Probing the Outer Mouth Structure of the hERG Channel with Peptide Toxin Footprinting and Molecular Modeling

Abstract

Previous studies have shown that the unusually long S5-P linker lining human ether a-go-go related gene's (hERG's) outer vestibule is critical for its channel function: point mutations at high-impact positions here can interfere with the inactivation process and, in many cases, also reduce the pore's K+ selectivity. Because no data are available on the equivalent region in the available K channel crystal structures to allow for homology modeling, we used alternative approaches to model its three-dimensional structure. The first part of this article describes mutant cycle analysis used to identify residues on hERG's outer vestibule that interact with specific residues on the interaction surface of BeKm-1, a peptide toxin with known NMR structure and a high binding affinity to hERG. The second part describes molecular modeling of hERG's pore domain. The transmembrane region was modeled after the crystal structure of KvAP pore domain. The S5-P linker was docked to the transmembrane region based on data from previous NMR and mutagenesis experiments, as well as a set of modeling criteria. The models were further restrained by contact points between hERG's outer vestibule and the bound BeKm-1 toxin molecule deduced from the mutant cycle analysis. Based on these analyses, we propose a working model for the open conformation of the outer vestibule of the hERG channel, in which the S5-P linkers interact with the pore loops to influence ion flux through the pore.

K+ Channel Modulators for the Treatment of Neurological Disorders and Autoimmune Diseases

“…47,49 Using an early molecular model of Shaker by Durrell and Guy 312 and the results from their mutant cycle analysis, this group then generated a molecular model of the S5-P-S6 region of the K V 1.3 channel. 47 In collaboration with Raymond Norton's group in Melbourne, KTX was manually docked in this model by guiding Lys 27 into the pore such that it would lie near the selectivity filter, and the toxin was then rotated about the central pore axis until Arg 24 in the toxin aligned with Asp p38 (386) in K V 1.3 in a salt bridge suggested by the mutagenesis. 47 Using highresolution solid-state NMR spectroscopy, Lange et al demonstrated that high-affinity binding of KTX to a chimeric KcsA-K V 1.3 channel involves structural rearrangements in both molecules.…”

APETx1 from Sea Anemone Anthopleura elegantissima Is a Gating Modifier Peptide Toxin of the Human Ether-a-go-go- Related Potassium Channel

“…1A) (Korolkova et al, 2002). BeKm-1 does not totally occlude ion flux through the hERG pore (Zhang et al, 2003), probably because it binds above the selectivity filter (Tseng et al, 2007).…”

“…Several such peptide toxins have been identified (Gurrola et al, 1999;Korolkova et al, 2001;Corona et al, 2002;Nastainczyk et al, 2002;Huys et al, 2004). Two peptide toxins purified from scorpions, BeKm-1 and CnErg1 (also called ErgTx1), are the best-studied cases (Korolkova et al, 2002;Pardo-López et al, 2002a,b;Zhang et al, 2003;Tseng et al, 2007). Both toxins bind to hERG's outer vestibule to suppress ion conduction through the pore.…”

“…The spatial relationship among Lys18, Tyr32, Phe33, and Leu34 on APETx1 bears some resemblance to the relationship among residues on BeKm-1 that are involved in binding to the hERG channel (Lys18, Phe14, and Tyr11) ( Fig. 1) (Korolkova et al, 2002;Tseng et al, 2007). However, the mechanism of APETx1's action and its binding site on the hERG channel have not been investigated.…”

In Silico Prediction of the Chemical Block of Human Ether-a-Go-Go-Related Gene (hERG) K+ Current

“…The sequence alignment of KcsA, KvAP, and hERG was generated by T-Coffee [93] and then manually adjusted to increase the accuracy of homology modeling. Although an extracellular loop between S5 and pore helix (between positions 572 and 610) is proposed to be structured and involved in ion conduction in hERG [94], this loop was omitted for simplicity. After the refinement of the structure by molecular mechanics, the model had no dihedral angle violation in the Ramachandran plot, with the exception of distal termini …”