Effects of Hydrophilic Residues and Hydrophobic Length on Flip-Flop

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B: Biomaterials, Surfactants, and Membranes

Effect of Hydrophilic Residues and Hydrophobic Length on Flip-Flop Promotion by Transmembrane Peptides Hiroyuki Nakao, Chihiro Hayashi, Keisuke Ikeda, Hiroaki Saito, Hidemi Nagao, and Minoru Nakano J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b00298 • Publication Date (Web): 28 Mar 2018 Downloaded from http://pubs.acs.org on March 30, 2018

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The Journal of Physical Chemistry

Effect of Hydrophilic Residues and Hydrophobic Length on Flip-Flop Promotion by Transmembrane Peptides Hiroyuki Nakao†,‡, Chihiro Hayashi†,‡, Keisuke Ikeda‡, Hiroaki Saito§,#, Hidemi Nagao§, and Minoru Nakano*,‡ ‡

Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630

Sugitani, Toyama 930-0194, Japan §

Institute of Science and Engineering, Kanazawa University, Kakuma, Kanazawa, Ishikawa

920-1192, Japan #

Laboratory for Computational Molecular Design, RIKEN Quantitative Biology Center, 6-2-4

Furuedai, Suita, Osaka 565-0874, Japan †

Both authors contributed equally to this work

*Corresponding author: Minoru Nakano Tel.: +81 76 434 7565 E-mail address: [email protected]

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ABSTRACT Peptide-induced phospholipid flip-flop (scrambling) was evaluated using transmembrane model peptides in which the central residue was substituted with various amino acid residues (sequence: Ac-GKK(LA)nXW(LA)nLKKA-CONH2). Peptides with a strongly hydrophilic residue (X = Q, N, or H) had higher scramblase activity than other peptides, and the activity was also dependent on the length of the peptides. Peptides with a hydrophobic stretch of 17 residues showed high flip-promotion propensity, whereas those of 21 and 25 residues did not, suggesting that membrane thinning under negative mismatch conditions promotes the flipping. Interestingly, a hydrophobic stretch of 19 residues intensively promoted phospholipid scrambling and membrane leakage. The distinctive characteristics of the peptide were ascribed by long-term molecular dynamics simulation to the arrangement of central glutamine and terminal four lysine residues on the same side of the helix. The combination of simulated and experimental data enables understanding of the mechanisms by which transmembrane helices, and ultimately, unidentified scramblases in biomembranes, cause lipid scrambling.


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INTRODUCTION Phospholipid transbilayer movement (flip-flop) in biological membranes is regulated in several ways for membrane functions.1-2 Amino phospholipid translocases and ATP-binding cassette transporters transport specific lipids unidirectionally using metabolic energy, and their action generates asymmetric distribution of phospholipids in the plasma membrane (PM) of eukaryotes.3-4 ATP-independent bidirectional transport, i.e., scrambling, is also catalyzed by membrane proteins. Phosphatidylserine (PS) is exposed to the cell surface by the activity of a scramblase in the PM.5 PS externalization is important in apoptotic cell death or platelet-dependent co-aggregation.5 Unlike in the PM, flip-flop occurs constantly and frequently in the endoplasmic reticulum (ER), with the half time ranging from seconds to minutes.6-9 Rapid flip-flop is necessary for the integrity of the ER membranes, because phospholipids are mainly synthesized only at its cytoplasmic side.10-12 ER scramblases have, however, not been identified yet.2 Although many studies to elucidate the scrambling mechanism have been conducted, structural features of the energy-independent lipid transport mediated by proteins still remain unknown. We have previously shown by time-resolved small-angle neutron scattering that while a transmembrane model peptide with Leu-Ala repetitive sequence (KALP2313) does not induce flip-flop of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),14 substitution of its central Leu with Lys or Glu residues does so.15 We have also reported that a peptide of a putative membrane-spanning sequence in the ER-resident protein EDEM1 promotes phospholipid flip-flop and that Arg and His residues in the sequence play a role.16 Brunner et al. recently presented the crystal structure of nhTMEM16, a lipid scramblase of Nectria haematococca.17 This protein possesses a hydrophilic cavity directly exposed to the hydrophobic region of the 3 ACS Paragon Plus Environment


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membrane, which is thought of as a catalytic site of the scrambling. These results suggest the possibility that hydrophilic residues in the transmembrane region have a critical role in scramblase activity. Hydrophobic stretches of the membrane-spanning domain may also be involved in flip-flop promotion by proteins. Negative mismatch, at which the length of protein’s hydrophobic segments is shorter than that of the hydrocarbon region of the membrane, causes membrane thinning in the vicinity of the proteins.18 Phospholipids are considered to flip more frequently in thinner membrane, since an energy cost for lipid headgroup to cross the hydrophobic passage becomes

lower.

Indeed,

phosphatidylcholine

flip-flop

has

been

observed

for

a

1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayer, but not for a thicker POPC bilayer.14 Thus, the membrane thinning induced by the negative mismatch might increase the flip-flop rates. Here, we studied the effect of hydrophilic residues in the membrane-spanning domain on the flip-flop of phospholipids in large unilamellar vesicles (LUVs) using 23-residue transmembrane model peptides with various kinds of amino acid residues (Gln, Asn, His, Pro, Tyr, Ser, Thr, or Leu) located at the center of a hydrophobic Leu-Ala repetitive sequence (TMP23X, X = Q, N, H, P, Y, S, T, or L, respectively; Figure 1A). We also compared the scramblase activities of Gln-containing peptides with hydrophobic stretches of 13, 17, 19, 21, and 25 residues (TMP19Q, 23Q, 25Q, 27Q, and 31Q, respectively; Figure 1B).


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Figure 1. Amino acid sequences of the peptides. (A) Peptides with different central residues. (B) Peptides with different Leu-Ala repeat sequences.

EXPERIMENTAL METHODS Materials.

POPC

was

purchased

from

NOF

Corporation

(Tokyo,

Japan).

1-Palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4yl)amino]hexanoyl}-sn-glycero-3-phosphocholine (C6NBD-PC) and 1-palmitoyl-2-stearoyl-(ndoxyl)-sn-glycero-3-phosphocholine (5-doxyl-PC, 10-doxyl-PC, and 16-doxyl-PC for n = 5, 10, and 16, respectively) were obtained from Avanti Polar Lipids (Alabaster, AL). Calcein was purchased from Dojindo Laboratories (Kumamoto, Japan). All other chemicals used were of the highest reagent grade.

Synthesis of the model peptides. The sequences of the model transmembrane peptides used in this study are shown in Figure 1. A Trp residue next to the central residue in the sequences was 5 ACS Paragon Plus Environment


utilized to characterize the peptides in the membrane by fluorescence. These peptides were synthesized using Fmoc-based chemistry and purified by reverse-phase high-pressure liquid chromatography (HPLC; Shimadzu, Kyoto, Japan) with an Inertsil WP300 C18 column (GL sciences, Tokyo, Japan). The purity of the peptides was more than 90%, which was confirmed by HPLC and matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF-MS; Bruker Daltonics K.K., Kanagawa, Japan). Concentrations of the peptides in methanol were determined from the absorbance at 280 nm, using the molar extinction coefficients of Trp and Tyr.19

Vesicle preparation. The required amounts of chloroform-methanol solutions containing POPC and methanol solutions containing the peptides were mixed in a round-bottomed flask. After the evaporation of organic solvents, the sample was dried overnight under vacuum. The film was hydrated with a Tris-HCl buffer (10 mM Tris, 150 mM NaCl, 1 mM EDTA, and 0.01% NaN3; pH 7.4) or a Tris-HCl buffer without NaCl (10 mM Tris and 1 mM EDTA; pH 7.4). For the preparation of LUVs, the suspension was freeze-thawed several times and extruded through a 100-nm pore polycarbonate filter, using a LiposoFast extruder (Avestin, Ottawa, Canada). Small unilamellar vesicles (SUVs), which were used for CD spectroscopy, were prepared by sonication of the suspension under a nitrogen atmosphere for 9 min (three cycles of 3 min) using a UD-200 probe-type sonicator (TOMY SEIKO, Tokyo, Japan). Metal debris from the titanium tip of the probe was removed by centrifugation at 10,000 × g for 10 min. The vesicle size of the LUV and SUV suspensions was confirmed to be approximately 110–150 and 40–80 nm, respectively, by dynamic light scattering analysis using an FPAR-1000 particle analyzer (Otsuka Electronics,


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Osaka, Japan). The concentrations of POPC were determined by using an enzymatic assay kit for choline (Wako, Osaka, Japan).

CD spectroscopy. For circular dichroism (CD) measurements, SUVs in Tris-HCl buffer without NaCl were utilized to eliminate the background. SUVs were prepared at a POPC/peptide molar ratio of 100/1. Each SUV suspension (1 mM) was placed in a quartz cell with a 0.5-cm path length. The CD spectra were recorded at 25 °C from 200 to 250 nm with a J-805 spectropolarimeter

(JASCO,

Tokyo,

Japan).

The

spectra

obtained

represented

the

signal-averaged accumulation of 8 scans. The peptide spectra were converted to mean-residue ellipticity values after subtracting the spectra of SUVs without peptides. The α-helix content was estimated from the following equation:20 α - helix content (%) =

−[θ222 ] − 2340 ×100 30300

(1)

where [θ222] is the mean molar residue ellipticity at 222 nm. €

Quenching of tryptophan fluorescence by n-doxyl-PC. LUVs were prepared using a Tris-HCl buffer at a POPC/peptide/n-doxyl-PC molar ratio of 100/1/20. Tryptophan fluorescence spectra were recorded on an F-2500 spectrofluorometer (Hitachi, Tokyo, Japan) at an excitation wavelength of 280 nm.

Flip assay. To LUVs with various peptide/lipid ratios in Tris-HCl buffer, an ethanol solution of C6NBD-PC was added at a molar concentration (fluorescent lipid/non-fluorescent lipid) of 0.2 mol% to enable asymmetric incorporation of the fluorescent lipids into the outer leaflets. After incubation at 37 °C for several different times (4–360 min) to allow the C6NBD-PC to 7 ACS Paragon Plus Environment


translocate (flip) to the inner leaflet, 80 mM sodium dithionite in 2 M Tris was added to 50 µM LUV suspension to obtain a final dithionite concentration of 8 mM, which led to the reduction (and fluorescence quenching) of C6NBD-PC in the outer leaflet of the LUVs. After the addition of dithionite, the fluorescence of NBD was monitored on an F-2500 spectrofluorometer for 500 s at excitation and emission wavelengths of 460 and 534 nm, respectively. The mole fraction of C6NBD-PC that translocated to the inner leaflet (Φinner) and flip rate of C6NBD-PC (kflip) was calculated according to a previous report.16 In this analysis, since the fluorescence decay data are fitted with a double exponential function and its slower decay is extrapolated to t = 0 to determine the Φinner value, artifacts that bring about the slower decay, i.e., the leakage of dithionite into vesicles and/or flop of C6NBD-PC back to outer leaflet, can be eliminated.16

Leakage of calcein encapsulated in LUVs. LUVs containing differing amounts of peptides were prepared with 50 mM calcein solution. Calcein solution outside the LUVs was exchanged for a Tris-HCl buffer using a spin column with Superdex 200 (GE Healthcare UK, Buckinghamshire), and the fluorescence of the calcein-containing LUVs was monitored at 25 °C on an F-4500 spectrofluorometer (Hitachi, Tokyo, Japan) at excitation and emission wavelengths of 490 and 520 nm, respectively. After 1 h, Triton-X 100 was added to obtain a final concentration of 0.25 v/v %, which caused membrane disruption.

Molecular dynamics simulations. Four molecular dynamics (MD) simulations of the POPC/peptide mixture bilayers (POPC/TMP23L, POPC/TMP23Q, POPC/TMP25Q, and POPC/TMP27Q) were carried out in this study. The model structures of the transmembrane peptides were prepared by Swiss-PdbViewer21. The initial structures of the POPC/peptide 8 ACS Paragon Plus Environment

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bilayers consist of a peptide, 128 POPC lipids, and 8192 water molecules, providing a hydration level of 64 water per lipid. The center of mass of the peptide was first placed at the center of the x-y plane of the bilayer center, and the molecular axis of the peptide was oriented to the bilayer normal. The pre-equilibrated POPC lipids were arranged around the peptide to match the area per lipid with the experimental data.22 Water molecules were filled in the space above the polar region of the bilayer. Four Cl ions were added to neutralize the systems. The initial coordinates of each system were prepared by CAHRMM-GUI.23 The energy minimizations for the prepared initial structures of the POPC/peptide systems were first performed, and then 10 ns MD simulation was done for the system equilibration. We confirmed that the membrane structures such as the area per lipid of the POPC/peptide systems were equilibrated for 10 ns. In the case of peptide, though the intra-structures of the peptides can be equilibrated for 10 ns, the fluctuation of the peptide orientation in the membrane cannot be observed in such shot-term MD simulation.24 We thus carried out five independent 1,500 ns MD simulations with different initial velocities for each system (i.e. total 7,500 ns MD simulations were run for each system). The results of structure analysis of the membrane and peptides were obtained from these statistically sufficient simulation data. The MD simulations of the POPC/peptide bilayers were run under the constant temperature (303 K) and pressure (1 atm) condition. Nose-Hoover thermostat25-26 and Parrinello-Rahman type barostat27 were used to control the system temperature and the pressure. CHARMM36 force field22 including CMAP correction28-29 and TIP3P water model30 were adopted for the POPC/peptide bilayers and water molecules, respectively. The cutoffs for the van der Waals interactions were applied using a switching scheme, within a range of radius from 10 to 12 Å. The particle mesh Ewald method31 was adopted for the calculation of the Coulomb electrostatic interactions. The time step for 9 ACS Paragon Plus Environment


numerical integration of equations of motion was 2 fs, and the snapshot coordinates were stored every 10 ps. All MD simulations were carried out by GROMACS 5.1.432.

RESULTS AND DISCUSSION In order to quantify the peptides in the extruded vesicle samples, POPC LUVs prepared with 0.1 mol% TMP23L, 23Q, 27Q, or 31Q were solubilized by adding methanol (2-fold in volume). Tryptophan fluorescence intensities of these samples were measured and compared with those of standard samples, which were the mixtures of known amount of the peptides in methanol and peptide-free LUVs. The measured peptide concentrations were 0.103, 0.107, 0.108, and 0.114 mol%, respectively, suggesting that the final peptide/lipid ratio in LUVs did not change largely from the initial mixing ratio. CD spectra indicated that all the peptides, except for TMP23P and TMP19Q, formed α-helix-rich structures in POPC membranes (Figure S1 and Table S1 in Supporting Information). A Pro residue in TMP23P is considered to disrupt the α-helix structure. TMP19Q is the shortest peptide used in this study, and its short hydrophobic segment is considered inadequate for the peptide to be inserted across the membrane.33 To confirm whether the peptides adopt a membrane-spanning conformation, we performed Trp fluorescence quenching experiments using n-doxyl-PC (n = 5, 10, 16) (Figure S2). The doxyl moiety is a collisional quencher of fluorescence,34 and n-doxyl-PC has been utilized to assess the depth of a fluorescent moiety in the membrane.35 n-Doxyl-PC with a higher “n” number localizes its quenching moiety at a deeper position in the lipid bilayer, and hence, quenches Trp fluorescence more efficiently if the peptide spans the membrane, because the Trp residue lies near the center of the sequences. The most effective quenching was observed with 10 ACS Paragon Plus Environment

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16-doxyl-PC in the case of TMP23Q, 23N, 25Q, and 31Q. Although Trp fluorescence spectra of TMP27Q with 10- and 16-doxyl-PC were indistinguishable, the quenching efficiency of 16-doxyl-PC was almost the same as those for TMP23Q, 23N, 25Q, and 31Q, still suggesting that the Trp residue was deeply buried in the membrane. On the other hand, all three n-doxyl-PC had little effect on Trp fluorescence of TMP19Q. Fluorescence spectra of Trp are sensitive to the change of its surroundings. Peptides containing a central residue with high hydrophilicity shifted the Trp fluorescence spectra to longer wavelengths (Figure S3A), reflecting the increase in the local polarity near the central residue in the bilayer core. TMP19Q showed the emission maximum at significantly longer wavelength (340 nm) compared with other Gln-containing peptides with longer sequences (TMP23Q, 25Q, 27Q, and 31Q; 327–334 nm) (Figure S3B). Taken together, CD, quenching, and fluorescence spectral data suggest that, except for TMP19Q, all the peptides prepared adopt a transmembrane conformation. To evaluate the effect of a hydrophilic residue on the scramblase activity, we compared the flip-promotion

ability

of

the

23-residue

peptides

in

LUVs

using

1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocho line (C6NBD-PC). In this experiment, C6NBD-PC molecules were first incorporated into the outer leaflet of LUVs, which gradually translocated to the inner leaflet by the flip-flop promotion ability of the peptides. The mole fraction of C6NBD-PC in the inner leaflet (Φinner) after several incubation times was determined by dithionite quenching assay.36 As shown in Figure 2A, Φinner increased and approaches ~50% after incubation, suggesting that both flip and flop occurred at a similar rate (scrambling). TMP23Q, 23N, 23H, 23P, and 23Y, which contain a central residue with lower values of the hydropathy index,37 exhibited the scramblase activity, while the other 11 ACS Paragon Plus Environment


peptides with more hydrophobic residues (Ser, Thr, or Leu) did not (Figure 2B). The flip rates of TMP23Q-, 23N-, and 23H-containing LUVs were 20-, 13-, and 6-fold higher than the rates of the peptide-free LUVs, respectively. These results indicate that the flip-promotion ability of the peptides increases with the increase in the hydrophilicity of a residue located at the center of the sequences.

Figure 2. (A) Flip assay for C6NBD-PC in LUVs without or containing 0.1 mol% TMP23X (X = Q, N, H, P, Y, S, T, or L). (B) Flip rate constants obtained by fitting the curves in (A). A letter “X” under each bar expresses a central residue of TMP23X and the numerical value below represents the hydropathy index37 of “X”. Error bars represent the mean ± SD from n = 3 or 4 experiments. *p < 0.05, **p < 0.01 compared with LUVs without peptides

Molecular dynamics (MD) simulations have shown that hydrophilic residues in the hydrocarbon region of the bilayer allowed the penetration of water molecules into the vicinity of the residues to hydrate the side chains.38-39 A significant correlation was evident between the 12 ACS Paragon Plus Environment

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number of penetrated water molecules and the hydrophilicity of amino acid residues.38 In the present study, hydrophilic residues in the transmembrane domain were found to increase the polarity in the vicinity of Trp located at bilayer center and generate scramblase activity. A positive correlation between flip rate of C6NBD-PC and the degree of red shift in the Trp fluorescence spectra (r = 0.82, Figure S4) suggests that increase in the local polarity at the bilayer center attenuated the energy barrier for the phospholipid headgroups to pass through the hydrocarbon region. Indeed, the scramblases of the plasma membrane (nhTMEM16 and PLSCR1) contain hydrophilic residues (Arg, Lys, Glu, Asp, Gln, Asn, His) in their transmembrane regions.17,

40

Our results suggest that these residues play a critical role in

phospholipid scrambling. To elucidate whether the hydrophobic length influences the scramblase activity of the peptides, we performed the flip assay using Gln-containing peptides with various Leu-Ala repeat lengths. Because the length of the helical axis of peptides is 1.5 Å/residue, the length of the transmembrane domain (LTM) of TMP23Q is 25.5 Å, which is shorter by only a small margin than the hydrocarbon region of the POPC bilayer (25.8 Å41). TMP19Q (LTM = 19.5 Å), which was shown not to span the bilayer, did not represent the scramblase activity as expected (Figure 3B). Unlike TMP23Q, peptides with longer hydrophobic length (TMP27Q and 31Q, LTM = 31.5 and 37.5 Å, respectively) little promoted the flip (Figure 3), suggesting that the hydrophobic length is critical for the flip-promotion ability of the peptides. Langer et al. observed that the shorter peptide enhances the flip-flop of fluorescent lipids more effectively.42 Peptides with negative mismatch decrease the mean bilayer thickness at a higher peptide/lipid ratio.43 Therefore, it is considered that phospholipids are forced to adapt to the mismatch in the vicinity of TMP23X. Combined with the central hydrophilic residue, membrane thinning by the peptides 13 ACS Paragon Plus Environment


would promote the flip-flop of phospholipids. Although hydrophobic mismatch has been reported to induce oligomerization of peptides to minimize the area of mismatched surface,18, 44 this would not be the case for the peptides in the present study because of the intermolecular electrostatic repulsion between terminal Lys residues. We have previously calculated that human ER membrane proteins contain 7.3% of hydrophilic residues in their transmembrane sequences.15 Sharpe et al. reported that hydrophobic region of single-pass membrane proteins in the ER of eukaryotes is shorter than that in the PM.45 Hence, it is possible that several ER membrane proteins, including EDEM116, function as ER scramblases.

Figure 3. (A) Flip assay for C6NBD-PC in LUVs without or containing 0.1 mol% TMP19Q, 23Q, 27Q, or 31Q. (B) Flip rate constants (kflip) obtained by fitting the curves in (A). Error bars represent the mean ± SD from n = 3 or 4 experiments. *p < 0.05 compared with LUVs without peptides


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Interestingly, TMP25Q, which has intermediate length (LTM = 28.5 Å) between TMP23Q and TMP27Q, presented distinctive features. Fluorescence quenching curves for 0.1 mol% TMP25Q showed monophasic decay (Figure S5A), which is a characteristic observed when dithionite leaks into vesicles to quench all C6NBD-PC molecules. Indeed, the TMP25Q-containing LUVs leaked almost all the encapsulated calcein molecules within 1 h (Figure S6), which was not observed for TMP23Q- and 23N-containing LUVs. Permeability of the TMP25Q-containing LUVs was minimized by decreasing the peptide concentration to 0.033 mol%, which restored biphasic decay in the quenching profiles (Figure S5B). Thus, the scramblase activity of TMP25Q was examined at concentrations not more than 0.033 mol%. At these concentrations TMP25Q had an ability equivalent to that of TMP23Q (Figure 4). The linear concentration dependence of the scramblase activity suggested that TMP23Q and 23N act as a monomeric form within the ranges below 0.1 mol%, while TMP25Q does so only below 0.033 mol%.

Figure 4. (A) Flip assay for C6NBD-PC in LUVs containing 0.033 mol% TMP23Q, 23N, or 25Q. (B) Flip rate constants (kflip) plotted against peptide concentration.



Why does TMP25Q exhibit such distinctive features? To clarify this, we performed long-term (1,500 ns) MD simulations of POPC bilayers containing TMP23L, 23Q, 25Q, or 27Q. Tilt angles of peptides are plotted as a function of MD time steps in Figure S7, where the tilt angle was defined as an angle between the molecular axis of the transmembrane peptide and the bilayer normal (i.e. z-axis) in this study. The tilt angles at which the maximum frequency occurs were obtained from Figure S7 and averaged for 5 MD simulation runs. The tilt angles (mean ± S.D.) of TMP23L, 23Q, 25Q, and 27Q were 22.8° ± 6.0°, 21.2° ± 3.8°, 31.4° ± 2.7°, and 43.0° ± 1.2°, respectively, indicating that the helices incline more greatly from the bilayer normal as the length of the peptide increases. This agrees with a previous report of the MD simulation in which the tilt angle of KALP peptides increases with the extent of positive mismatch.46 The tilt angle properties of the peptides can also be seen in the snapshot structures of those systems (Figure 5). Figure S8 represents depth distributions of phosphorus, acyl chain carbonyl carbon, side chain groups of central Gln and four Lys residues. Second (K(3)) and third (K(21)) lysines of TMP23L and 23Q, but not 25Q, and 27Q, are located more deeply into the POPC bilayer than the position of the acyl chain carbonyl carbons, which demonstrates the negative mismatch. It is interesting to note that outermost Lys residue (K(2) or K(24)) of TMP25Q is arranged more deeply in the bilayer than inner Lys (K(3) or K(23), respectively) (Figure S8). This arrangement is not seen with the other peptides. Geometrically, the “inverted” arrangement of the Lys residues at each end is attainable only when all these residues are located on the same side of the inclined helix. Indeed, the snapshot of TMP25Q clearly shows that four Lys residues, together with central Gln, face the front side (Figure 5). Figure S9 represents the probability distributions of angles between the side chains of central Gln and four Lys of each peptide. The data revealed that all the Lys residues of TMP25Q turn to the same side as Gln. This feature is less evident for the 16 ACS Paragon Plus Environment

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other peptides. We have previously demonstrated with transmembrane peptides based on the EDEM1 protein that the same-side positioning of Arg and His in α-helix structure is critical for the flip-flop promotion.16 Therefore, the peculiar alignment of Lys and Gln residues in TMP25Q is considered crucial for its potential to cause the phospholipid flip-flop and calcein leakage.



Figure 5. Snapshot structures of (A) TMP23L, (B) TMP23Q, (C) TMP25Q, and (D) TMP27Q in the POPC bilayers at the last MD time step. Lys and Gln residues are represented by a CPK model.

CONCLUSIONS Our study using transmembrane model peptides demonstrated that hydrophilicity of the central residues, length of the membrane-spanning domain, and the position of Lys residues anchoring at each bilayer surface relate to the presentation of the scramblase activity by the peptides. Peptides with a strongly hydrophilic residue at the center of the sequences and negative mismatching peptides exhibited the scramblase activity. MD simulation showed that central Gln and terminal four Lys residues of TMP25Q were arranged on the same side of the helix, which could plausibly promote the phospholipid scrambling and membrane leakage. This study would provide clues for shedding light on the mechanism by which scramblases catalyze the lipid flipping (flopping) motion, and uncovering which proteins are responsible for the rapid flip-flop in the ER or other membranes where scramblases have not yet been identified.


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ASSOCIATED CONTENT Supporting information The Supporting Information is available free of charge. Experimental methods and supporting results (PDF) AUTHOR INFORMATION *E-mail: [email protected] Notes The authors declare no competing financial interests. ACKNOWLEDGEMENTS This study was supported by JSPS KAKENHI (Grant numbers JP17H06704 (to H.N.), JP16K18860 (to K.I.), JP16K05648 (to H.S.), and JP17H02941 (to M.N.)), JSPS Core-to-Core Program, B. Asia-Africa Science Platforms, and Takeda Science Foundation. The computations in this study were performed using the advanced center for computing and communication of RIKEN, the research center for computational science of Institute for Molecular Science (IMS), the research center for advanced computing infrastructure of Japan Advanced Institute of Science and Technology (JAIST), and the center for computational science of Tsukuba university.



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Effects of Hydrophilic Residues and Hydrophobic Length on Flip-Flop

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