Structure of the C-Terminal Region of an ERG Channel and Functional Implications

Structure of the C-terminal region of an ERG channel and functional implications

Tinatin I. Brelidzea,1, Elena C. Gianulisb, Frank DiMaioc, Matthew C. Trudeaub, and William N. Zagottaa,2

Departments of aPhysiology and Biophysics and cBiochemistry, University of Washington School of Medicine, Seattle, WA 98195; and bDepartment of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201

Edited by Richard W. Aldrich, University of Texas at Austin, Austin, TX, and approved May 31, 2013 (received for review April 11, 2013) The human ether-à-go-go–related gene (hERG) encodes a K+ of related cyclic nucleotide-gated (CNG) and hyperpolarization- channel crucial for repolarization of the cardiac action potential. activated cyclic nucleotide-modulated (HCN) channels (12, 19). EAG-related gene (ERG) channels contain a C-terminal cyclic nu- However, unlike the CNBD of CNG and HCN channels, the cleotide-binding homology domain coupled to the pore of the CNBHD of KCNH channels does not appreciably bind cyclic channel by a C-linker. Here, we report the structure of the C- nucleotides (18, 23). Therefore, paradoxically, despite the linker/cyclic nucleotide-binding homology domain of a mosquito presence of a CNBHD, the KCNH channels are not regulated ERG channel at 2.5-Å resolution. The structure reveals that the by direct binding of cyclic nucleotides (23–26).BasedonFör- region expected to form the cyclic nucleotide-binding pocket is ster resonance energy transfer (FRET) experiments, the negatively charged and is occupied by a short β-strand, referred CNBHDs and PAS domains are located in close proximity (27). to as the intrinsic ligand, explaining the lack of direct regulation Moreover, it has been proposed that the C-linker/CNBHD di- of ERG channels by cyclic nucleotides. In hERG channels, the in- rectly interacts with the PAS domain (28–30), forming an trinsic ligand harbors hereditary mutations associated with long- intersubunit interaction (28). In hERG channels, both the QT syndrome (LQTS), a potentially lethal cardiac arrhythmia. C-linker/CNBHD and PAS domain harbor genetically occur- Mutations in the intrinsic ligand affected hERG channel gating ring mutations that are linked to LQTS (11, 31–34) (reviewed in and LQTS mutations abolished hERG currents and altered trafﬁck- ref. 32). These LQTS mutations have been found to speed up ing of hERG channels, which explains the LQT phenotype. The deactivation of hERG channels or lead to defects in protein

structure also reveals a dramatically different conformation of trafficking and folding, precluding formation of functional PHARMACOLOGY the C-linker compared with the structures of the related ether-à- channels (35–43). + go-go–like K and hyperpolarization-activated cyclic nucleotide- Here, we present the crystal structure of the C-linker/CNBHD modulated channels, suggesting that the C-linker region may be of an Anopheles gambiae ERG channel (agERG) at 2.5-Å reso- highly dynamic in the KCNH, hyperpolarization-activated cyclic nu- lution. The agERG and hERG channels share 78% amino acid cleotide-modulated, and cyclic nucleotide-gated channels. identity and 90% similarity in the C-linker/CNBHD (Fig. 1A and Fig. S1). The structure of the C-terminal region of ERG chan- agERG | KCNH2 | cyclic nucleotide-binding domain | Kv11.1 channels nels reveals the structural basis of ERG channel regulation by the CNBHD. The C-linker in the ERG structure is in a dramat- he human ether-à-go-go-related gene (hERG) ion channel ically different conformation in comparison with the C-linkers in fi related zELK (18) and mHCN2 (21) channels. The diversity of Tunderlies the fast delayed recti er current (IKr) in the heart and is the major contributor to the repolarization phase of the the C-linker conformations observed in the ERG, ELK, and ventricular action potential (1–4). Disruption of the function of HCN structures suggests that the C-linker region may be highly this channel, either genetically or as an unintended consequence dynamic in the KCNH, HCN, and CNG channels. of prescription medication, causes lengthening of the ventricular Results action potential, which can lead to a condition known as long-QT syndrome (LQTS) (5–8). LQTS is characterized by a prolonged Structure of the C-Linker/CNBHD of an ERG Channel. To gain mo- QT interval on an electrocardiogram, cardiac arrhythmias, and lecular insight into the gating and regulation of ERG channels, predisposition to sudden cardiac death. hERG channel dysfunction we crystallized the C-linker/CNBHD of Anopheles gambiae is responsible for ∼45% of LQTS-related mutations identified (mosquito) ERG (agERG) channels. The C-linker/CNBHD of fi so far and the vast majority of drug-induced LQTS (9–11). agERG was identi ed as a suitable candidate for crystallization fl The EAG-related gene (ERG) channel subfamily belongs to the by using a screen based on the uorescence-detection size- + KCNH family of voltage-gated K channels, which also includes exclusion chromatography (FSEC) (44). The C-linker/CNBHD of + the ether-à-go-go (EAG) and EAG-like K (ELK) channel sub- agERG channels crystallized in the space group P3221 with a sin- families (Fig. 1A) (12). EAG and ELK channels are key regulators gle molecule in the asymmetric unit and diffracted X-rays to 2.5-Å of tumor progression (13, 14) and neuronal excitability (15, 16). resolution (Table S1). The structure was solved with molecular + fi Similar to other K selective channels, KCNH channels are as- replacement, using the structure of the CNBHD of zebra sh ELK sembled from four subunits around a central ion conducting pore. Each of the four subunits contains six membrane spanning segments (S1–S6) and an intervening pore-forming loop (Fig. 1B). Author contributions: T.I.B., M.C.T., and W.N.Z. designed research; T.I.B., E.C.G., and M.C. – T. performed research; F.D. contributed new reagents/analytic tools; T.I.B., E.C.G., and The S1 S4 segments comprise a voltage sensing domain, whereas M.C.T. analyzed data; and T.I.B., M.C.T., and W.N.Z. wrote the paper. – the S5 S6 segments together with the intervening loop form a pore The authors declare no conflict of interest. domain (17). This article is a PNAS Direct Submission. A characteristic feature of the channels in the KCNH family is Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, the presence of a Per-Arnt-Sim (PAS) domain in their cyto- www.pdb.org (PDB ID code 4L11). plasmic amino-terminal region, and a cyclic nucleotide-binding 1Present address: Department of Pharmacology and Physiology, Georgetown University homology domain (CNBHD) in their cytoplasmic carboxyl-terminal Medical Center, Washington, DC 20057. region coupled to the pore of the channel by a C-linker (12, 18–22) 2To whom correspondence should be addressed. E-mail: [email protected]. (Fig. 1B). The CNBHD of KCNH channels shares general This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. architecture with the cyclic nucleotide-binding domains (CNBD) 1073/pnas.1306887110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1306887110 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 A B cyclic nucleotides, explaining, in part, the absence of direct ELK regulation of KCNH channels by cyclic nucleotides (23–26). ERG hELK1 hELK2 The β9-strand is also unique to the KCNH family and is not hERG3 mELK3 mERG2 agERG present in other CNBD-containing proteins. It is part of a short hERG1 sequence of amino acids following the αC-helix that occupies the β-roll cavity where cyclic nucleotides normally bind in canonical cyclic nucleotide-binding proteins (Fig. 2C). We refer to this re- “ ” mEAG1 gion as the intrinsic ligand, a portion of the protein that occupies mEAG2 EAG the ligand binding site whose displacement regulates the channel. The sequence and structure of the intrinsic ligand is well conserved C D in the KCNH family of channels (18, 22) (Fig. 2 A and B and Fig. Elbow C-linker S1). In agERG, residues Y727 and M729 in the intrinsic ligand B’ form a network of direct interactions with the residues in the β-roll Shoulder A’ cavity of agERG channels (Fig. 2D). Y727 is located where the purine ring of cAMP is located in the CNBD of HCN2 channels, C’ D’ whereas M729 is located where the cyclic phosphate of cAMP is in roll the HCN2 structure. A Effect of Mutations in the Intrinsic Ligand on hERG Channel Gating. B To investigate whether the intrinsic ligand regulates the gating of C ERG channels, we mutated the intrinsic ligand and measured the effects on channel function. Expression of agERG channels in Xenopus oocytes did not produce detectable voltage-activated + K currents so we mutated the intrinsic ligand in hERG chan- Fig. 1. Topology and similarity of KCNH channels and the structure of the nels. In hERG channels, the intrinsic ligand includes conserved agERG C-linker/CNBHD. (A) Phylogenetic tree of the KCNH family of ion residues F860, N861, and L862 (Fig. S1). The mutant channels channels computed with Cobalt. (B) Cartoon of two opposing subunits of were expressed in Xenopus oocytes, and currents were recorded a tetrameric KCNH channel. The pore-forming loop and S5–S6 trans- by using two-electrode voltage clamp. All of the electrophysio- membrane domains are gray. The N-terminal α-helix and PAS domain are logical studies in this section were performed in the background magenta. The elbow and shoulder regions of the C-linker are represented by of S620T mutation in the pore region that removes the C-type the red and pink cylinders, respectively. The β9-strand is represented by a green rectangle. The rest of the CNBHD is blue. The dashed lines con- inactivation in hERG channels (47). necting the PAS domain and S1 transmembrane domain indicate inter- To explore the role of the intrinsic ligand in the function of subunit nature of the interactions between the PAS domain of one subunit hERG channels, we examined the effect of mutating the con- and the CNBHD of the adjacent subunit (28). (C) Ribbon representation of served residues to alanine. Currents were elicited by a series of the structure of the C-linker/CNBHD of agERG channels. (D) Electrostatic voltage pulses from −100 to 40 mV in 20-mV increments fol- potential surface of the C-linker/CNBHD of agERG channels viewed in the lowed by a pulse to −60 mV (or −100 mV; Fig. 3 A–D, Inset). All same orientation as in C. The electrostatic potential surface was calculated three mutant hERG channels (F860A, N861A, and L862A) gave ﬁ + by using the APBS plugin for PyMol with the PARSE force eld and colored rise to robust voltage-activated K currents in Xenopus oocytes (Fig. from red (−3 kT/e) to blue (+3 kT/e). 3 A–D). The conductance–voltage relationships for the wild-type

(zELK) channels (18) as a search model, combined with energy- and electron density-guided structure optimization with Rosetta (45). agERG / mEAG1 agERG / zELK The C-linker region in the crystal structure consists of four AB α-helices (αA′-αD′) with αA′-αB′ and αC′-αD′ helices forming two antiparallel helix-turn-helix motifs (Fig. 1C). This arrangement is markedly different from the topology of the C-linkers of zELK (18) and mHCN2 (21) channels that consist of six α-helices. The CNBHD of agERG is formed by eight β-strands forming an antiparallel β-roll, three α-helices (αA-αC), and a short β-strand (β9) D following the αC-helix (Fig. 1C). The structure of the CNBHD is C I656 similar to the crystallized CNBHDs from the two other subfamilies I671

of KCNH channels, mouse EAG (mEAG) (rmsd of 1.9 Å) (22) Y727 K658 F672 Y727 and zELK (rmsd of 2.4 Å) (18) (Fig. 2 A and B). With the ex- M729 M729 G673 ception of the β9-strand, the general fold of the CNBHD is N686 N728 N728 similar to the fold of canonical cyclic nucleotide-binding proteins, including channels, kinases, transcription factors, and S685 guanine nucleotide-exchange factors (46). Fig. 2. Structural comparison of the C-linker/CNBHDs of KCNH channels and Unlike canonical cyclic nucleotide-binding proteins, however, the close-up view of the agERG intrinsic ligand. (A and B) Structural align- the electrostatic proﬁle of the C-linker/CNBHD of agERG chan- ment of the CNBHD of agERG (the β9-strand is green and the rest is blue) and nels displays a negatively charged β-roll cavity in the CNBHD mEAG1 (gray) channels (22) (A) and agERG and zELK (yellow) channels (18) (Fig. 1D). The β-roll cavity is the site of cyclic nucleotide binding (B). The rmsd for the α carbons of the agERG and mEAG1 structures is 1.9 Å, and the rmsd for the α carbons of the agERG and zELK structures is 2.4 Å. (C) in canonical cyclic nucleotide-binding proteins (46). In HCN2 − channels, which are directly regulated by cyclic nucleotides, the A2Fo Fc omit electron-density map of residues Y727, N728, and M729 of the intrinsic ligand bound inside the β-roll cavity of the CNBHD of agERG β-roll cavity is positively charged, favoring binding of negatively σ β β channels. The map is contoured at 1.0 .(D) Residues in the -roll cavity charged cyclic nucleotides (21). In contrast, for agERG the -roll interacting with residues Y727 and M729 of the intrinsic ligand. Dashed lines cavity is negatively charged (Fig. 1D). The negatively charged β-roll show both polar and nonpolar interactions. cAMP from the HCN2 structure cavity would be expected to impede binding of negatively charged (21) structurally aligned with the agERG is shown in yellow.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1306887110 Brelidze et al. Downloaded by guest on September 26, 2021 A D more depolarized voltages (18). Surprisingly, for EAG1 channels,

0 mutation of the conserved residues Y672 and L674 to alanine in the WT L862A . 5

0.1 s A –

1 A intrinsic ligand shift the conductance voltage relationship to more

1 s hyperpolarized voltages (22, 48). Therefore, although all KCNH channels studied so far are regulated by the intrinsic ligand, the B E manner in which they are regulated differs for different channels. F860A 1.0 WT LQTS Mutations in the Intrinsic Ligand. In hERG channels, the in- F860A 0.8 N861A trinsic ligand harbors hereditary mutations N861I and N861H, L862A which were associated with LQTS (11, 31). To investigate the 0.6 C effect of the N861H and N861I mutations on channel functional N861A max 0.4 properties, mutant hERG channels were transiently expressed in

G/G mammalian human embryonic kidney 293 (HEK293) cells and 0.2 currents were recorded in the whole-cell patch-clamp configu- 0.0 ration. Whereas we measured robust currents from cells with 40 mV -80 -40 0 40 -80 mV WT hERG channels, no measurable currents were recorded -60 mV Voltage (mV) from cells with N861H or N861I mutant hERG channels (Fig. 5 Fig. 3. Mutations in the intrinsic ligand have no measurable effect on the A–D). Western blot analysis showed that all of the channels had conductance–voltage relationship of hERG channels. (A–D) Representative a robust band at ∼135 kDa, which represents an immature form currents recorded from whole oocytes expressing WT (A), F860A (B), N861A of the protein, but only WT hERG channels had a band at ∼155 (C), and L862A (D) hERG channels elicited by a series of voltage pulses from kDa, which represents the mature (N-glycosylated) form of the −100 to 40 mV in 20-mV increments followed by a pulse to −60mV (or −100 mV; Inset). (E) Conductance–voltage relations for WT (filled squares), F860A protein (Fig. 5E). Our results indicate that N861H and N861I (open squares), N861A (open triangles), and L862A (open circles) hERG channels had a defect in maturation, similar to what was shown channels measured from the tail currents at −100 mV. Data points were fit for LQTS mutations in the CNBHD, including N861I, which was with a Boltzmann equation to determine the V1/2 and s values. The number proposed to be retained in the endoplasmic reticulum (41–43). of cells n ≥ 3 for each of the channels. (Scale bars: 1 μA and 1 s; Inset,0.5μA The lack of hERG currents is consistent with an LQTS pheno- and 0.1 s.) type. Taken together, our findings suggest that the intrinsic ligand regulates the deactivation rate and surface expression of PHARMACOLOGY (WT) channels and the three mutant channels were generated hERG channels. from the tail currents at −100 mV (Fig. 3 A–D, Inset) and were not Conformations of the C-Linker Region. Interestingly, the crystal significantly different (P > 0.05, ANOVA) from one another [WT: structures of the C-linker/CNBHDs of agERG, zELK, and mHCN2 the half-maximal activation voltage (V1/2) = −18.3 ± 3.2 mV, the slope of the relation (s) = 12.4 ± 2.1 mV; F860A: V1/2 = −22.5 ± 2.2 mV, s = 11.3 ± 2.4 mV; N861I: V1/2 = −22.1 ± 1.7 mV, s = 10.5 ± = − ± = ± 0 0.9 mV, and L862A: V1/2 20.0 0.7 mV, s 9.9 0.5 mV) A C (Fig. 3E). WT N861A To investigate the effect of the mutations in the intrinsic ligand on the deactivation kinetics, we examined tail currents (Fig. 4). fi B D The tail currents were elicited by rst activating the channels with F860A L862A a pulse to 20 mV and then applying a voltage pulse to −100 mV and −120 mV. The time constant of deactivation was determined 2 A fi 0.1 s by tting the tail currents with a single exponential function (Fig. 4 E 20 mV – -100 mV A D). The F860A and L862A mutations caused an increase in the 140 rate of deactivation, whereas N861A had a deactivation time con- -120 mV stant (τ) that was statistically similar to the WT channels (at −100 120 mV, WT: τ = 108 ± 13 ms; F860A: τ = 49 ± 5ms;N861A:τ = 94 ± 5 ms; L862A: τ = 66 ± 3 ms. At −120 mV, WT: τ = 45 ± 4 ms; 100 τ = ± τ = ± τ = ± F860A: 26 4 ms; N861A: 40 2 ms; L862A: 31 2 80 ** ms) (Fig. 4E). Residues F860 and L862 in hERG correspond to ** residues Y727 and M729 in the agERG structure that form 60 multiple direct polar and nonpolar interactions with the residues ** in the β-roll cavity (Fig. 2D). In contrast, residue N861 in hERG 40 * β corresponds to N728 in agERG that is facing away from the -roll Time constant of deactivation (ms) 20 cavity and forms virtually no direct interactions with residues on WT F860A N A268LA168 WT A068F N861A 268L A the β-roll. These results indicate that the intrinsic ligand stabilizes the open state of hERG channels, similar to an agonist of a li- -100 mV -120 mV gand-gated channel. Interestingly, these effects of mutations of Fig. 4. Mutations in the intrinsic ligand accelerate deactivation of hERG the intrinsic ligand are similar to the effects of many LQTS channels. Tail currents for WT (A), F860A (B), N861A (C), and L862A (D) hERG mutations in the amino-terminal PAS domain (32, 35, 36), sug- channels were elicited by first activating the channels with a pulse to 20 mV gesting that the PAS domain might regulate the channel via the and then applying a voltage pulse to −100 mV and −120 mV. (E) Box plot of CNBHD and perhaps through the intrinsic ligand. deactivation time constants at −100 mV and −120 mV for WT, F860A, N861A, fi These effects of mutations of the intrinsic ligand are somewhat and L862A hERG channels. The time constants were derived by tting the tail currents at −100 mV and −120 mV with a single exponential function as different from the effects previously seen in zELK and EAG1 depicted by the overlaid gray traces in A–D. The median is represented by channels. For zELK channels, mutation of the tyrosine residue the middle line, the bottom and top lines are the 25th and 75th percentiles, in the intrinsic ligand to alanine or deletion of the conserved and the vertical lines are the 10th and 90th percentiles. **P < 0.01, *P < 0.05, YNL tripeptide shift the conductance–voltage relationship to by ANOVA. n ≥ 4 for each of the channels. (Scale bar: 2 μA and 0.1 s.)

Brelidze et al. PNAS Early Edition | 3of6 Downloaded by guest on September 26, 2021 A D the loop between the αB′ and αC′-helices is much longer in the 70 agERG structure, 12 amino acids in agERG compared with just 2 WT 60 in zELK and 6 in mHCN2, and the αD′-helix is also much longer in WT 50 N861H the agERG structure, 18 amino acids in agERG compared with 8 N861I 40 in zELK and 11 in mHCN2. As a result, despite the length and sequence similarity to the C-linkers of zELK and mHCN2 chan- 30 nels, the C-linker of agERG channels contains only four α-helices, 20 as opposed to six in the C-linkers of zELK and mHCN2. The B Current density (pA/pF) 10 manifestation of these differences in the fold of the C-linker is N861H 0 that the conserved elbow-on-the-shoulder interface, which is -80 -60 -40 -20 0 20 40 60 Voltage (mV) the primary region of intersubunit interactions in the zELK and mHCN2 structures, is instead an intrasubunit interface in the E agERG structure. N861I Control C WT N861H Discussion N861I kD With this X-ray crystal structure of the C-linker/CNBHD of 0.5nA 150 agERG channels, there is now a structure of the C-terminal region 1s of channels from each of the three KCNH subfamilies: ELK 110 60 mV (zELK; ref. 18), EAG; (mEAG; ref. 22), and ERG (agERG). -50-50 mVmV These structures share some features in common that are unique -80 mVV to the KCNH family. In each structure, the β-roll cavity is negatively charged in contrast to the positively charged cavity, where Fig. 5. LQT2 mutations in the intrinsic ligand prevent functional expression of hERG channels. Whole-cell patch-clamp recordings from cells transfected the cyclic nucleotides bind in canonical cyclic nucleotide-binding with WT hERG (A), hERG N861H (B), and hERG N861I (C) channels by using proteins. In addition, each structure contains an intrinsic ligand the indicated voltage protocol. (D) Current–voltage relationship indicating after the C-helix that occupies the β-roll cavity at the position robust current from WT hERG (squares), but no measurable currents from where cyclic nucleotides would normally bind. These signature hERG N861H (circles) or hERG N861I (triangle). (Scale bar: 1 s and 0.5 nA.) n ≥ features of KCNH channels reveal why the KCNH channels are 3 for each of the channels. (E) Representative immunoblot of whole-cell not regulated by the direct binding of cyclic nucleotides (23–26). lysates from control (untransfected) HEK293 cells or cells expressing WT Mutations of residues in the intrinsic ligand affect gating of hERG, hERG N861H, or N861I, as indicated. Similar results were obtained in channels in all three KCNH subfamilies, but the effects are dif- three independent experiments. ferent depending on the subfamily (refs. 18, 22, and 48 and Fig. 4). There are several plausible ways in which the intrinsic ligand channels have different oligomeric assembly. The C-linker/CNBHD could be dynamically regulated in the cell. Phosphorylation, 2+ of agERG channels crystallized as a monomer, zELK channels Ca -calmodulin, and PIP2 binding sites have all been reported crystallized as a dimer (18), and mHCN2 channels crystallized as a to occur near the intrinsic ligand of different KCNH channels – tetramer (21) (Fig. 6A). The differences in the oligomeric assembly (50 54). Another possibility is that the intrinsic ligand might stem from differences in the folding and orientation of the C-linker compete with an unknown extrinsic ligand for the binding site on regions of the three structures. Structural alignment of the entire the CNBHD. Therefore, the binding of the extrinsic ligand would C-linker/CNBHDs of agERG with zELK and mHCN2 gives an displace the intrinsic ligand from the site. Because the intrinsic α rmsd of 13 Å and 12 Å, respectively, and alignment of just the ligand is covalently attached to the C-helix, displacing it could α C-linkers gives an rmsd of 17 Å and 13 Å, respectively. Alignment of move the C-helix. This mechanism is reminiscent of what the β-rolls of the C-linker/CNBHDs of agERG, zELK, and mHCN2 happens in CNG and HCN channels when cyclic nucleotides reveals that the C-linkers are in dramatically different conformations in the three structures (Fig. 6B). The αA′ helix of agERG is ∼ ∼ α ′ agERG zELK mHCN2 rotated by 180° and shifted by 30 Å relative to the A helix in A monomer dimer tetramer mHCN2. The αA′ helix of zELK is rotated by ∼55° relative to its orientation in mHCN2. These results reveal that the C-linker can adopt different conformations in the agERG, zELK, and mHCN2 channel fragments. Whether these conﬁgurations correspond pre- cisely to native conformations of the channel is unknown, but it seems likely that they reﬂect a dynamic nature of the C-linker region, consistent with its proposed role in channel gating (49). B C The C-linker contains an “elbow” and “shoulder” region. The elbow is formed by the αA′ and αB′-helices, whereas the shoulder is formed by the αC′ and αD′-helices (Fig. 1C). Despite the struc- agERG o tural differences in the C-linkers of agERG, zELK, and mHCN2, in mHCN2 90 all three structures the elbows rest on the shoulder, and the elbow- zELK on-the-shoulder interface is very similar (Fig. 6C). For the zELK Fig. 6. The C-linker adopts different orientations in the structures of the and mHCN2 structures, the elbow-on-the-shoulder interface con- C-linker/CNBHDs of different KCNH and HCN channels. (A) A monomer of tains the primary intersubunit interactions in the C-linker/CNBD the C-linker/CNBHD of agERG (red; Left); a dimer formed by the C-linker/ fragment and is formed by the elbow of one subunit resting on the CNBHDs of zELK channels (18) viewed parallel to the twofold axis (blue and shoulder of the neighboring subunit. In the zELK structure, the yellow; Center); and a tetramer formed by the C-linker/CNBDs of HCN2 subunits forming the elbow-on-the-shoulder interface are related channels (21) viewed parallel to the fourfold axis (green and gray; Right). (B) The C-linkers of agERG (red), zELK (yellow), and HCN2 (green) with super- by a twofold symmetry (18), whereas in HCN2, the subunits are imposed α-carbons of the corresponding β-rolls. The CNBHDs were removed related by a fourfold symmetry (21) (Fig. 6A). for clarity. (C) Superposition of the elbow-on-the-shoulder interface of Surprisingly, however, in the agERG structure, the elbow is agERG (red), zELK (yellow), and HCN2 (green) channels shown from two resting on its own shoulder (Fig. 1C and Fig. S2). This is because different perspectives rotated by 90°.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1306887110 Brelidze et al. Downloaded by guest on September 26, 2021 bind to the CNBD and draw in the αC-helix (49). Regulation of a Mosquito (TTP LABTECH). The final protein solution contained the fol- mEAG1 channels by flavonoids was recently shown to occur by lowing: ∼250 mM KCl, 1 mM TCEP, and 30 mM Hepes at pH 7.0. The reservoir this mechanism (48). solution contained the following: 9 mM urea, 18.2% (wt/vol) PEG 3350, and The hERG channel, however, is specialized for its role in re- 7.3% (vol/vol) tacsimate at pH 7.0. The crystals were cryoprotected in reservoir solution supplemented with 25% (vol/vol) glycerol before being flash polarizing the cardiac action potential. This specialization includes frozen in liquid nitrogen. a slow rate of channel deactivation, allowing the channels to re- Diffraction data were collected at the ALS (beamline 8.2.1) at Lawrence main open during the repolarization of the cardiac action poten- Berkeley National Laboratory in Berkeley. Data were analyzed with Mosfilm tial. Here, we show that the slow deactivation arises, in part, from (55) and HKL2000 (56) software. The structure of the agERG C-linker/CNBHD the intrinsic ligand. We found that, unlike in ELK (18) and EAG was solved by molecular replacement using the structure of the CNBHD of (22) channels, in hERG channels mutations in the intrinsic ligand zELK channels (PDB ID code 3UKN) (18) as a search model. The molecular did not measurably affect the conductance–voltage relationship. replacement was carried out by using Phaser in PHENIX (57) followed by the Instead, these mutations accelerated the deactivation rate of energy- and electron density-guided structure optimization with Rosetta (45). fi hERG channels, similar to the effects of N-terminal mutations The quality of the model was improved by numerous cycles of re nement in PHENIX and manual model building in Coot (58). The asymmetric unit con- that cause LQTS, a condition characterized by a delayed retained one molecule in the P3221 space group. The crystallographic data and polarization of the ventricular action potential (32). refinement statistics are summarized in Table S1. Analysis with molprobity of hERG channels harbor hereditary mutations in the intrinsic the final model indicated 1.5% ramachandran outliers. Figures were made ligand that are associated with LQTS (11, 31). We found that using PyMOL. The electrostatic potential surface calculations were carried LQTS mutations N861I and N861H drastically decreased currents out by using the APBS plugin for PyMOL and colored from red (−3 kT/e) to from and altered trafficking of hERG channels. LQTS-associated blue (+3 kT/e). mutations in the C-linker/CNBHD have been shown to abolish hERG currents by interfering with the hERG channel expression Channel Expression in Oocytes and Cultured Cells. The full-length agERG (GI: at the cell surface (41–43). Together these findings indicate that 158285159) with a C-terminal FLAG epitope was generated by Bio Basic and theintrinsicligandisanimportant structural element for a subcloned into the pGEMHE oocyte expression vector (a gift of E. Liman, University of Southern California, Los Angeles, CA). The hERG1 expression LQTS phenotype. clone (GI: 487737) containing the S620T mutation to remove C-type in- The structure of agERG also revealed another difference from activation (47) was fused at position 1159 to a monomeric citrine fluorescent the structures of other related channels: The C-linker region is in protein (gift from R. Y. Tsien, University of California, San Diego, CA) and a dramatically different conformation compared with the structures subcloned into the pGH19 oocyte expression vector as described (37). The

of the ELK and HCN channels. Although the elbow-on-the-shoul- cRNA was transcribed by using the mMessage mMachine kit (Ambion). PHARMACOLOGY der interface is conserved in all three structures, the interface is Xenopus laevis oocytes were defolliculated and injected with 50 nL of agERG intrasubunit in agERG and intersubunit in ELK and HCN despite or hERG cRNA per oocyte. The oocytes were incubated for 3–10 d at 16 °C μ the high degree of sequence similarity between these channels. It in ND96 with 50 g/mL gentamicin. remains unclear whether the different conformations and oligomeric For electrophysiological recordings in HEK293 cells, WT and mutant (N861H fl and N861I) hERG expression clones were subcloned into the pcDNA3.1 mam- assembly seen in the structures from different channels re ect dif- malian expression vector. HEK293 cells were cultured and transiently trans- ferences in conformational state, differences in channel type, or arise fected with hERG as described (36). The cells were incubated for 24–48 h from working with channel fragments. However, these differences before analysis. almost certainly reflect that the C-linker is a dynamic structure. In CNG and HCN channels, the C-linker couples the binding of Electrophysiology and Data Analysis. Currents from whole oocytes or HEK293 cyclic nucleotide to the opening of the pore, a process thought to cells expressing the WT or mutant hERG channels were recorded with a two- involve a rearrangement of the elbow-on-the-shoulder interface electrode voltage-clamp (OC-725C; Warner Instruments) connected to an an- (49). It seems likely that the C-linker is also involved in coupling alog to digital converter (ITC-18; Instrutech) or a patch-clamp (EPC10; HEKA). the intrinsic ligand of KCNH channels to opening (or closing) of Data were recorded by using Patchmaster software (HEKA) and analyzed using Igor Pro software (WaveMetrics). The solutions for electrophysiological the pore. recordings from oocytes (37) and HEK293 cells (36) were described. − Materials and Methods Currents were elicited by a series of 1-s voltage pulses from 100 to 40 mV in 20-mV increments followed by a 3-s repolarizing pulse to −60 mV. For more fi FSEC. The C-linker/CNBHD of agERG channels [gene identi er (GI): 158285159, detailed investigation of the tail currents, the channels were first activated – amino acids S535 Q734] was covalently fused to a C-terminal GFP in the with a pulse to 20 mV followed by the application of voltage pulses between pCGFP-BC bacterial expression vector kindly provided by T. Kawate and −120 and 20 mV in 20-mV increments. The holding potential was −80 mV for E. Gouaux, Vollum Institute, Portland, OR (44). The constructs were transformed all experiments. Experiments were performed at room temperature. Error bars into BL21 (DE3) cells. The cell cultures were induced with IPTG and harvested by indicate the SEM. Statistical analysis was performed by using one-way ANOVA. centrifugation. The cell pellets were resuspended in a lysis buffer and sonicated. AvalueP < 0.05 was considered statistically significant. n represents the Insoluble protein was separated by centrifugation, and the supernatant was number of recordings. analyzedwithFSEConaSuperdex20010/300GLcolumn(GEHealthcare). To obtain conductance–voltage relationships, peak tail current amplitudes at −100 mV (Fig. 3 A–D, Insets) were normalized to the largest peak conduc- fi Scale-Up Protein Puri cation. The C-linker/CNBHD of agERG (amino acids tance amplitude, which followed a step to 40 mV. These normalized data were S535–Q734) was subcloned into a modified pMALc2T vector (New England then plotted against the test voltage and were fit with a Boltzmann equation. Biolabs) containing an N-terminal maltose-binding protein (MBP) affinity Deactivating currents were fit with a single-exponential equation. tag followed by a thrombin cleavage site. The protein was expressed in BL21 (DE3) Escherichia coli cells as described (23). The cells were harvested by Cell Lysis and Western Blot Analysis. Cells were homogenized and lysed in lysis centrifugation, resuspended in a lysis buffer [500 mM KCl, 1 mM Tris(2-car- buffer as described (36). Lysates were cleared by centrifugation. The protein boxyethyl)phosphine (TCEP), 30 mM Hepes, 1 mM PMSF, and 2.5 mg/mL concentration of the supernatant was quantified by using a Bradford assay fl DNase at pH 8.0] and lysed in an Emulsi ex-C5 (Avestin). Insoluble protein (Pierce). Protein samples (20 μg) were incubated with equal amounts of fi was separated by centrifugation. The C-linker/CNBHD of agERG was puri ed Laemmli sample buffer with 5% (vol/vol) β-mercaptoethanol (Bio-Rad) for 30 fi on an amylose af nity column and then was loaded on a HiTrap SP FF ion- min at room temperature, subjected to 7.5% SDS/PAGE, and electrophoreti- exchange column following an overnight cleavage with thrombin at 4 °C. cally transferred onto nitrocellulose membranes. Membranes were immuno- – The protein was eluted with a linear KCl gradient and concentrated to 5 10 blotted with an anti–hERG-KA antibody followed by an HRP-linked secondary mg/mL for crystallization. antibody (Jackson Labs) and developed by using an ECL detection kit (Thermo).

Crystallization and Structure Determination. Crystals were grown at 20 °C by ACKNOWLEDGMENTS. We thank S. Camp and S. Cunnington for excellent using the sitting-drop vapor diffusion method. One hundred ﬁfty nanoliter technical assistance and the beamline staff at the Advanced Light Source drops of the concentrated protein and reservoir solution were mixed 1:1 by (ALS) for help with data collection. This work was supported by the Howard

Brelidze et al. PNAS Early Edition | 5of6 Downloaded by guest on September 26, 2021 Hughes Medical Institute (HHMI) and National Institutes of Health (NIH) Institute of General Medical Sciences, and the HHMI. The ALS is supported Grants R01 EY010329 (to W.N.Z.) and R01 HL083121 (to M.C.T.). The Berkeley by the Director, Ofﬁce of Science, Ofﬁce of Basic Energy Sciences, of the Center for Structural Biology is supported in part by the NIH, National Department of Energy under Contract DE-AC02-05CH11231.

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