Structural properties of PAS domains from the KCNH potassium channels

Abstract

KCNH channels form an important family of voltage gated potassium channels. These channels include a N-terminal Per-Arnt-Sim (PAS) domain with unknown function. In other proteins PAS domains are implicated in cellular responses to environmental queues through small molecule binding or involvement in signaling cascades. To better understand their role we characterized the structural properties of several channel PAS domains. We determined high resolution structures of PAS domains from the mouse EAG (mEAG), drosophila ELK (dELK) and human ERG (hERG) channels and also of the hERG domain without the first nine amino acids. We analyzed these structures for features connected to ligand binding and signaling in other PAS domains. In particular, we have found cavities in the hERG and mEAG structures that share similarities with the ligand binding sites from other PAS domains. These cavities are lined by polar and apolar chemical groups and display potential flexibility in their volume. We have also found that the hydrophobic patch on the domain β-sheet is a conserved feature and appears to drive the formation of protein-protein contacts. In addition, the structures of the dELK domain and of the truncated hERG domain revealed the presence of N-terminal helices. These helices are equivalent to the helix described in the hERG NMR structures and are known to be important for channel function. Overall, these channel domains retain many of the PAS domain characteristics known to be important for cell signaling.

Figure 1. Cavity in the PAS domain of hERG channel.

a) Cartoon representation of high resolution structure of PAS domain from hERG channel. Cavity is shown as wireframe surrounded by several residues shown in stick. Arrow indicates residue H70 at the entrance to cavity. b) PAS domain structure of phototropin Phot-LOV1 (PDB code 1N9N). Flavin mononucleotide, shown as red stick, is bound within binding site cavity, shown as wireframe. c) Detailed view on hERG PAS domain cavity. The molecular surface of the domain is shown as wireframe and extends to the domain cavity. Water molecules in cavity are shown as red spheres. Some of the residues lining cavity are shown and labeled. Residues that in other channel PAS domain are substituted by polar residues are shown in gold. d) Polar and apolar chemical groups lining the cavity of the hERG PAS domain are shown. Wireframe delimiting cavity is colored according to the atoms that compose the cavity lining, red for oxygen, blue for nitrogen and white for carbon; the polar face (predominantly colored red and blue) of the cavity is towards the reader and the apolar face is at the opposite side.

Figure 2. Cα representation of PAS domains from three different channels.

a) View of the helical face of PAS domains. b) View of the β-sheet face of PAS domains. Colors are: hERG in redbrick, mEAG in green and dELK in blue. The N-terminal helix in dELK PAS domain (absent in the other two structures) is shown in light blue. N- and C- terminal are labeled in b). Several structural features discussed in the text are labeled.

Figure 3. Cavities in the PAS domain from dELK channel.

a) Cα representation of structure of domain. Cavities detected by CASTp server are shown as blue wireframe representations. b) Histidine 129 and surrounding residues.

Figure 4. Cavities in the PAS domain from the mEAG channel.

a) Cα representations of two of the models (in green and gray) generated by combinations of side-chain conformers present in the asymmetric unit molecules of the mEAG PAS domain crystal. The cavities shown in gray and green wireframe correspond to the largest and smallest cavities, respectively, detected by CASTp server. b) Residues lining the largest cavity are shown as stick and are labeled. Cavity represented as wireframe. Colors on wireframe correspond to atoms lining the cavity: red for oxygens, yellow for sulfurs, white for carbons. The face of the cavity away from the reader is apolar and only colored white. c) Superposition of the 4 molecules in the asymmetric unit of mEAG PAS domain crystal. Serine in the hydrophobic core (S65) and surrounding residues are shown in stick, cavity is shown as wireframe.

Figure 5. Crystal contacts formed by hydrophobic patch.

a) Lattice packing of two molecules of hERG PAS domain. The molecules are related by 2 fold crystallographic axis indicated by arrow. b) Packing of two molecules in the asymmetric unit of the mEAG PAS domain. The two molecules have a ∼2-fold relation that is roughly perpendicular to page. In both panels the two molecules on the right are shown in a similar orientation. Residues from the hydrophobic patches are shown as stick.

Figure 6. N-terminal helices in channel PAS domain structures.

a) View of N-terminal helix (light blue) packed against hydrophobic patch of dELK PAS domain. Residues forming the patch are shown as stick. b) View of Δ9-hERG PAS structure with N-terminal helix packed against the β-sheet. Dotted line represent a possible linker connection between N-terminal helix and the body of the Δ9-hERG PAS domain. c) Superposition of structures of Δ9-hERG PAS (redbrick) and dELK (blue). N-terminal helices are indicated. Dotted line as in b).

Figure 7. Superposition of three (in red, yellow and cyan Cα trace) of the NMR model structures of the hERG PAS domain (PDB code 1L0W).

Cavities detected in the three models are shown as red, yellow or cyan wireframe.