Conductance of P2X4 Purinergic Receptor Is Determined by Conformational Equilibrium in the Transmembrane Region

Conductance of P2X4 purinergic receptor is determined by conformational equilibrium in the transmembrane region

Yuichi Minatoa,1, Shiho Suzukia,1, Tomoaki Haraa, Yutaka Kofukua, Go Kasuyab, Yuichiro Fujiwarac, Shunsuke Igarashid, Ei-ichiro Suzukid, Osamu Nurekib, Motoyuki Hattorie,f, Takumi Uedaa,f, and Ichio Shimadaa,2

aGraduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; bDepartment of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan; cDepartment of Physiology, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; dInstitute for Innovation, Ajinomoto, Kawasaki 210-8681, Japan; eState Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Yangpu District, Shanghai 200438, China; and fPrecursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Chiyoda-ku, Tokyo 102-0075, Japan

Edited by Richard W. Aldrich, The University of Texas at Austin, Austin, TX, and approved March 15, 2016 (received for review January 15, 2016)

Ligand-gated ion channels are partially activated by their ligands, The P2X receptors are a family of cation channels gated by resulting in currents lower than the currents evoked by the physio- extracellular ATP (1, 7–9) and are involved in many physiological logical full agonists. In the case of P2X purinergic receptors, a cation- and pathophysiological processes (10–12). Seven subtypes of the selectiveporeinthetransmembrane region expands upon ATP P2X receptors have been identified in mammals (13), and they ∼ binding to the extracellular ATP-binding site, and the currents share 40% sequence identity. The P2X4 receptor is involved in evoked by α,β-methylene ATP are lower than the currents evoked the pathogenesis of chronic neuropathic, inflammatory pain and by ATP. However, the mechanism underlying the partial activation the endothelial cell-mediated control of vascular tone (11, 14, 15). α β α β of the P2X receptors is unknown although the crystal structures of Compared with ATP, , -methylene ATP ( , -meATP), in which the oxygen atom linking the α-andβ-phosphorous atoms of ATP zebrafish P2X4 receptor in the apo and ATP-bound states are avail- A able. Here, we observed the NMR signals from M339 and M351, is replaced by a methylene group (Fig. S1 ), reportedly induces a which were introduced in the transmembrane region, and the en- lower maximum current in cells expressing the mouse, rat, and human P2X4 receptors and other P2X receptors (16, 17). dogenous alanine and methionine residues of the zebrafish P2X4 α β The crystal structures of zebrafish P2X4 receptor (zfP2X4) (18, purinergic receptor in the apo, ATP-bound, and , -methylene 19), together with mutational analyses (20–26), provided the ATP-bound states. Our NMR analyses revealed that, in the structural basis for the channel opening of P2X receptors upon α,β-methylene ATP-bound state, M339, M351, and the residues that ATP binding. In the crystal structures, zfP2X4 forms a homotrimer connect the ATP-binding site and the transmembrane region, M325 (27, 28), in which the transmembrane region of each subunit is and A330, exist in conformational equilibrium between closed and composed of two helices (19). In the crystal structure of zfP2X4 in open conformations, with slower exchange rates than the chemical the ATP-bound state, three ATP molecules are bound to the −1 shift difference (<100 s ), suggesting that the small population of intersubunit nucleotide binding pockets. In addition, the region the open conformation causes the partial activation in this state. that connects the ATP-binding site and the transmembrane re- Our NMR analyses also revealed that the transmembrane region gion, which is referred to as the “lower body” (Fig. 1 A and B), is adopts the open conformation in the state bound to the inhibitor trinitrophenyl-ATP, and thus the antagonism is due to the closure Significance of ion pathways, except for the pore in the transmembrane region: i.e., the lateral cation access in the extracellular region. Partial agonists of ligand-gated ion channels reportedly offer clinical advantages over antagonists and full agonists in anti- NMR | membrane proteins | ligand-gated ion channels | depressant and smoking-cessation treatment. In the cases of insect cell expression system | purinergic receptors P2X purinergic receptors, the currents evoked by α,β-methylene ATP are lower than the currents evoked by ATP. Here, our NMR n chemical neurotransmission, various neurotransmitters bind to analyses revealed that the transmembrane region and the Iligand-gated ion channels expressed in the plasma membrane of membrane side of the lower body exist in conformational postsynaptic cells, such as the NMDA, AMPA, and P2X receptors, equilibrium between the closed and open conformations, with

leading to changes in membrane potential and the concentration slower exchange rates than the chemical shift difference BIOPHYSICS AND -1 of intracellular ions. Each ligand for a ligand-gated ion channel has (<100 s ), and that the small population of the open confor- COMPUTATIONAL BIOLOGY

a distinct ability to evoke currents (1), and the ligands are classified mation of zebrafish P2X4 purinergic receptor causes the partial according to the evoked current level: such as, full agonists, partial activation in the α,β-methylene ATP-bound state. These find- agonists, and antagonists. Partial agonists of ligand-gated ion chan- ings provide insights into the mechanism underlying the par-

nels reportedly offer clinical advantages over antagonists and full tial activation of P2X4 receptors and other ligand-gated agonists in antidepressant and smoking-cessation treatment (2, 3). ion channels. Two mechanisms have been proposed for the partial activation of the ligand-gated ion channels: the equilibrium between the Author contributions: Y.M., S.S., T.H., Y.K., G.K., O.N., M.H., T.U., and I.S. designed research; open and closed conformations and the distinct conformation of Y.M., S.S., T.H., Y.K., G.K., O.N., M.H., T.U., and I.S. performed research; Y.F., S.I., and E.-i.S. the partial agonist-bound states from the closed and open con- contributed new reagents/analytic tools; Y.M., S.S., T.H., Y.K., G.K., O.N., M.H., T.U., and I.S. formations (4, 5). In the crystal structures of the extracellular analyzed data; and Y.M., S.S., T.H., Y.K., G.K., O.N., M.H., T.U., and I.S. wrote the paper. region of the AMPA receptor, in which the distances between The authors declare no conflict of interest. the two extracellular domains are changed upon agonist binding, This article is a PNAS Direct Submission. the interdomain distances in the partial agonist-bound states 1Y.M. and S.S. contributed equally to this work. correlated with the conductance level, suggesting that the AMPA 2To whom correspondence should be addressed. Email: [email protected]. receptor adopts specific intermediately permeable conforma- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tions (4, 6). 1073/pnas.1600519113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1600519113 PNAS | April 26, 2016 | vol. 113 | no. 17 | 4741–4746 Downloaded by guest on September 26, 2021 expanded by ∼10 Å in the ATP-bound state, and a pore is formed receptor is embedded in lipid bilayers under physiological condi- in the transmembrane region, which is proposed to expand by the tions. It was recently reported that reconstituted high-density li- iris-like movement of the transmembrane helices (18). However, poproteins (rHDLs), which are also known as nanodiscs (29), can the mechanism underlying the partial activation of P2X receptors accommodate membrane proteins within a 10-nm-diameter disk- is unknown because the structures of the P2X receptors have not shaped lipid bilayer (30). The rHDLs reportedly provide a lipid environment with more native-like properties, compared with li- been examined in the partial agonist-bound states. posomes, in terms of the lateral pressure and curvature profiles The P2X receptor used in the previous crystallographic 4 because detergent micelles have strong curvature and different studies was solubilized by detergents, which are widely used for lateral pressure profiles from lipid membranes (31). Our NMR structural investigations of membrane proteins, but the P2X4 analyses of a G protein-coupled receptor (GPCR) and an ion channel in rHDL lipid bilayers revealed that the population and the exchange rates of the conformational equilibrium determine their signal transduction and ion transport activities (32–34) and that the population of the active conformation of the GPCR in rHDLs correlated better with the signaling levels than that in detergent micelles (32). Therefore, NMR investigations of membrane proteins in the lipid bilayer environments of rHDLs are necessary for accurate measurements of the exchange rates and the populations in conformational equilibrium. Here, we used NMR to observe the conformational equilibrium of the alanine and methionine residues of zfP2X4 bound to α,β-meATP in rHDLs. Based on the conformational equilibrium, we discuss the mechanism underlying the partial activation of P2X receptors. Results Steady-State Current Evoked by α,β-meATP. The amplitude of the plateau current determines the responsiveness of the P2X receptors in the sustained presence of ATP (11). In addition, NMR spectra are recorded after prolonged (>30 min) exposure to the ligands. Therefore, the currents in the sustained presence of ATP or α,β-meATP were determined by whole cell two-electrode voltage-clamp (TEVC) analyses using Xenopus oocytes expressing the rat P2X4 receptor. As a result, the currents in the sustained presence of α,β-meATP were ∼20% of the currents in the sustained presence of ATP (Fig. S1 B and C).

Preparation and Characterization of Zebrafish P2X4 Embedded in Reconstituted High-Density Lipoproteins. A truncated zebrafish P2X4 receptor (zfP2X4) construct (35, 36) with the M364L mutation, with residues S28 through K365, was expressed in a baculovirus–insect cell expression system. Hereafter, this construct is referredtoasΔzfP2X4-A′. TEVC analyses using Xenopus oocytes expressing ΔzfP2X4-A′ revealed that ΔzfP2X4-A′ retains the full ATP-dependent channel activity (Fig. S1D). To examine the conformational dynamics of ΔzfP2X4-A′ embedded in lipid bilayers, purified ΔzfP2X4-A′ in n-dodecyl-β-D-maltopyranoside (DDM) micelles was reconstituted into rHDLs, and further purified with three chromatography steps. The size exclusion chromatography revealed that purified ΔzfP2X4-A′ in rHDLs is monodisperse, with aStokesdiameterof12nm(Fig. S1E), in good agreement with the previously reported rHDL size (37). As judged from the SDS/ PAGE analysis, the purity of ΔzfP2X4-A′ in rHDLs was >90%, andtheratioofzfP2X4 and MSP1 was consistent with the 3:2 stoichiometry that would be expected if each rHDL particle is Fig. 1. NMR resonances from the endogenous methionine residues of zfP2X4 in composed of two MSP1 molecules and a trimer of zfP2X4 (Fig. Δ – ′ 3 rHDL. (A and B) Distribution of the methionine residues in the zfP2X4 A .One S1F). The dose-dependent curve observed in the H-ATP binding subunit from the crystal structure of zfP2X4 in the apo form (A)(PDBIDcode assays revealed that the EC50 value and the Hill coefficient were 4DW0) and one from the ATP-bound form (B) (PDB ID code 4DW1) are shown in 93 nM and 1.5, respectively (Fig. S1G), which are comparable with ribbons. The lower body and the right flipper are yellow. The A330 residues, the the previously reported affinity and cooperativity of the interaction methionine residues, and the residues in which methionine mutations were in- between zfP2X4 and ATP (18, 19). troduced, L339 and L351, are depicted by green sticks. ATP is depicted by red sticks. Dummy atoms generated by Orientations of Proteins in Membranes (OPM), which NMR Resonances from Endogenous Methionine Residues in the represent membrane boundary planes, are gray. (C)Overlaid1H-13CHMQCspectra 2 2 13 Extracellular Region. ΔzfP2X4-A′ possesses five methionine resi- of [ H-11AA, α, β- H, methyl- C-Met]ΔzfP2X4-A′, embedded in rHDLs, in the apo state (black) and the ATP-bound state (red). The regions with resonances from dues in the extracellular region, and they adopt distinctly different conformations between the apo and ATP-bound crystal structures methionine residues are shown, and the assigned resonances are indicated. The A B centers of the resonances are indicated with dots. Cross-sections at lines through (Fig. 1 and ). M108, M249, and M256 exist in the region the centers of each resonance in the ATP-bound state and the cross-sections of the previously referred to as the “right flipper” or the extracellular spectra using [α, β-2H, methyl-13C-Met]ΔzfP2X -A′ are shown on the top of the side of the lower body, and M325 exists on the membrane side of 4 13 overlaid spectra. The intensities of the cross-sections were normalized by the con- the lower body (Fig. 1 A and B). Therefore, we used the Cse-

centration of ΔzfP2X4-A′ and the conditions of the NMR measurements. lective labeling of methionine methyl groups to investigate the

4742 | www.pnas.org/cgi/doi/10.1073/pnas.1600519113 Minato et al. Downloaded by guest on September 26, 2021 structure of ΔzfP2X4-A′. To observe the NMR resonances from the huge trimeric ΔzfP2X4-A′ (∼250 kDa in rHDLs), ΔzfP2X4-A′ was deuterated for sensitivity enhancement. We selected the deuterated amino acids, based on the previously reported labeling efficiencies (32) and the 1H–1H distances between the observed methionine methyl groups and each amino acid residue in the crystal structures of zfP2X4. Our calculation revealed that, with the deuteration of the alanine, phenylalanine, glycine, isoleucine, leucine, proline, arginine, serine, threonine, valine, and trypto- phan residues, the 1H–1Hdipole–dipole interactions of the methyl groups in ΔzfP2X4-A′ would be ∼30% of the dipole–dipole interactions of nondeuterated ΔzfP2X4-A′ (Fig. S1 H and I), and the sensitivities of the resonances from these residues would be in- creased by about fivefold upon deuteration. Therefore, we pre- pared ΔzfP2X4-A′ in which the methionine methyl groups were labeled with 13C and the 11 types of amino acid residues were deuterated. Hereafter, the ΔzfP2X4-A′ obtained by this method is 2 2 13 referred to as [ H-11AA, αβ- H-, methyl- C-Met] ΔzfP2X4-A′. In the 1H-13C HMQC spectra of [2H-11AA, αβ-2H-, methyl- 13 C-Met] ΔzfP2X4-A′ in rHDLs in the apo state, signals that apparently correspond to the five methionine residues were observed (Fig. 1C). In the spectra of the ATP-bound states, all resonances markedly shifted, compared with the resonances in the apo state (Fig. 1C). For the assignment of the methionine resonances, we mutated the methionine residues. For example, M108 was assigned by introducing the M108L mutation. Assignments were established using the spectra in DDM micelles, considering that chemical shifts Fig. 2. NMR resonances from A330, M339, and M351. (A and B) Distribution of

in rHDLs were similar to the chemical shifts in DDM micelles. In the alanine residues in ΔzfP2X4–A′. One of the subunits from the crystal structure the apo and ATP-bound states, one resonance was absent in the of zfP2X4 in the apo form (A) (PDB ID code 4DW0) and one from the ATP-bound spectra of the M108L mutant, revealing that these resonances are form (B) (PDB ID code 4DW1) are shown in ribbons. Alanine and tyrosine residues from M108 (Fig. S2 A and B). The resonances from the other are depicted by green and white sticks, respectively. (C)Overlaid1H-13CHMQC 2 α 2 13 Δ – ′ methionine residues in the apo and ATP-bound states were spectra of [ H-5AA, - H, methyl- C-Ala] zfP2X4 A without the M364L mutation, assigned in a similar manner (Fig. 1C and Fig. S2 C–J). embedded in rHDLs, in the apo state (black) and the ATP-bound state (red). (D and 1 13 2 E) Position of L339 in the zfP2X4.(F)Overlaid H- CHMQCspectraof[H-11AA, α, β 2 13 Δ – ′ Observation and Assignments of the NMR Resonances from A330. - H, methyl- C-Met] zfP2X4 A /L339M, embedded in rHDLs, in the apo state Δ ′ (black) and the ATP-bound state (red). (G and H)PositionofL351inzfP2X4.(I) zfP2X4-A possesses 17 alanine residues, and A330, as well as 1 13 2 α β 2 13 Δ – M325, exists on the transmembrane side of the lower body. It is Overlaid H- C HMQC spectra of [ H-11AA, , - H, methyl- C-Met] zfP2X4 A′/L351M, embedded in rHDLs, in the apo (black) and ATP-bound (red) states. possible that the resonance from A330 will be separated from the In D, E, G,andH, the transmembrane helices of the crystal structures of zfP2X4 other resonances from the alanine residues because the Y198 in the apo and ATP-bound states, respectively, viewed from the intracellular ring current should induce a strong upfield shift in the A330 side, are shown in ribbons. One of the subunits is colored blue, and L339 (D methyl group, based on the crystal structures of zfP2X4 in the and E)orL351(G and H) in this subunit is depicted by a green stick model. In C, apo and ATP-bound states (Fig. 2 A and B). Therefore, we used F,andI, only the regions with the A330, M339, and M351 methyl resonances the alanine methyl groups to investigate the conformation of the are shown, respectively, and the centers of the resonances from these residues extracellular region of zfP2X4. are indicated with dots. The full spectra are shown in Figs. S3 and S4. The preparation of the deuterated and alanine selectively labeled protein, using the insect cell–baculovirus expression system, was accomplished by modification of the aforementioned A330 corresponds to G325. As a result, the most upfield-shifted method for the preparation of the deuterated and methionine resonance was absent in the spectra of the A330G mutant, revealing selectively labeled protein. We added deuterated amino acids and that this resonance is from A330 (Fig. S3C). The resonance from algal amino acid mixtures, as well as [α-2H-, methyl-13C]-alanine, A330 markedly shifted upon ATP binding (Fig. 2C). to the amino acid-deficient medium. The 13C labeling efficiencies were calculated from NMR analyses of thioredoxin, which was NMR Resonances from Methionine Residues Introduced in the used as a test case. As a result, the alanine methyl groups were Transmembrane Region. To investigate the structure of the trans- selectively labeled with 13C with an efficiency of ∼30%. membrane region, we introduced methionine residues to the ex- BIOPHYSICS AND For the observation of the A330 methyl resonances of zfP2X4,we tracellular and intracellular sides of the transmembrane helix 2 COMPUTATIONAL BIOLOGY selected the deuterated amino acids in the same manner as described (TM2) by the L339M and L351M mutations, respectively (Figs. 1 A above for the methionine methyl groups. Our calculation revealed and B and 2 D and E and G and H), according to the comparison that, in the case of the deuteration of the cysteine, phenylalanine, with the human P2X4 receptor, in which L339 and L351 correspond glycine, leucine, and tyrosine residues and alanine Hα,the1H–1H to M336 and M348, respectively. L351 is a pore-forming residue – Δ ′ dipole dipole interactions of the A330 methyl groups in zfP2X4-A (Fig. 2H) and is close to a glycine residue, G350 in zfP2X4,which would be ∼25% of the dipole–dipole interactions of nondeuterated was proposed to function as a gating hinge (18, 38). We confirmed ΔzfP2X4-A′. Hereafter, the ΔzfP2X4-A′ obtained by this method is that the L339M and L351M mutants retained full ATP-binding and 2 2 13 referred to as [ H-5AA, α- H-, methyl- C-Ala] ΔzfP2X4-A′. ATP-dependent cation channel activities (Fig. S4 A and D). In the 1H-13C heteronuclear multiple quantum coherence In the 1H-13CHMQCspectraof[2H-11AA, αβ-2H-, methyl- 2 α 2 13 13 (HMQC) spectra of apo [ H-5AA, - H-, methyl- C-Ala] C-Met] ΔzfP2X4-A′/L339M and ΔzfP2X4-A′/L351M in the apo ΔP2X4-A′ in rHDLs, the signals that apparently correspond to and ATP-bound states, one resonance was additionally observed in the 17 alanine residues were observed (Fig. S3A). In the spectra each spectrum, revealing that these resonances are from M339 and of the ATP-bound states, several resonances markedly shifted, M351 (Fig. S4 B and C and E and F). The remaining signals did not compared with the resonances in the apo state (Fig. S3B). For the exhibit significant changes upon introducing the L339M and L351M resonance assignments, the spectra of the A330G mutant were mutations, suggesting that the conformation of the extracellular re- recorded, according to the comparison with rat P2X7,inwhich gion of ΔzfP2X4-A′ was not affected by these mutations. Both of the

Minato et al. PNAS | April 26, 2016 | vol. 113 | no. 17 | 4743 Downloaded by guest on September 26, 2021 resonances from M339 and M351 remarkably shifted upon ATP resonances with chemical shifts almost identical to the chemical shifts binding (Fig. 2 F and I and Fig. S4 B and C and E and F). in the apo state and the chemical shifts in ATP-bound state were also observed for M268 (Fig. 3E). In the spectra of [2H-11AA, αβ-2H-, α β – 13 Conformational Equilibrium in the , -meATP Bound State. We used methyl- C-Met] ΔzfP2X4-A′/L339M with a lower concentration of the methionine and alanine methyl groups to investigate the con- α,β-meATP, the M339 signals were observed at the same formation of the zfP2X4 bound to a partial agonist, α,β-meATP. chemical shifts as the signals at higher ligand concentrations Our TEVC analyses revealed that the α,β-meATP–evoked currents (Fig. S5 H–J), suggesting that the NMR signals are not signifi- from ΔzfP2X4-A′ expressed in Xenopus oocytes were ∼30% of the cantly affected by the exchange between the free and bound states currents induced by ATP (Fig. S5 A–C)andthatα,β-meATP or the nonspecific effects of the ligands. 3 competed with H-ATP binding to ΔzfP2X4-A′ in a concentration- To examine the resonances from M325, M339, and M351 in the dependent manner, with a Ki of 2.7 μM(Fig. S5D). α,β-meATP–bound state undergoing conformational exchange, we To investigate the structure in the transmembrane region of also recorded the spectra at a higher temperature, 318 K (Fig. 3 B– 1 13 zfP2X4 bound to α,β-meATP, the H- CHMQCspectraof D). As a result, the intensity ratios of the resonances from M325, 2 α 2 13 Δ ′ 2 αβ 2 [ H-5AA, - H-, methyl- C-Ala] P2X4-A ,[H-11AA, - H-, M339, and M351 with chemical shifts identical to the chemical 13 2 2 methyl- C-Met] ΔzfP2X4-A′/L339M, and [ H-11AA, αβ- H-, shifts in the apo state, relative to the resonances with chemical 13 Δ ′ methyl- C-Met] zfP2X4-A /L351M in rHDLs were observed, shifts identical to the chemical shifts in ATP-bound state, were > Δ ′ under the condition where 99% of zfP2X4-A bound to decreased with the increase of temperature. α,β-meATP (Fig. 3 and Fig. S5 E–G). As a result, two resonances In the cases of the resonances from M108, M249, and M256, each were observed for A330, M339, and M351, which exist in the which are on the right flipper or the extracellular side of the lower transmembrane region and on the membrane side of the lower body (Fig. 1 A and B), the chemical shifts in the α,β-meATP– body (Fig. 1 A and B), and their chemical shifts were almost bound state were almost identical to the chemical shifts in the identical to the chemical shifts in the apo and ATP-bound ATP-bound state (Fig. 3 F–H). states (Fig. 3 A–C). Two resonances were also observed for M325, which exists on the membrane side of the lower body NMR Resonances in the TNP-ATP–Bound State. To examine how (Fig. 1 A and B), and the chemical shift of one of the resonances was different ligands produce distinct structural changes in different almost identical to that in the ATP-bound state (Fig. 3D). Two parts of P2X receptors, we recorded the NMR spectra of zfP2X4

Fig. 3. Resonances from M268, M325, A330, M339, and M351 in the α,β-meATP–bound state. 1H-13C HMQC spectra of [2H-5AA, α-2H, methyl-13C-Ala] 2 2 13 ΔzfP2X4–A′ (A), [ H-11AA, α,β- H, methy- C-Met] 2 ΔzfP2X4–A′/L339M (B and D–H), and [ H-11AA, 2 13 α,β- H, methy- C-Met] ΔzfP2X4–A′/L351M (C), embedded in rHDL, in the α,β-meATP–bound state recorded at 310 K (cyan) and the spectra recorded at 318 K (purple) are shown. The centers of the resonances from these residues are indicated with dots. Only the regions with A330, M339, M351, M325, M268, M108, M249, and M256 methyl signals are shown in A, B, C, D, E, F, G,andH, respectively. Dashed lines represent the 1Hor 13C chemical shifts of the resonances in the α,β-meATP– bound states, observed at 310 K. The full spectra in the α,β-meATP–bound state recorded at 310 K are shown in Fig. S5.InE, the chemical shifts of the M268 signals shown in the Middle in the apo state are similar to the chemical shifts in the ATP-bound state, and the M268 signal in the apo state was not observed in the Bottom.

4744 | www.pnas.org/cgi/doi/10.1073/pnas.1600519113 Minato et al. Downloaded by guest on September 26, 2021 bound to trinitrophenyl ATP (TNP-ATP), which is composed of In the crystal structure of zfP2X4 in the ATP-bound state, the ATP and a trinitrophenyl group attached to the 2′ and 3′ hydroxyl regions previously referred to as the “dorsal fin” and the “left of the ribose moiety (Fig. S6A) and is reportedly a representative flipper” (Fig. S8A) directly bind to ATP and adopt markedly non–subtype-selective antagonist of P2X receptors that competes different conformations from the conformations in the apo state, with ATP (10, 39–41). We confirmed that the ATP-evoked currents and the conformation of the left flipper is closely associated with Δ ′ of zfP2X4-A were inhibited by TNP-ATP and that TNP-ATP did the conformations of the membrane side of the lower body and Δ ′ B not evoke currents in oocytes expressing zfP2X4-A (Fig. S6 and the transmembrane region (Fig. S8B). The residues in the left 1 13 2 2 13 C). In the H- CHMQCspectraof[H-5AA, αβ- H-, methyl- C- flipper (N296 and R298) bind to the phosphates of ATP, in which 2 13 Met, α- H-, methyl- C-Ala] ΔP2X4-A′/L339M in the TNP-ATP– one of the oxygen atoms is replaced by a methylene group in bound state, two resonances from A330 with 1Hchemical α,β-meATP (Fig. S1A), whereas the ribose and adenine base of shifts different from that in the ATP-bound state were observed ATP bind to the residues on the dorsal fin (L217 and L232) and 1 13 2 2 (Fig. S7A). In the H- C HMQC spectra of [ H-11AA, αβ- H-, the extracellular side of the lower body (T189 and L191) (Fig. 13 Δ ′ 2 αβ 2 methyl- C-Met] zfP2X4-A /L339M and [ H-11AA, - H-, S8A). Therefore, in the α,β-meATP–bound state, the perturbation 13 Δ ′ – methyl- C-Met] zfP2X4-A /L351M in the TNP-ATP bound of the interaction between the left flipper and ATP would induce state, the chemical shifts of the resonances from M339 and changes in the conformational equilibrium in the left flipper, the M351, as well as the resonances from the endogenous methi- membrane side of the lower body, and the transmembrane region, onine residues, were almost identical to the chemical shifts in whereas the ATP-binding mode and the conformations of the B C the ATP-bound state (Fig. S7 and ). dorsal fin and the extracellular side of the lower body would be similar to the conformations in the ATP-bound state (Fig. S8B). Discussion A330 is located near D61 and Q329, which were proposed to In this study, we established methods for alanine residue-selective form the lateral cation pathway, because the ATP-evoked cur- – labeling and deuteration in the insect cell baculovirus system. rents were affected by the chemical modification of these resi- These methods, along with recently developed methods for me- dues (42). Therefore, the difference between the 1H chemical thionine residue selective labeling (32), enabled us to observe the shift of A330 in the ATP-bound state and chemical shift in the resonances from ΔzfP2X4-A′ in rHDLs in the apo, ATP-bound, and α,β-meATP–bound states (Figs. 1–3). The 1H chemical shifts of methionine methyl signals depend on the ring current effects from the neighboring aromatic rings: Y45 in the case of M339 (Fig. 2 D and E). The 1H chemical shift of M339 in the apo state (1.82 ppm) exhibits a larger upfield ring current shift than that in the ATP-bound state (1.91 ppm) (Fig. 2F and Fig. S4 B and C), implying the close contact of the M339 methyl group with Y45 in the apo state. In the crystal structure of apo zfP2X4, Y45 is in close proximity to L339 whereas no aromatic residue contacts the sidechain of L339 in the crystal structure of zfP2X4 with ATP (Fig. 2 D and E). These characteristics of the structure are in good agreement with the above-described conformations indicated by the 1H chemical shifts. Therefore, we conclude that the resonances observed in the apo and ATP-bound states correspond to the closed and open conformations, respectively (Fig. 4 A and B). The ATP-induced chemical shift changes observed for the other methionine resonances (Figs. 1 and 2) are in agreement with the conformational differences of the crystal structures in the apo and ATP-bound states. In the α,β-meATP–bound state, the observation of two resonances each for A330, M325, M339, and M351 in the α,β-meATP– bound state (Fig. 3), together with the temperature-dependent population shift, suggests that the transmembrane region and the membrane side of the lower body exist in conformational equilibrium between the closed and open conformations, with slower − exchange rates than the chemical shift difference (<100 s 1)(Fig. 4C). The population of the open conformation in the state bound to α,β-meATP at 293 K, which was calculated from the signal intensities of the resonances from M339, is ∼35% (see SI Text BIOPHYSICS AND for details), in good agreement with the currents evoked by COMPUTATIONAL BIOLOGY α,β-meATP relative to the currents evoked by ATP in the TEVC recordings of ΔzfP2X4-A′ (Fig. S5C). These results suggest that the small population of the open conformation of zfP2X4 causes the partial activation in the α,β-meATP–bound state. The small chemical shift difference between the apo and α,β-meATP–bound states observed for M325 (Fig. 3D) suggests that the closed conformation of the membrane side of the lower body in the α,β-meATP–bound state is slightly different from that in the apo state. The chemical shifts of the resonances from Fig. 4. Schematic diagrams of the conformational equilibrium of the lower the other methionine resonances suggest that, even in the α β – body and the transmembrane region of zfP2X4 in the apo (A), ATP-bound , -meATP bound state, the conformation of the extracellular (B), and α,β-meATP–bound (C) states. The conformations of the lower body side of the lower body is almost identical to that in the ATP- and the transmembrane region change upon ATP binding. In the bound state (Fig. 4C). Therefore, the conformational changes of α,β-meATP–bound state, the transmembrane region and the region con- the extracellular and transmembrane sides, which are observed necting the transmembrane region and the membrane side of the lower in the crystal structures of zfP2X4 in the apo and ATP-bound body exist in equilibrium between the closed and open conformations, with states, are uncoupled in the α,β-meATP–bound state. exchange rates slower than the chemical shift differences (<100 s−1).

Minato et al. PNAS | April 26, 2016 | vol. 113 | no. 17 | 4745 Downloaded by guest on September 26, 2021 TNP-ATP–bound state (Fig. S7A) suggests that the conforma- DDM micelles demonstrate that the exchange rate and/or the tion of the lateral cation pathway in the ATP-bound state is population of the conformational equilibrium in the α,β-meATP– different from that in the TNP-ATP–bound state. In contrast, the bound state in DDM micelles are markedly different from the ex- chemical shifts of the resonances from M339 and M351, as well as change rate and/or the population in rHDLs, and not in agreement the resonances from the endogenous methionine residues, were with the current evoked by α,β-meATP in the TEVC recordings almost identical to the chemical shifts in the ATP-bound state (Fig. S9) (see SI Text for details). Therefore, we conclude that NMR (Fig. S7 B and C), suggesting that the antagonism induced by investigations of zfP2X4 in the lipid bilayer environments of rHDLs TNP-ATP is not due to the closure of the pore in the trans- are necessary for accurate measurements of the exchange rates and membrane region. In the crystal structure of zfP2X4 in the ATP- the populations in conformational equilibrium. bound state, the 2′ and 3′ hydroxyl groups of ATP, to which a trinitrophenyl group is attached in the case of TNP-ATP, are Materials and Methods recognized by the dorsal fin (Fig. S8A), and the dorsal fin is See SI Materials and Methods for the reagents (44, 45) and the methods for

proposed to form a lateral cation access pathway through the the expression and purification of zfP2X4, preparation of zfP2X4 in rHDLs, fenestrations above the ion channel pore (18, 19, 42, 43). There- [3H] ATP binding assay (46), NMR experiments (47), and the two-electrode fore, in the TNP-ATP–bound state, the conformation and the voltage clamp electrophysiology (48–50). ATP-binding mode of the dorsal fin would be perturbed by the trinitrophenyl group, leading to the closure of the lateral cation ACKNOWLEDGMENTS. We thank Dr. Eric Gouaux for kindly providing zfP2X4 access pathway (Fig. S8C). plasmids, Dr. Kazunari Tohara for kindly providing Xenopus oocytes, and Drs. Previous NMR analyses of membrane proteins embedded in the Masanori Osawa and Noritaka Nishida for helpful advice. This work was supported in part by grants from the Japan New Energy and Industrial Technology Develop- lipid bilayers of rHDLs revealed that the conformational equilibria ment Organization (NEDO) and the Ministry of Economy, Trade and Industry and the functions of membrane proteins are affected by the lipid (METI), and by a Grant-in-Aid for Scientific Research on Priority Areas from the bilayer environments (32, 33). Our NMR spectra of ΔzfP2X4-A′ in Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT).

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