Phospholipid Membrane Fluidity Alters Ligand Binding Activity of a G

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Phospholipid membrane fluidity alters ligand binding activity of a G protein-coupled receptor by shifting conformational equilibrium Kouhei Yoshida, Satoru Nagatoishi, Daisuke Kuroda, Nanao Suzuki, Takeshi Murata, and Kouhei Tsumoto Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01194 • Publication Date (Web): 08 Jan 2019 Downloaded from http://pubs.acs.org on January 8, 2019

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Biochemistry

Phospholipid membrane fluidity alters ligand binding activity of a G protein-coupled receptor by shifting conformational equilibrium Kouhei Yoshida†, Satoru Nagatoishi†,‡, Daisuke Kuroda†,§, Nanao Suzuki∥, Takeshi Murata∥, Kouhei Tsumoto†,‡,§,* †

Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 1138656, Japan ‡

The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan

§

Medical Device Development and Regulation Research Center, School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan ∥

Graduate School of Science, Chiba University, 1-33, Yayoi-cho, Inage-ku, Chiba 263-8522, Japan

*Corresponding Author: (Phone) +81 3 6409 2129. (Fax) +81 3 6409 2129. (E-mail) [email protected]

ABSTRACT: The affinity of a ligand for a receptor on the cell surface will be influenced by the membrane composition. Herein, we evaluated the effects of differences in membrane fluidity, controlled by phospholipid composition, on the ligand binding activity of the G protein-coupled receptor human serotonin 2B. Using Nanodisc technology to control membrane properties, we performed biophysical analysis and employed molecular dynamics simulations to demonstrate that increased membrane fluidity shifted the equilibrium toward an active form of the receptor. Our quantitative study will enable development of more realistic in vitro drug discovery assays involving membrane-bound proteins such as G protein-coupled receptors.

The activities of G protein-coupled receptors (GPCRs) located in plasma membranes are influenced by the surrounding fluidic membranes.1,2 Membrane physical properties are mainly affected by lipid composition, which is constantly changing due to disease, eating habits, and aging. Changes of lipid composition influence signaling mediated by GPCRs.3–6 Therefore, understanding effects of membrane physical properties on GPCRs is essential for the development of drugs targeting these receptors. Phospholipids are the main constituents of most cell membranes.7 The polar head group of a phospholipid involves in physical property of the membrane surface, and the lipid tail group influences membrane fluidity.8,9 Accordingly, considering the relationship between phospholipid composition surrounding GPCRs and their functional activities, quantitative in vitro analysis of ligand binding to GPCR using biophysical technologies is necessary for precise understanding of GPCR function. The Nanodisc is a homogeneous, flat membrane model system.10,11 Interactions of GPCRs with their ligands or G proteins have been studied using the Nanodisc technology.11–13 Previous studies have reported on effects of phospholipid head groups on

homogeneity of GPCR-Nanodiscs and the ligand binding activity of GPCRs.14–17 On the other hand, influence of phospholipid tails on ligand binding activity of GPCRs has not been characterized. Here we analyzed conformation of a GPCR as a function of membrane fluidity and used molecular dynamics (MD) simulations to visualize the conformational alterations induced. We demonstrate that the equilibrium of GPCR activation is shifted by the phospholipid membrane fluidity. We selected human serotonin receptor 2B (5-HT2BR) as a model GPCR to analyze effects of phospholipid composition on ligand binding activity; the ligand for this GPCR is 5-hydroxytryptamine (5-HT) also known as serotonin (Figure 1A).18 5-HT2BR is a class A GPCR and a member of the 5-HT2 subfamily of 5-HT receptors. As with other GPCRs, 5-HT2BR is activated in signal transduction via conformational changes in the transmembrane (TM) region

Figure 1. (A) Crystal structure of human serotonin receptor 2B (PDB ID: 4IB4, BRIL region is not shown) and the ligand 5-HT. (B-C) SEC profiles of Empty Nanodiscs and 5-HT2BR-embedded Nanodiscs. Nanodiscs were composed of POPC, DLPC, DOPC, and DMPC, and corresponding curves are colored in green, red, orange, and blue, respectively. Each main peak corresponds to the main volume of the reconstituted Nanodiscs, respectively.

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Figure 2. (A-D) Fluorescence plots of each 5-HT2BR-Nanodisc in the MST assay. The normalized fluorescence Fnorm is plotted for different concentrations of the ligand (black). Fitted curves are represented as red lines. Error bars mean standard error (n=3). (E-H) Ligand binding curves for 5-HT2BR-Nanodiscs of indicated compositions in the SPR assay. Black and red curves represented raw data and fitted data, respectively. The SPR measurements were performed twice, and the result of a measurement is shown as a representative. upon ligand binding. To vary membrane fluidity, we used four phospholipids, 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC), and 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC) (Figure S1). Since these phospholipids have the same head group, we assumed that each membrane surface condition was identical. The tails of these phospholipids have different numbers of alkyl chains and unsaturated bonds, which affect the membrane fluidity.19 First, we estimated the membrane fluidity of each preparation using MD simulations. Phospholipid bilayer membranes were constructed by using each phospholipid and explicit solvent in the simulations. The computed B-factors of phospholipids varied depending on the lipid compositions (Figure S2). We also calculated the lateral diffusion coefficient D of C2 atom of each phospholipid molecule in parallel to the membrane surface plane (Table 1, Figure S3). The C2 atom of the DLPC showed the largest D, whereas that of DMPC was smaller than the others. As in previous studies,20,21 these results suggested that the membrane of DLPC possessed the highest fluidity of the four phospholipids, and also suggested that the DMPC membrane was more rigid than those composed of the other three phospholipids. In support of this conclusion, the gel-liquid crystal transition temperature of DMPC is higher than that of the others.22 Moreover, these calculated D values are in the range of the experimentally determined D values of human biomembrane.23 This indicates that our MD simulations are reliable. We constructed 5-HT2BR-free Nanodiscs (Empty-Nanodiscs) and 5-HT2BR-embedded Nanodiscs (5-HT2BR-Nanodiscs) of each phospholipid composition (Figure 1B-C). To examine binding affinities between the different 5-HT2BR-Nanodiscs and the ligand 5-HT, we performed a binding analysis using microscale thermophoresis (MST). We observed binding of 5-HT to 5-HT2BRNanodiscs composed of POPC, DLPC, and DOPC as fluorescence shifts as a function of the ligand concentration in the nM region (Figure 2A-C). No binding of 5-HT to 5-HT2BR was observed when the phospholipid was DMPC (Figure 2D). There was also no shift of fluorescence for the interactions between an EmptyNanodisc and 5-HT (Figure S4). 5-HT2BR showed the highest ligand binding affinity in the Nanodisc composed of the DLPC

Figure 3. Thermal denaturation monitored by CPM of 5-HT2BR in indicated Nanodiscs. Analysis of inflection points is shown in the main figure. Inset shows normalized fluorescence intensity at each temperature. Each curve is an average of three measurements. (Table 1). These results indicate that the fluidity of phospholipid membrane drastically alters the affinity of 5-HT2BR for 5-HT. This was not expected since 5-HT2BR itself have not been modified, and the only difference among the systems was in the lipid compositions. To provide more convincing evidence that phospholipid composition alters the binding activity of 5-HT2BR, we carried out a kinetic binding analysis using surface plasmon resonance (SPR). In the SPR assay, 5-HT2BR in POPC-, DLPC-, and DOPCNanodiscs specifically bound ligand (Figure 2E-G). No binding was observed in the context of the DMPC-Nanodisc (Figure 2H). Also in this assay, 5-HT2BR in the DLPC-Nanodisc bound ligand with the highest affinity (Table 1). It is noteworthy that the ligand Table 1. Physicochemical properties of 5-HT2BR-Nanodiscs with indicated lipid compositions. Lipid bilayer membrane D (×10-8 cm2·s-1) 5-HT binding in Nanodisc KD, MST (nM) kon, SPR (×107 M-1·s-1) koff, SPR (×10-1 s-1) KD, SPR (nM) Tm, CPM (oC)

POPC

DLPC

DOPC

DMPC

6.4 ± 0.5

9.3 ± 0.3

7.2 ± 1.2

4.3 ± 0.3

40.6 ± 0.6 0.98 ± 0.03 1.25 ± 0.13 12.8 ± 0.99 65.4 ± 0.5

3.9 ± 0.3 1.25 ± 0.04 0.46 ± 0.01 3.7 ± 0.19 60.2 ± 0.8

23.3 ± 1.0 0.93 ± 0.11 0.86 ± 0.08 9.3 ± 0.31 65.2 ± 0.5

N/A N/A N/A N/A 69.4 ± 0.5

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Biochemistry

Figure 4. Distances between Cα atoms of K531.32 and E3637.36 of the 5-HT2BR in (A) POPC, (B) DLPC, (C) DOPC, and (D) DMPC membranes. Each 50 ns run was performed three times, indicated in red, blue, and green lines. (E) Average distances of Cα atom between K531.32 and E3637.36 in the trajectories with standard errors calculated from three independent simulations. binding affinities depend mainly on the dissociation rate (koff), suggesting that increased membrane fluidity may stabilize the activated 5-HT2BR-ligand complex. Based on these biophysical analyses, we hypothesize that membrane fluidity influences the equilibrium between active and inactive forms of 5-HT2BR. To evaluate the conformational equilibria of 5-HT2BR in Nanodisc composed of different phospholipids, we performed a thermal shift assay using a thiol-reactive fluorescent dye (CPM assay; Figure S5). Whereas membrane scaffold protin 1 (MSP1) of Nanodisc lacks cysteine residues, 5-HT2BR contains three free cysteine residues in its TM helices and the C terminal region (C1473.44, C3386.49, and C397), enabling specific detection of GPCR denaturation by monitoring fluorescence intensity. 5HT2BR in the DLPC-Nanodisc was less thermally stable than in the other three 5-HT2BR-Nanodiscs; the Tm in the DMPCNanodisc was the highest (69.4 ± 0.47 oC) (Figure 3, Table 1). It was previously reported that the active form of GPCRs without the ligand is generally less stable than the inactive form.24 The results of the CPM assay implied that the increased membrane fluidity shifted the equilibrium to favor the active form of the receptor. In contrast, the decreased membrane fluidity, observed in the DMPC-Nanodisc would prevent the shift of conformational equilibrium toward the active form of the receptor. This would be the reason that we could not observe binding responses in the MST and SPR assays (Figure 2D, H). To further understand how membrane fluidity influences the conformation of the GPCR, we performed multiple 50 ns MD simulations of 5-HT2BR embedded in each of the phospholipid membranes. Since hydrophobic mismatch might have some effects on the activity of GPCR,25 we first examined each phospholipid membrane thickness (Figure S6). There seems to be no correlations between the membrane thickness in the simulation and the ligand binding activities of 5-HT2BR in vitro. In addition, DLPC membrane was thinner (25.3 ± 0.08 Å) than the estimated membrane boundary of 5-HT2BR (34.0 ± 2.3 Å), and hence DLPC would not be optimal to embed 5-HT2BR. However, we were still able to see the binding activity of 5-HT2BR in the DLPC membrane. These results suggest that a primary factor in the activity of 5-HT2BR is not hydrophobic mismatch, but rather membrane fluidity. We also performed principal component analysis (PCA) to evaluate the differences in conformations of the TM helices. We extracted Cα atoms of the TM helices and helix 8 (H8) of the receptor from the last 40 ns of each trajectory. We detected differ-

ences in conformation in the PC3 and PC4 (10% and 6% of the total variation, respectively). PCA plots of the receptor in the DLPC and the DOPC membranes were clustered together and PCA plots of the receptor in the POPC and the DMPC membranes were clustered (Figure S7). In both PC3 and PC4, the most mobile region was the extracellular side of the TM1 (Figure S8). The simulations suggest that K531.32 may make electrostatic contact with E3637.36 to tether the TM1 to the TM7. The Cα atoms of the K531.32 and E3637.36 in the DLPC and the DOPC membranes were closer than those in the POPC and the DMPC membranes (Figure 4), suggesting that the electrostatic interaction contributing to formation of the active conformation is more likely to be present when the membrane fluidity is increased. We then prepared three variants of 5-HT2BR in which K531.32 was mutated to alanine, glutamic acid, or arginine (K531.32A, K531.32E, and K531.32R, respectively), reconstituted them into the DLPC-Nanodiscs, and measured the ligand binding affinities using SPR. The K531.32A abolished the electrostatic attraction and decreased the affinity of ligand by approximately 5 fold compared to the wild-type 5-HT2BR (Figure S9A). This binding affinity was close to that in the SPR assay of the POPC-Nanodisc; the simulations show no evidence of the electrostatic interaction in this membrane. The K531.32E should cause charge-charge repulsion to E3637.36, and it decreased the affinity by approximately 10 fold (Figure S9B). In contrast, the K531.32R showed similar binding affinity to the DLPC-Nanodisc, as expected because the electrostatic interaction with E3637.36 should be maintained (Figure S9C). Although the electrostatic interaction between K531.32 and E3637.36 had not been confirmed in previous crystal structures due to the unclear electron density,18 our analysis demonstrates that it influences the function of the receptor. The molecular details revealed by MD simulations were verified by biophysical analyses with Nanodisc. The sequence alignment of 5-HTR subtypes reveals that most 5-HT receptors have the potential to form an electrostatic interaction or a hydrogen bond at this location (Figure S10). Taken together, our results show that the TM1-TM7 interaction on the extracellular side of the membrane plays an important role in the activation of 5-HT2BR. Further, our data suggest that the fluidity of the phospholipid membrane would lower the energy barrier between conformational states of this GPCR by forming the intramolecular interaction of TM1-TM7. This interaction in the receptor would in turn make 5-HT binding more favorable, and make the receptor-ligand complex more sta-

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ble, leading to the slower koff in the SPR. The conformational change of TM1-TM7 has caused the activation of A2A and β2A receptors in previous reports,26 and our finding reveal the close relationship between conformations of GPCRs and membrane fluidity at atomic resolution. Changes in membrane physical properties in humans may cause the disruption of conformational equilibrium of GPCRs and consequently alter the functional activities. Our biophysical and computational studies show that membrane fluidity can impact receptor activation. Further, our study provides valuable guidelines that will enable precise characterization of ligand binding during drug discovery efforts targeting GPCRs.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Information related to methods, additional experimental and analytical details (PDF)

AUTHOR INFORMATION Corresponding Author *Phone: +81 3 6409 2129. Fax: +81 3 6409 2129. E-mail: [email protected].

Author Contributions This study was conceived and designed by K.Y., S.N., and K.T.. K.Y., and N.S. performed the experiments. K.Y., S.N., D.K., N.S., and T.M. analyzed the data. K.Y., S.N., and D.K. wrote the manuscript. All the authors approved the manuscript.

Funding This work was supported by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED of Japan under Grand Number JP18am0101094 (to K.T.) and by JSPS KAKENHI-A grants 16H02420 (to K.T.) and 18H05425 (to T.M. and S.N.).

Notes The authors declare no competing interests.

ACKNOWLEDGMENT The authors would like to thank Hiroyuki Hanzawa and Shohei Kawasaki (DAIICHI SANKYO RD NOVARE CO., LTD.) for technical assistance with the MST experiments. We thank also the staff of NanoTemper Technologies and KC Central Trading Co., Ltd. for backup of MST measurements. The super-computing resources used in this study were provided by the Human Genome Center at the Institute of Medical Science, The University of Tokyo, Japan.

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Biochemistry

For Table of Contents Use Only Phospholipid membrane fluidity alters ligand binding activity of a G proteincoupled receptor by shifting conformational equilibrium Kouhei Yoshida†, Satoru Nagatoishi†,‡, Daisuke Kuroda†,§, Nanao Suzuki∥, Takeshi Murata∥, Kouhei Tsumoto†,‡,§,*

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