Philippine E-Journals

In silico Characterization of GSPXII: A Novel γ-Conotoxin-Like Turritoxin Targeting the Cardiac Pacemaker Channel

Marian Gayle Angela C. Guevara | Neil Andrew D. Bascos | Cynthia P. Saloma

 

Abstract:

Turritoxins are venomous peptides from the Family Turridae. Gene mining performed within our laborato-ry revealed that Gemmula speciosa has conotoxin-like and non-conotoxin-like peptides that may similarly target ion channels and receptors with high specificity similar to that of the Conus peptides which have been harnessed for use in pharmacology. One of the peptides from this study was GspXII, a 46 AA turritoxin that shares the γ-conotoxin framework XII present among published γ-conotoxins and γ-conotoxin-like peptides based on multiple sequence alignment. γ-conotoxins target the pacemaker channels; however, none of these have elicited a phenotype in verte-brate models. The intravenous and intracranial injection of GspXII in mouse models resulted in a decrease in heart rate and back stiffening. Based on previous experiments, GspXII may target the cardiac pacemaker channel, HCN4. To test this hypothesis, in silico based docking studies were done using structural models of the peptide, and the target receptor (HCN4). Structures of the GspXII peptide were modeled through I-TASSER (zhanglab.ccmb.med.umich.edu/I-TASSER/) with and without disulfide constraints predicted by DiANNA (clavius.bc.edu/~clotelab/DiANNA/). These models, together with the prototype of the γ-family of conotoxins, PnVIIA, were used to dock to HCN4 (PDB ID: 6GYO, 6GYN) via ClusPro 2.0 (cluspro.bu.edu/). GspXII was observed to target HCN4 in both hyperpolarized and cAMP-bound states. The predicted binding of GspXII to the HCN4 pore is ex-pected to prevent the flow of ions through the channel. Notably, PnVIIA was not observed to dock onto the pore of the HCN4 channel; hence, this data supports that PnVIIA is unable to elicit a phenotype in vertebrates (Fainzilber et al., 1998). GspXII was also not observed to dock to the pore of the HCN1 channel (PDB ID: 5U6O, 5U6P, 6UQF) despite the similarity of HCN4 and HCN1, suggesting a specific GspXII-HCN4 interaction. GspXII remains the only member of the γ-conotoxin family of peptides that elicits a phenotype in vertebrate models. Our results suggest this mechanism involves the specific targeting of the HCN4 channel by this pore-blocking peptide.