Structure of KCNH2 cyclic nucleotide-binding homology domain reveals a functionally vital salt-bridge

Abstract

Human KCNH2 are key channels governing cardiac repolarization. Here, a 1.5 Å resolution structure of their cyclic nucleotide-binding homology domain is presented. Structural analysis and electrophysiological validation reveal a novel salt-bridge, playing an important role in hKCNH2 functional regulation.3 AbstractHuman KCNH2 (hKCNH2, Ether-à-go-go (EAG)-Related Gene, hERG) are best known for their role in cardiac action potentials repolarization and have key roles in various pathologies. As other KCNH family members, hKCNH2 contains a unique intracellular complex crucial for channel function, consisting of an N-terminal eag domain and a C-terminal cyclic nucleotide-binding homology domain (CNBHD). Previous studies demonstrated that the CNBHD is occupied by an intrinsic ligand motif (ILM), in a self-liganded conformation, providing a structural mechanism for the lack of KCNH channels regulation by cyclic nucleotides. While significant advancements in structural and functional characterizations of the CNBHD of KCNH channels have been made, a high-resolution structure of the hKCNH2 intracellular complex was missing. Here, we report the 1.5 Å resolution structure of the hKCNH2 channel CNBHD. The structure reveals the canonical fold shared by other KCNH family members, where the spatial organization of the ILM is preserved within the b-roll region. Moreover, measurements of small-angle X-ray scattering profile in solution, as well as comparison with a recent nuclear magnetic resonance (NMR) analysis of hKCNH2, revealed high agreement with the structure, indicating an overall low flexibility in solution. Importantly, we identified a novel salt-bridge (E807-R863), which was not previously resolved in the NMR and cryogenic electron microscopy (cryo-EM) structures. Strikingly, electrophysiological analysis of charge reversal mutations revealed its crucial role for hKCNH2 function. Moreover, comparison with other KCNH members revealed the structural conservation of this salt-bridge, consistent with its functional significance.Together with the available structure of the mouse KCNH1 intracellular complex, and previous electrophysiological and spectroscopic studies of KCNH family members, we propose that this salt-bridge serves as a strategically positioned linchpin to support both the spatial organization of the ILM and the maintenance of the intracellular complex interface.

Gating and regulation of KCNH (ERG, EAG, and ELK) channels by intracellular domains

“…New details provided by this structure are 1) a new, very detailed picture showing a meshwork of hydrogen bonds associated with the intrinsic ligand, including I804-F860, G806-L862, N819-L862, T859-I804, R863-N861, N819-N861, and N819-R863 (black dashed lines, Figure 7b; underlined residues, Figure 7c) and 2) the identification of a new salt bridge (green-dashed line, Figure 7b) between a residue (E807) in the CNBHD and a residue (R863) adjacent to the intrinsic ligand (red font, Figure 7c). Electrophysiology experiments show a functional role for the salt bridge in hERG channel gating and sequence alignments (Figure 7c) show the conservation of the salt bridge in other KCNH channels [4].…”

“…Recently, Haitin and colleagues solved the X-ray crystal structure of the hERG CNBHD at high (1.5 Angstrom) resolution [4]. New details provided by this structure are 1) a new, very detailed picture showing a meshwork of hydrogen bonds associated with the intrinsic ligand, including I804-F860, G806-L862, N819-L862, T859-I804, R863-N861, N819-N861, and N819-R863 (black dashed lines, Figure 7b; underlined residues, Figure 7c) and 2) the identification of a new salt bridge (green-dashed line, Figure 7b) between a residue (E807) in the CNBHD and a residue (R863) adjacent to the intrinsic ligand (red font, Figure 7c).…”

“…In summary, recent reports have increased our understanding of KCNH channel regulation by the intracellular PAS and CNBHDs (Figure 9). These include the identification of a small molecule, chlorpromazine, that binds to the EAG channel PAS domain and inhibits EAG channel gating [3], the high-resolution structure of the hERG CNBHD, which identifies new hydrogen bond contacts for the intrinsic ligand within the CNBHD and identifies a new salt bridge adjacent to the intrinsic ligand that is common in all KCNH channels [4] and the finding that mutations in the intrinsic ligand disrupt the PAS-CNBHD interaction in hERG channels [1,2]…”

“…In this review, we discuss the discovery that point mutations in the intrinsic ligand disrupt the PAS-CNBHD interaction as measured with electrophysiology and FRET in hERG channels [ 1 , 2 ], the identification of a small molecule (chlorpromazine) that targets the PAS and disrupts its regulatory function in EAG channels [ 3 ] and a high (1.5 Angstrom) resolution X-ray crystal structure of the hERG CNBHD which reveals new hydrogen bond contacts for the intrinsic ligand and identifies a salt bridge near the intrinsic ligand that is necessary for hERG channel function [ 4 ].…”

Long QT Syndrome Type 2: Emerging Strategies for Correcting Class 2 KCNH2 (hERG) Mutations and Identifying New Patients

“…Nonetheless, a gold standard for surmising structure/function relationships in proteins and how they might be perturbed by missense mutations is interpreting atomistic-scale structural data of intact proteins, when available. To date, such information for Kv11.1a is incomplete and comprises only Angstrom resolution models of its PAS, EAG and CNBD domains but not of the whole channel [ 92 , 93 , 94 ]. However, a significant advance in this regard is the recent availability of sub-nanometer resolution structural data of the Kv11.1a channel via cryo-electron microscopy [ 27 ].…”