. C, cartoon representation of the SSP-2 structure in the SSP-2–triflin complex. The orientation of SSP-2 is a view rotated 180° around a vertical axis, as shown in A. Conserved disulfide bonds are shown as ball and stick models with residue numbers in purple. See also Fig. S1 and Table S1.

Figure 2.

Binding interface between SSP-2 and triflin. SSP-2 is shown as a cartoon model, whereas triflin is shown as a surface model. The complex structure is the same view as in the top right panel of Fig. 1A, showing the binding mode of SSP-2 in the cleft of triflin. A–D, the boxed regions are shown as detailed views. A, focused view of the β1 strand of SSP-2. Black dotted lines indicate hydrogen bonds. B, detailed view of the charge-charge interactions. The ion pair and hydrogen bonds are indicated with black dotted lines. A weak ion pair (>4.0 Å distance) between Asp⁴⁸ of SSP-2 and Lys⁷² of triflin is also shown. C, detailed view of the C-terminal β-sheet formed by the SSP-2 β5 strand and the triflin β4 strand. Black dotted lines indicate hydrogen bonds, and related residues are shown as the stick model. The residues involved in the hydrophobic interaction are also indicated with the stick model. D, detailed view of the cation-π interaction between Lys¹³⁸ and Tyr¹³⁹ of triflin. Black dotted lines indicate hydrogen bonds. See also Fig. S2 and Table S2.

Figure 3.

PSP94–CRISP–binding model. A, the partial N-terminal structure of the PSP94 family protein human PSP94 (PDB ID 3IX0) is superimposed on the structure of SSP-2 in complex with triflin, as determined in this study. The color of each molecule is the same as in Fig. 2 and Fig. S1A. The enlarged view shows which β-strands align to form interaction surface. The PSP94–CRISP-3 model based on NMR titration experiments show that the N-terminal Greek key motif and C-terminal β8 strand of PSP94 interact with the N-terminal PR-1 domain and hinge region of CRISP-3, respectively, in a parallel manner (20). In contrast, our structure shows an upside-down, anti-parallel orientation, although the same side of the PSP94-family protein interacts with the same concave surface of the CRISP protein (Fig. 1A). B, the triflin-interacting residues are mapped on the SSP-2 with the stick model in yellow. C, CRISP-interacting residues identified by the NMR titration experiment (20) are mapped on PSP94 (PDB ID: 3IX0) with the stick model in pink. D, CRISP-interacting residues identified by the coimmunoprecipitation experiment using alanine or deletion mutants (21) are mapped on PSP94 with the stick model in purple. Disrupting the disulfide bond between the N- and C-terminal domains of PSP94 alters the unusual domain orientation, which indirectly abolishes the CRISP-interacting activity. The binding interface is located on the same side of the PSP94-family protein molecule, mainly composed of β1, β4, and β5 (for SSP-2) or β8 (for PSP94). E, the natural variants reported on the UniProt database (http://www.uniprot.org/) are mapped on PSP94 with the stick model in yellow. (Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site.) Two of the residues, I5M and D60A, are located within the CRISP-binding interface. It is of note that seven other SNPs are found at the opposite side of the CRISP-binding interface. See also Figs. S3 and S5.