The Target Selects the Toxin: Specific Amino Acids in Snake-Prey Nicotinic Acetylcholine Receptors That Are Selectively Bound by King Cobra Venoms

Abstract

Snake venom is an adaptive ecological trait that has evolved primarily as a form of prey subjugation. Thus, the selection pressure for toxin diversification is exerted by the prey’s physiological targets, with this pressure being particularly acute for specialist feeders, such as the King Cobra species, all of which are snake-prey specialists. However, while extensive research has been undertaken to elucidate key amino acids that guide toxin structure–activity relationships, reciprocal investigations into the specific sites guiding prey-lineage selective effects have been lacking. This has largely been due to the lack of assay systems amenable to systematic amino acid replacements of targeted proteins in the prey’s physiological pathways. To fill this knowledge gap, we used a recently described approach based upon mimotope peptides corresponding to the orthosteric site of nicotinic acetylcholine receptor alpha-1 subunits, a major binding site for snake venom neurotoxins that cause flaccid paralysis. We investigated the venoms of four different types of King Cobra (Cambodian, Javan, Malaysian, and Thai). This approach allowed for the determination of the key amino acid positions in King Cobra snake prey that are selectively bound by the toxins, whereby replacing these amino acids in the snake-prey orthosteric site with those from lizards or rats resulted in a significantly lower level of binding by the venoms, while conversely replacing the lizard or rat amino acids with those from the snake at that position increased the binding. By doing such, we identified three negatively charged amino acids in the snake orthosteric site that are strongly bound by the positively charged neurotoxic three-finger toxins found in King Cobra venom. This study, thus, sheds light on the selection pressures exerted by a specialist prey item for the evolution of lineage-selective toxins.

High-Voltage Toxin’Roll: Electrostatic Charge Repulsion as a Dynamic Venom Resistance Trait in Pythonid Snakes

“…To test this hypothesis, using our validated biolayer interferometry protocols [ 6 , 26 , 27 , 28 , 42 ], we constructed peptidic mimotopes corresponding to the orthosteric sites ( Figure 2 ) and tested for the relative binding by a diversity of α-neurotoxic snake venoms ( Figure 3 and Figure 4 ). The relative binding affinity followed the relative presence of lysines: A. melanocephalus was bound the strongest by all venoms, consistent with this species retaining both the Pythonidae family’s negatively charged amino acids (191D and 195E) and the lack of any positive charges in the orthosteric site.…”

“…Venom in snakes is a pivotal trait that originally evolved for the incapacitation of prey [ 5 ]. This evolution, driven by the need to overcome the defensive mechanisms of prey species, has resulted in the complex diversification of venom components [ 6 , 7 ]. The selective forces at play are closely linked to the specific physiological systems of the prey, which in turn have spurred the evolution of countermeasures in these species to mitigate the effects of these powerful toxins [ 8 ].…”

“…Steric hindrance arises from structural changes to the orthosteric site of the α-1 subunit of the nAChR. The earliest recognized example of steric hindrance, N-glycosylation, was detected in the Egyptian Mongoose ( Herpestes ichneumon ), a predator of certain cobras [ 6 ]. This trait was later identified as a basal trait of the mongoose/meerkat family (Herpestidae), which includes several snake-consuming species [ 37 , 38 ].…”

“…The prey species Burmese Python has evolved resistance through the replacement of negatively charged amino acid (aspartic acid (D) or glutamic acid (E)) at positions 191 or 195 in the orthosteric site with a positively charged lysine (K) [ 26 , 28 , 39 ]. Introducing positive charges on the key receptor sites leads to electrostatic repulsion [ 26 ] due to the high concentration of positive charges on the surface of the neurotoxic peptides, which themselves evolved, in response, to facilitate the binding to the negatively charged orthosteric site residues [ 6 ]. However, even the elimination of the negativecharge without the replacement by positive charge but with neutral charge, is enough to significantly decrease the binding efficiency of the α-neurotoxins of the snake venom [ 28 ].…”

Resistance Is Not Futile: Widespread Convergent Evolution of Resistance to Alpha-Neurotoxic Snake Venoms in Caecilians (Amphibia: Gymnophiona)

“…The orthosteric site typically contains a negatively charged amino acid (aspartic acid (D) or glutamic acid (E)) at either position 191 or 195 (or both), thus conferring an inherent negatively charged state to the orthosteric site. This adaptation has, in turn, exerted a selection pressure for alpha-neurotoxins to evolve with strongly positively charged molecular surfaces, thereby facilitating the docking of the toxin via an opposite-charge attraction effect [53]. Mutations that reverse the charge at one or both positions by encoding for a positively charged amino acid (such as lysine (K)) confer resistance as the neurotoxins are now electrostatically repulsed by the same-charge interaction, analogous to the repelling effect of attempting to bring the same sides of two magnets together [37] (Figure 1C).…”