Structures of pseudechetoxin and pseudecin, two snake-venom cysteine-rich secretory proteins that target cyclic nucleotide-gated ion channels: implications for movement of the C-terminal cysteine-rich domain

Abstract

Cyclic nucleotide-gated (CNG) ion channels play pivotal roles in sensory transduction by retinal photoreceptors and olfactory neurons. The elapid snake toxins pseudechetoxin (PsTx) and pseudecin (Pdc) are the only known protein blockers of CNG channels. These toxins belong to a cysteine-rich secretory protein (CRISP) family containing an N-terminal pathogenesis-related proteins of group 1 (PR-1) domain and a C-terminal cysteine-rich domain (CRD). PsTx and Pdc are highly homologous proteins, but their blocking affinities on CNG channels are different: PsTx blocks both the olfactory and retinal channels with approximately 15-30-fold higher affinity than Pdc. To gain further insights into their structure and function, the crystal structures of PsTx, Pdc and Zn2+-bound Pdc were determined. The structures revealed that most of the amino-acid-residue differences between PsTx and Pdc are located around the concave surface formed between the PR-1 domain and the CRD, suggesting that the concave surface is functionally important for CNG-channel binding and inhibition. A structural comparison in the presence and absence of Zn2+ ion demonstrated that the concave surface can open and close owing to movement of the CRD upon Zn2+ binding. The data suggest that PsTx and Pdc occlude the pore entrance and that the dynamic motion of the concave surface facilitates interaction with the CNG channels.

Figure 1

Sequence alignment of known structures of snake-venom CRISP-family proteins. Identical residues are shaded in grey. The residues involved in divalent-ion binding and Na⁺ binding are highlighted in red and in blue, respectively. All cysteine residues are shown as inverse characters and residues involved in disulfide bonds are connected by lines. The locations of the regular secondary-structure elements identified in PsTx are depicted at the top (α-helices and β-strands). The numbering for PsTx is indicated above the sequence. The seven different residues between PsTx and Pdc are shown in pink and the residues important for CNG-channel blockage are marked with dots.

Figure 2

(a) Stereoview of the structural comparison between PsTx and Pdc. Ribbon models of PsTx and Pdc are shown in cyan and magenta, respectively. Amino-acid residues that differ between PsTx and Pdc are indicated by side chains and are labelled. The Na⁺ ion-binding site at the bottom is shown with the side chains of the coordinated residues (labelled) and the bound Na⁺ ion as a purple sphere. (b) A close-up view of the Na⁺ ion-coordination geometries in Pdc with 2F _o − F _c electron-density map contoured at 1.5σ.

Figure 3

Domain movement and Zn²⁺-binding sites. (a) Stereoview of the superposition of snake-venom CRISP-family proteins (PsTx, cyan; Pdc, magenta; triflin, green; stecrisp, yellow; natrin, orange). When the only PR-1 domains are superposed, changes or movement of the CRD domains are visible relative to the PR-1 domain. (b) Pdc (magenta) is superposed with Zn-Pdc (molecule B, light grey; molecule D, dark grey) and Zn-1 is shown as a green sphere. (c) As (b) but superposed with Zn-Pdc (molecule A, light grey; molecule C, dark grey). The Zn-2 bound to molecule A and to molecule C is shown as a green and a dark green sphere, respectively. (d) Zn-1 coordination (left) and a close-up view with 2F _o − F _c electron-density map contoured at 1.0 in Zn-Pdc (right). (e) Zn-2 coordination between molecules A and C in Zn-Pdc (left) and a close-up view with 2F _o − F _c electron-density map (right).

Figure 3

Domain movement and Zn²⁺-binding sites. (a) Stereoview of the superposition of snake-venom CRISP-family proteins (PsTx, cyan; Pdc, magenta; triflin, green; stecrisp, yellow; natrin, orange). When the only PR-1 domains are superposed, changes or movement of the CRD domains are visible relative to the PR-1 domain. (b) Pdc (magenta) is superposed with Zn-Pdc (molecule B, light grey; molecule D, dark grey) and Zn-1 is shown as a green sphere. (c) As (b) but superposed with Zn-Pdc (molecule A, light grey; molecule C, dark grey). The Zn-2 bound to molecule A and to molecule C is shown as a green and a dark green sphere, respectively. (d) Zn-1 coordination (left) and a close-up view with 2F _o − F _c electron-density map contoured at 1.0 in Zn-Pdc (right). (e) Zn-2 coordination between molecules A and C in Zn-Pdc (left) and a close-up view with 2F _o − F _c electron-density map (right).

Figure 4

Sequence comparison of CNG with K⁺ and NaK channels. The amino-acid residues of CNGA2, containing the pore turret, are compared with those of KcsA, KvAP, Kv1.2 and NaK, which have known structures. The turret sequence is shown in red; the outer helix and the pore helix are located on the left and right sides of the turret region, respectively. Hydrophobic residues with some homology are shown in blue. The SF (selectivity filter) sequence is shown in orange. Asp316, Tyr321 and Glu325 in CNGA2, shown in green, are important residues for the interaction with PsTx.

Figure 5

Interaction between PsTx and the CNG pore turret. (a) Surface-potential model of PsTx compared with that of Pdc. Amino-acid residues that differ between PsTx and Pdc are indicated by labels. (b) Docking model of the interaction between the CNG channel and PsTx. Helical models (cylinders) in the CNG channel were prepared by using those of the Kv1.2 channel and the turret regions are shown by dotted lines, in which acidic residues are shown in red. Thus, this CNG-channel model is likely to correspond to the homotetrameric CNGA2 model. The concave surface of PsTx is shown at the bottom for the interaction with the pore turret, where only the different amino-acid residues between PsTx and Pdc are highlighted in green, with labels for clarity.

Figure 6

The isolated CRD of PsTx is not an effective blocker of CNGA2. The currents elicited by −50 mV pulses were recorded from HEK-293 cells expressing CNGA2. PsTx (100 nM) or its isolated CRD (12 µM) was bath applied. The application of Mg²⁺ blocks >98% of the current through CNGA2, indicating the ‘leak’ current through the seal resistance. This trace is representative of results from four cells.