The spider venom peptide π-TRTX-Pc1a (Pc1a) is still one of the most potent and selective modulators of acid-sensing ion channel (ASIC) 1a known. It has centrally acting analgesic activity and is a potent neuroprotective agent in models of ischemic stroke. Thus, understanding the molecular basis of the Pc1a:ASIC1a interaction should facilitate development of therapeutically useful ASIC1a modulators. We previously determined a high-resolution solution structure of Pc1a and carried out a limited SAR study which revealed that most of the key pharmacophore residues reside in a b-hairpin loop. The structure of the Pc1a/ASIC1 complex was subsequently solved using X-ray crystallography. However, the extensive list of peptide/channel contacts in the crystal structure does not inform us about their functional importance.
In this study we carried out more extensive mutagenesis of the peptide and rat ASIC1a in combination with molecular-dynamics simulations of the Pc1a/cASIC1 complex in order to compare contacts identified in the crystal structure with functional and in silico data. 15 Pc1a residues were identified as close contacts in the crystal structure and MD simulations, and we found that mutation of eight of these residues to alanine had substantial effects on Pc1a inhibition of ASIC1a. NMR studies revealed that the reduced efficacy of some mutants is due to structural perturbation of primary pharmacophore residues. Most importantly, we found that several Pc1a residues that make close contacts with the channel in the Pc1a:ASIC1 crystal structure are not important for Pc1a inhibition of ASIC1a. These results highlight that data from crystal structures and in silico studies need to be coupled with functional experiments in order to obtain detailed insight into the molecular mechanism of ligand:target interactions.