Crystal structure of the PAS domain of the hEAG potassium channel

The structure of the hEAG PAS domain is presented and compared with similar segments of human and insect ion channels.

Similar to other K + channels, the members of the KCNH family are organized with four subunits surrounding a central pore (Becchetti et al., 2002). Each subunit contains six transmembrane helices (S1-S6), and the opening and closing of the channels depends on the S4 helix. In the cytosolic regions, KCNH channels contain a Per-Arnt-Sim (PAS) domain at the N-terminus (Morais Cabral et al., 1998) and a cyclic nucleotidebinding homology (CNBH) domain at the C-terminus (Brelidze et al., 2013;Marques-Carvalho et al., 2012), which has little affinity for cyclic nucleotides (Brelidze et al., 2009). The cytosolic regions harbour phosphorylation sites (Wang et al., 2002), with potential for interaction with kinases (Sun et al., 2004), integrins (Cherubini et al., 2005) and calmodulin (Schö nherr et al., 2000). It is proposed that these cytosolic regions can potentially regulate the channel activity and cell signalling; however, the precise functions of the cytosolic regions remain unclear.

ISSN 2053-230X
Voltage-gated potassium channels have been associated with a number of diseases, including cancers, in previous studies. Among these channels, EAG channels have been identified to play fundamental roles because of their restricted distributions, their regulatory roles and their oncogenic and pharmacological properties (Camacho, 2006). On one hand, EAG channels have been defined in a series of cancer cells, such as prostate, colon, ovary, melanoma, liver and thyroid cancer cells (Camacho, 2006;Farias et al., 2004;Meyer et al., 1999;Pardo et al., 2005;Ousingsawat et al., 2007). EAG has been identified as a potential tumour marker (Ludwig et al., 1994). In addition, various studies have associated EAG with the cell cycle and transformation (Arcangeli et al., 1995). Inhibition of the EAG channel activity reduces tumour-cell proliferation, indicating its potential role as a therapeutic target (Pardo et al., 2005). Despite its demonstrated role in cancers, little is known about the regulation of EAG.
Per-Arnt-Sim (PAS) domains are widespread in prokaryotes and eukaryotes (McIntosh et al., 2010;Henry & Crosson, 2011). In mammals, PAS domains are involved in the regulation of cardiac rhythm, hormone secretion and kinetic activity. They either act as sensors to mediate cellular responses to environmental stimuli, such as light, ligands and action potential, or directly participate in the response processes (McIntosh et al., 2010). The sensing roles of the PAS domains depend on their interaction with small molecules, for example haem, carboxylic acids and flavin mononucleotide (Mö glich et al., 2009;Henry & Crosson, 2011). It has been reported that some PAS domains mainly mediate protein interactions independent of ligand stimulation (Henry & Crosson, 2011). However, the definite functional roles of the PAS domains in EAG channels still need to be clarified.
In this study, we first determined the crystal structure of the PAS domain found at the N-terminus of human EAG (N-PAS domain of hEAG; PDB entry 5j7e) and compared it with the structures of Homo sapiens ERG (hERG) and Drosophila ELK (dELK). We present the structural details and discuss the implications for the functional roles of the PAS domain in the hEAG channel.

Protein expression and purification
The DNA sequence encoding residues 1-146 of the N-terminus of the human EAG channel, named the N-PAS domain, was amplified from a human cDNA library by PCR. The gene was cloned into the pET-GST vector (Invitrogen), which adds a glutathione transferase (GST) tag and a PreScission protease cleavage site at the N-terminus. The reconstructed plasmids were transformed into Escherichia coli BL21 (DE3) cells for expression.
The E. coli cells were cultured until the OD 600 reached $0.6 at 310 K and overexpression of the fusion protein was then induced using 0.2 mM isopropyl -d-1-thiogalactopyranoside (IPTG) at 298 K for 16 h in LB medium (10 g l À1 NaCl, 10 g l À1 tryptone, 5 g l À1 yeast extract). The cells were harvested by centrifugation, resuspended in buffer A (20 mM Tris-HCl pH 7.5, 300 mM NaCl) and lysed by sonication. The lysate was centrifuged at 18 000g for 40 min and the research communications  supernatant was loaded onto a GST affinity column which had been equilibrated with buffer A. The column was washed with buffer A and eluted using 10 mM reduced glutathione. The fusion protein was cleaved with 2 mg ml À1 PreScission protease at 277 K for 16 h to remove the N-terminal GST tag. A HiTrap Q column (GE Healthcare) equilibrated with 20 mM Tris-HCl pH 7.5 was then used to remove the N-terminal GST tag. The column was eluted with an NaCl concentration gradient from 5 to 700 mM, which was applied to the column over a 75 min period, and the target protein eluted between 200 and 260 mM NaCl. Finally, the eluate was concentrated to 10 ml by ultrafiltration using a 10 kDa cutoff membrane. The eluate was then loaded onto a HiLoad 26/60 Superdex 200 size-exclusion column in the presence of 20 mM Tris-HCl pH 7.5, 200 mM NaCl, 5 mM DTT and was immediately subjected to crystallization trials. SDS-PAGE was used to determine the purity. The calculated molecular mass of the N-PAS domain is 16 933.16 Da. The eluted peak containing the N-PAS domain from the HiLoad 26/60 Superdex 200 sizeexclusion column corresponded to a dimer. Samples were applied to SDS-PAGE and showed a single band at $17 kDa for the N-PAS domain (Fig. 1a). Finally, a UV spectrophotometer was used to determine the concentration, and the molar extinction coefficient was calculated using Vector NTI (Thermo Fisher). The molar extinction coefficient was 18 260 and one A 280 unit corresponds to 0.93 mg ml À1 protein. The sample was concentrated to 20 mg ml À1 for crystallization.

Crystallization and data collection
Crystallization screening of the N-PAS domain was performed using the sitting-drop vapour-diffusion method. Up to 14 different series of screening solutions were prepared, including Index, Index 2, Crystal Screen, Crystal Screen 2, PEG/Ion, PEG/Ion 2, SaltRx, SaltRx 2, PEGRx and PEGRx 2 (Hampton Research, California, USA) as well as Wizard I and II (Emerald Bio). For crystallization, 1 ml protein solution (20 mg ml À1 N-PAS domain, 20 mM Tris-HCl pH 7.5, 200 mM NaCl, 5 mM DTT) was mixed with 1 ml precipitant solution (Table 1). Different combinations of precipitant, pH and salt were tested and several optimization screens were used, including Detergent Screen, Additive Screen and Silver Bullets (Hampton Research). After optimization, crystals for data collection were obtained at 291 K. The crystals were recovered and immediately flash-cooled in liquid nitrogen.
Data were collected from a single flash-cooled crystal, which was a long rod, using 25%(v/v) glycerol as a cryoprotectant on beamline BL17U at Shanghai Synchrotron Radiation Facility (SSRF; Table 2). The data were processed, integrated and scaled using HKL-2000 (Otwinowski & Minor, 1997).

Structure solution and crystallographic refinement
The structure of the N-PAS domain was determined by molecular replacement using Phaser    jF obs j À jF calc j = P hkl jF obs j. § R free is calculated in the same way as R cryst but using a test set containing 5% of the data which were excluded from the refinement calculations.
sequence identity between the two proteins was $38%, and the LOV-PAS dimer of Arabidopsis phototropin 1 was present in the asymmetric unit. Refinement was performed in REFMAC5 (Murshudov et al., 2011) from the CCP4 suite. Model building was performed using Coot (Emsley et al., 2010). Manual model adjustment to improve the fit to the electron-density maps was also performed using Coot. The stereochemistry and the agreement between the model and the X-ray data were verified using Coot. After the initial refinement, solvent molecules were added based on standard geometrical and chemical restraints. Residues 1-26 and 136-146 in the structure were not built in the final model because of the poor quality of the electron density. PROCHECK (Laskowski et al., 1993)   overall refinement and final quality of the models are shown in Table 3. Molecular comparisons were performed at PBIL (https://npsa-prabi.ibcp.fr/) and the figures were prepared using PyMOL (http://www.pymol.org). consisting of 0.03 M citric acid pH 7.6, 0.07 M bis-tris propane pH 7.6, 20%(v/v) PEG 3350. These conditions were optimized, and after a week crystals were obtained in a condition consisting of 20%(v/v) PEG 3350, 0.1 M bis-tris propane pH 7.0. The crystals were suitable for X-ray analysis and diffracted to $3.5 Å resolution on beamline BL-17U1 at SSRF. The Additive Screen, Detergent Screen and Silver Bullets kits (Hampton Research, California, USA) were used for further optimization. Larger crystals were obtained after 7 d using condition No. 57 of the Detergent Screen kit: 20%(v/v) PEG 3350, 0.1 M bis-tris propane pH 7.0, 244.0 mM n-octanoylsucrose (Fig. 1c). The N-PAS domain crystals used for X-ray  diffraction were flash-cooled in liquid nitrogen with a cryoprotectant consisting of 20%(v/v) PEG 3350, 0.1 M bis-tris propane pH 7.0, 25%(v/v) glycerol and diffracted to $1.9 Å resolution on beamline 17U at SSRF (Fig. 1b).

Structure determination of the hEAG N-PAS domain
The crystal belonged to space group C2, with unit-cell parameters a = 213.974, b = 39.058, c = 106.802 Å , = 118.93 .
Resolution-dependent Matthews coefficient probability analysis suggested the presence of six molecules per asymmetric unit, with around 50% solvent content and a V M of 2.54 Å 3 Da À1 .  consisting of residues 28-137 was refined to 1.9 Å resolution, while the first 27 amino acids could not be detected in the electron-density map. Crystallographic statistics are shown in Table 1.
In the crystal structure of the N-PAS domain, there are six molecules per asymmetric unit. Each molecule displays the canonical fold of a PAS domain comprising a central -sheet with five strands labelled 1-5. Four -helices, 1-4, decorate the -sheet (Fig. 2a). The topological order of -strands is 2-1-5-4-3. The secondary structure from 1 to 5 is referred to as the core region of the N-PAS domain and the extensions to the N-terminus and C-terminus are referred to as flanking regions.
In order to investigate the evolutionary conservation of PAS domains in EAG channels, we aligned the amino-acid sequences of the human, mouse, fruit-fly and zebrafish proteins. The amino-acid sequences are highly conserved (Fig. 2c). The crystal structure of the PAS domain from mouse EAG (mEAG; PDB entry 4hoi; Adaixo et al., 2013) was superposed with the N-PAS structure through main-chain alignments and shows that apart from the N-terminal and C-terminal loops there are no obvious differences (Fig. 2b). The root-mean-square deviation (r.m.s.d.) of the aligned structures is 0.362 Å . The sequence alignment shows that there are two conservative differences between the hEAG PAS domain and the mEAG PAS domain, The68 and Ile80, which were not present in either structure. These results show that the PAS-domain structures of EAG channels are highly conserved.

Structures of PAS domains from KCNH channels
To better understand the structural features of PAS domains from KCNH channels, we compared the structures of PAS domains from hEAG, mERG and dELK. We superposed these structures using their main-chain atoms. The overall structures show high similarity, apart from the N-terminal helix, which is not defined in the hEAG structure. The main differences appear in the 1-2, 4-5, 5-3 and 4-5 loop regions (Fig. 3a). To analyze the differences in the amino-acid sequences, we performed a multiple sequence alignment of PAS domains from hEAG, mERG and dELK. The amino-acid sequences, including the core -sheet region, have low conservation (Fig. 3b). These results indicate that while the amino-acid sequences share little similarity among KCNH channels, the secondary structures are highly conserved.

Functional structures of the PAS domain of hEAG
One of the interesting regions in the structure of the N-PAS domain of hEAG is a hydrophobic patch on the outer side of the -sheet (Fig. 4b). The patch is thought to mediate the interactions between the PAS domain and other channel regions or those among PAS domains (Gustina & Trudeau, 2011). In the ERG channel, Forster resonance energy transfer (FRET) results suggest that the PAS domain directly interacts with other parts of the channel (Gustina & Trudeau, 2009 (Gustina & Trudeau, 2011). The interaction is crucial for cellular functions, which serves as the molecular basis underlying long QT2 syndrome (Spector et al., 1996). The hydrophobic patches in PAS domains are strongly conserved in position, size and chemical features. In the crystal structure of the N-PAS domain of hEAG, we note that the hydrophobic patches mediate interaction between molecules C and E and between molecules A and D (Fig. 4c). In the hERG and dELK structures, the patches are found in the same region and also mediate the intermolecular interaction (Adaixo et al., 2013). The hydrophobic patch on the hEAG N-PAS domain includes 12 apolar residues (Fig. 4a) and is highly conserved in hERG and dELK. These results suggest the possibility that the functions of KCNH channels may be regulated by the interactions between this hydrophobic patch on the PAS domain and other entities. Based on the previously reported structures, the PAS domain is a monomer in hERG, while it is a dimer in dELK. It is possible that the dimerization of PAS domains may have little effect on the assembly of the channels. However, another crucial region regulating the functions of the hEAG channel, residues 1-27, at the N-terminus was not defined in the structure (Schö nherr & Heinemann, 1996).
Despite many years of study, the functional roles of the PAS domain in KCNH channels still remain unclear. In particular for EAG, which has been determined as a diagnosis marker or a therapeutic target, clarification of the functional roles of the PAS domain is urgently required. In our study, we determined the structure of the PAS domain at the N-terminus of hEAG. The overall structure fits the conserved fold of the domain family. Alignment with previously determined structures of PAS domains from hERG and dELK indicates that the hydrophobic patch on the outer surface of the -sheet may mediate both the interaction between homodimers and the interaction between the PAS domain and other channel regions in order to regulate the channel function.