Mambalgin-1 Pain-relieving Peptide, Stepwise Solid-phase Synthesis, Crystal Structure, and Functional Domain for Acid-sensing Ion Channel 1a Inhibition

Abstract

Mambalgins are peptides isolated from mamba venom that specifically inhibit a set of acid-sensing ion channels (ASICs) to relieve pain. We show here the first full stepwise solid phase peptide synthesis of mambalgin-1 and confirm the biological activity of the synthetic toxin both in vitro and in vivo. We also report the determination of its three-dimensional crystal structure showing differences with previously described NMR structures. Finally, the functional domain by which the toxin inhibits ASIC1a channels was identified in its loop II and more precisely in the face containing Phe-27, Leu-32, and Leu-34 residues. Moreover, proximity between Leu-32 in mambalgin-1 and Phe-350 in rASIC1a was proposed from double mutant cycle analysis. These data provide information on the structure and on the pharmacophore for ASIC channel inhibition by mambalgins that could have therapeutic value against pain and probably other neurological disorders.

Acid-sensing ion channel (ASIC) structure and function: Insights from spider, snake and sea anemone venoms

“…Two high-resolution NMR structures have been published and are in good agreement with one another, with the lower part of loop 2 showing some flexibility within the NMR ensemble (Pan et al, 2014;Schroeder et al, 2014). More recently crystal structures of two Ma-1 polymorphs were determined, showing significant structural variations compared to the NMR structures (Mourier et al, 2016) (see Figure 2B). In both crystal structures the lower part of loop 2 is extended and well defined and there was a large degree of conformational variability in loop 3.…”

“…However several elegant strategies have been developed to synthesise the peptide for structural and functional studies (Mourier et al, 2016;Pan et al, 2014;Schroeder et al, 2014). Two high-resolution NMR structures have been published and are in good agreement with one another, with the lower part of loop 2 showing some flexibility within the NMR ensemble (Pan et al, 2014;Schroeder et al, 2014).…”

“…The pharmacophore of Ma-1 has also been partially determined with alanine mutants only made so far in loop 2. Mutating residues Phe27, Arg28, Leu32, Ile33, and Leu34 induced a significant decrease in potency, and this face of the peptide was determined to be the ASIC-interacting surface (Mourier et al, 2016) (Figure 3F), in contrast to the results of a prior docking-based prediction by the same group (Salinas et al, 2014).…”

“…A third peptide, Ma-3, from the green mamba (Dendroaspis augusticeps) was also isolated and has a single mutation at position 23 when compared to Ma-1 (Baron et al, 2013) (Figure 2A). These peptides conform to the threefinger toxin fold ( Figure 2B) and inhibit homomeric ASIC1a (rat and human IC 50 3-55 and 127 nM, respectively) and rASIC1b , as well as ASIC1a-and ASIC1b-containing heteromers with weaker potency (Diochot et al, 2012;Mourier et al, 2016;Schroeder et al, 2014). Mambalgins inhibit ~60% of ASIC currents in rat sensory neurons, whereas PcTx1 only inhibits ~40%, most likely due to the broader activity profile of mambalgins through ASIC1b containing channels (Diochot et al, 2012).…”

Conformational dynamics and role of the acidic pocket in ASIC pH-dependent gating

“…Although mutation of AcP Glu and Asp residues shifts the pH dependence of ASIC activation to more acidic values (5,(10)(11)(12), H + -sensing residues have also been identified outside the AcP (10,12,13), indicating that the AcP is not the only extracellular pH-sensing domain. Its importance is, however, underlined by the fact that it constitutes the binding site of several ASIC-specific toxins (7,14).…”