Crystal structure of Natratoxin, a novel snake secreted phospholipaseA2 neurotoxin from Naja atra venom inhibiting A‐type K⁺ currents

Abstract

Snake secreted phospholipasesA2 (sPLA2s) are widely used as pharmacological tools to investigate their role in diverse pathophysiological processes. Some members of snake venom sPLA2s have been found to block voltage-activated K(+) channels (K(v) channels). However, most studies involved in their effects on ion channels were indirectly performed on motor nerve terminals while few studies were directly done on native neurons. Here, a novel snake sPLA2 peptide neurotoxin, Natratoxin, composed of 119 amino acid residues and purified from Naja atra venom was reported. It was characterized using whole-cell patch-clamp in acutely dissociated rat dorsal root ganglion (DRG) neurons. It was found to effectively inhibit A-type K(+) currents and cause alterations of channel gating characters, such as the shifts of steady-state activation and inactivation curves to hyperpolarization direction and changes of V(1/2) and slope factor. Therefore, Natratoxin was suggested to be a gating modifier of K(v) channel. In addition, this inhibitory effect was found to be independent of its enzymatic activity. These results suggested that the toxin enacted its inhibitory effect by binding to K(v) channel. To further elucidate the structural basis for this electrophysiological phenomenon, we determined the crystal structure of Natratoxin at 2.2 A resolution by molecular replacement method and refined to an R-factor of 0.190. The observed overall fold has a different structural organization from other K(+) channel inhibitors in animal toxins. Compared with other K(v) channel inhibitors, a similar putative functional surface in its C-terminal was revealed to contribute to protein-protein interaction in such a blocking effect. Our results demonstrated that the spatial distribution of key amino acid residues matters most in the recognition of this toxin towards its channel target rather than its type of fold.

Crystal structure of phospholipase PA2‐Vb, a protease‐activated receptor agonist from the Trimeresurus stejnegeri snake venom

“…In the vessel tension measurements, PA2-Vb was able to induce a contractile response, in both high K + and normal solutions. This vasoconstriction effect was also found in other snake toxins, such as natratoxin, a secreted PLA 2 from Naja atra venom which can effectively inhibit A-type K + currents [12] and AhV_TL-I, a thrombin-like enzyme from A. halys pallas which acts on RyRs to evoke Ca 2+ release [37]. In this study, the results show that the intracellular Ca 2+ release mediated by IP 3 R results in vasoconstriction.…”

“…Functionally, PLA 2 specifically recognizes the sn-2 acyl bond of phospholipids and catalytically hydrolyzes the bond to release arachidonic acid and lysophospholipids involved in many critical biological processes [14,30]. In snake venom, PLA 2 (svPLA 2 ) also inhibits A-type K + currents in rat dorsal root ganglion neurons [12]. Several crystal structure studies suggest that most svPLA 2 s share a common set of structural features, including their small molecular mass (14-18 kDa) and a compact structure with seven conserved disulfide bonds and an important Ca 2+ -loop [21].…”

Snake Venom: A Promising Source of Neurotoxins Targeting Voltage-Gated Potassium Channels

“…Additionally, there are three helical short turns including residues 19–21 (SH4), 107–109 (SH5), and 114–116 (SH6). The residues spanning from 69 to 72 and 75 to 78 exhibit a double-stranded antiparallel β-sheet structure [ 140 ].…”

Kalium 3.0 is a comprehensive depository of natural, artificial, and labeled polypeptides acting on potassium channels

“…Several proteins and enzymes were also claimed to present activity on K + channels. For instance, two high‐molecular‐mass components of Naja atra snake venom, natrin and natratoxin (snake‐secreted phospholipases A2) target K Ca 1.1 and K V 1 channels, respectively (Hu et al, 2008; Wang et al, 2006). Depending on the source, polypeptides can also be post‐translationally modified (Wilkins et al, 1999).…”