Molecular dynamics and functional studies define a hot spot of crystal contacts essential for PcTx1 inhibition of acid‐sensing ion channel 1a

Abstract

BACKGROUND AND PURPOSEThe spider-venom peptide PcTx1 is the most potent and selective inhibitor of acid-sensing ion channel (ASIC) 1a. It has centrally acting analgesic activity and is neuroprotective in rodent models of ischaemic stroke. Understanding the molecular details of the PcTx1 : ASIC1a interaction should facilitate development of therapeutically useful ASIC1a modulators. Previously, we showed that several key pharmacophore residues of PcTx1 reside in a dynamic β-hairpin loop; conclusions confirmed by recent crystal structures of the complex formed between PcTx1 and chicken ASIC1 (cASIC1). Numerous peptide : channel contacts were observed in these crystal structures, but it remains unclear which of these are functionally important. EXPERIMENTAL APPROACHWe combined molecular dynamics (MD) simulations of the PcTx1 : cASIC1 complex with mutagenesis of PcTx1 and rat ASIC1a. KEY RESULTSCrystal structures of the PcTx1 : cASIC1 complex indicated that 15 PcTx1 residues form a total of 57 pairwise intermolecular contacts (<5 Å) with 32 channel residues. MD simulations, however, suggested that about half of these interactions do not persist in solution. Mutation to alanine of only eight of 15 PcTx1 contact residues substantially altered ASIC1a inhibition by PcTx1. Our data reveal that many of the peptide-channel interactions observed in the PcTx1 : cASIC1 crystal structures are not important for PcTx1 inhibition of rat ASIC1a. CONCLUSIONS AND IMPLICATIONSWe identified the atomic interactions that are critical for PcTx1 inhibition of ASIC1a. Our data highlight the value of combining structural information, MD and functional experiments to obtain detailed insight into the molecular basis of protein : protein interactions. Non-standard AbbreviationsASIC, acid-sensing ion channel; βHL, β-hairpin loop; cASIC1, chicken acid-sensing ion channel, isoform 1; ECD, extracellular domain; HMQC, heteronuclear multiple quantum coherence; HSQC, heteronuclear single quantum coherence; MALDI-TOF, matrix-assisted laser desorption/ionization/time-of-flight; MBP, maltose binding protein; MD, molecular dynamics; PcTx1, psalmotoxin 1 (also known as π-theraphotoxin-Pc1a); rASIC1a, rat acid-sensing ion channel, isoform 1a; RP-HPLC, reversed-phase HPLC; TEV, tobacco etch virus; TEVC, two-electrode voltage clamp BJP British Journal of Pharmacology

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

“…Using alanine-scanning mutagenesis of PcTx1 we determined the primary functional pharmacophore at rat (r) ASIC1a to comprise the residues Trp7, Trp24, Phe30, Arg26, Arg27, and Arg28 (Saez et al, 2015;Saez et al, 2011). These findings agree with the co-crystal structures of PcTx1:cASIC1, however the structures showed many more close contacts between the peptide and channel (Baconguis and Gouaux, 2012;Dawson et al, 2012) ( Figure 3A-C).…”

“…charged, basic cluster of Arg26, Arg27, and Arg28, that form interactions with proton-sensing residues in the acidic pocket (Saez et al, 2015) ( Figure 3B), however, this theory requires some direct binding experiments to confirm. An unexpected outcome of these peptide mutagenesis studies was the discovery of some potent ASIC1a potentiating molecules, the PcTx1 mutants R26A and F30A.…”

“…A combination of approaches employing radioligand binding and electrophysiology with chimeric channels (Chen et al, 2006;Salinas et al, 2006), mutagenesis (Saez et al, 2015) and co-crystal structures (Baconguis and Gouaux, 2012;Dawson et al, 2012) has shown that PcTx1 binds to the acidic pocket with the majority of contacts on the thumb domain, with residues on α helix 5 being particularly critical. The thumb region is known to be important for proton binding and subsequent gating of the channel (Ramaswamy et al, 2013), consistent with PcTx1 mimicking the action of protons and inhibiting ASIC1a via stabilising the desensitised state (Chen et al, 2005).…”

Biotechnological applications of spider venom peptides

“…Molecular dynamics and mutagenesis studies based on the co-crystal structure further refined our knowledge on the interaction of PcTx1 with ASIC1a by identifying all the peptide residues that are important for functional activity 110 .…”

“…Although PcTx1 contains a histidine residue that could potentially be iodinated, the reaction is 30-100 times slower than with tyrosine, requiring harsher conditions, longer reaction times, and excessive iodine 253 . Due to the lack of tyrosine in the sequence of PcTx1, a variant containing a non-native tyrosine was produced, based on the success of this approach for radioligand binding assays where the authors demonstrated that incorporating a tyrosine residue at either N-or C-terminus and iodination of PcTx1 had minimal effects on its ability to inhibit ASIC1alab has conducted extensive structure-activity relationship studies showing that the Nterminal region of PcTx1 is not involved in its interaction with ASIC1a 104,110 .…”