Structural Basis of Neutralization of the Major Toxic Component from the Scorpion Centruroides noxius Hoffmann by a Human-derived Single-chain Antibody Fragment

Abstract

It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 Å resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complex reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of ␤-toxins to its Na ؉ channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs.The most harmful components of scorpion venoms are toxins that can selectively bind to voltage-gated ion channels and affect their modulation. A subgroup among these toxins, the long chain Na ϩ channel toxins, are formed by 60 -70-amino acid peptides, which adopt a highly packed core with a ␤␣␤␤-fold (1). These Na ϩ channel toxins have further been classified into two major classes (␣ and ␤) based on their effects in the gating mechanism of Na ϩ channels and their properties (2-4). The old world toxin II from Androctonus Australia Hector (AaHII) and the new world toxin II from Centruroides suffusus suffusus (Css2) 4 are considered to be the archetypes of ␣-and ␤-toxins active against mammals, respectively (3, 5).The buthid scorpion, C. suffusus suffusus, is one of the most poisonous to the rural human population in the Northwest of Mexico. The most noxious and abundant molecule found in the venom of this scorpion is Css2 (LD 50 of 0.7 g/20 g of mice of the strain CD1) (6). In addition, the Mexican scorpion Centruroides noxius Hoffmann produces toxin Cn2, one of the most abundant and noxious peptides against mammals (LD 50 of 0.25 g/20 g of mice of the strain CD1) (7). Sequence comparison shows that Cn2 presents a high similarity with other ␤-toxins from C. suffusus suffusus, such as Css2 and Css4 (around 90%, see below). Both toxins, Cn2 and Css2, are specific to Na ϩ channel subtype Na v 1.6 (6, 8).Due to the fact that scorpion stings are a considerable public health issue in a number of countries (9), efforts have been put forth to explore alternative technologies to generate more specific antibodies against the venom of scorpions harmful to humans (reviewed in Ref. 10). One of the most promising alternatives to classical...

Optimal Neutralization of Centruroides noxius Venom Is Understood through a Structural Complex between Two Antibody Fragments and the Cn2 Toxin

“…3A). The structure of the Cn2 toxin in the ternary complex is virtually the same as that in the 9004G-Cn2 crystallographic structure (14), with a root mean square deviation between both Cn2 toxin structures of only 0.507 Å. This is also similar to the NMR solution structure already reported for this toxin (38).…”

“…2), which explained the complementary neutralization activity of the two scFvs (RU1 and LR), prompting us to identify the second interaction site on the Cn2 toxin. The first binding site on the Cn2 toxin, which was recognized by scFv LR and was confirmed in this work, allowed us to reinforce the available information related to the toxin areas that interact with the sodium channel and the proposed neutralization mechanisms (14,40). The new binding site on the Cn2 toxin, which was structurally opposite the one recognized by scFv LR, is composed of the N-terminal segment, including residues Leu 5 , Val .…”

“…scFv LR (derived from 9004G) maintained the same binding site on the Cn2 toxin in the ternary complex compared with that in the binary complex scFv 9004G-Cn2, as reported previously (14). A new epitope, which was recognized by the scFv RU1, was identified.…”

“…3A). An important observation is that the contribution of the V H domains is far superior to that of the V L domains for both scFv interactions with the The scFv LR Binding Site on the Cn2 Toxin-scFv LR is located at the same place on the Cn2 toxin as its predecessor (9004G) (14). Specifically, the binding site of scFv LR is located near the N terminus of the ␣-helical region of the Cn2 toxin, and the scFv RU1 binding site is located near the C terminus of this helix (Figs.…”

Evaluation of three different formats of a neutralizing single chain human antibody against toxin Cn2: Neutralization capacity versus thermodynamic stability

“…The interactions in the V H -V L interface area were analyzed in all the models and compared with the structure of the scFv 9004G, an antibody that neutralizes the Cn2 toxin, which present high homology with the scFv 6009F [43]. In this interface zone, all the structures from the scFv 9004G showed that two residues of the CDR L3 (Q221 and R228) as well as one of the framework-2 (Y168) interact, by hydrogen bonds, with two residues of the CDR H3 (G102 and F104), when the Cn2 toxin is present (Fig.…”