Binding of a Truncated Form of Lecithin:Retinol Acyltransferase and Its

Article pubs.acs.org/Langmuir

Binding of a Truncated Form of Lecithin:Retinol Acyltransferase and Its N- and C-Terminal Peptides to Lipid Monolayers Sylvain Bussières,† Line Cantin,† Bernard Desbat,‡ and Christian Salesse*,† †

LOEX/CUO-recherche, Centre hospitalier affilié universitaire de Québec, Hôpital du Saint-Sacrement, 1050 Chemin Ste-Foy, and Département d’ophtalmologie, Faculté de médecine, and PROTEO, Université Laval, Québec (Québec), Canada ‡ CBMN, Université de Bordeaux, UMR CNRS 5248, IPB, allée Geoffroy Saint Hilaire, 33600 Pessac, France S Supporting Information *

ABSTRACT: Lecithin:retinol acyltransferase (LRAT) is a 230 amino acid membraneassociated protein which catalyzes the esterification of all-trans-retinol into all-trans-retinyl ester. A truncated form of LRAT (tLRAT), which contains the residues required for catalysis but which is lacking the N- and C-terminal hydrophobic segments, was produced to study its membrane binding properties. Measurements of the maximum insertion pressure of tLRAT, which is higher than the estimated lateral pressure of membranes, and the positive synergy factor a argue in favor of a strong binding of tLRAT to phospholipid monolayers. Moreover, the binding, secondary structure and orientation of the peptides corresponding to its N- and C-terminal hydrophobic segments of LRAT have been studied by circular dichroism and polarization-modulation infrared reflection absorption spectroscopy in monolayers. The results show that these peptides spontaneously bind to lipid monolayers and adopt an α-helical secondary structure. On the basis of these data, a new membrane topology model of LRAT is proposed where its N- and C-terminal segments allow to anchor this protein to the lipid bilayer.

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targeting.11 The secondary structure and membrane binding of the hydrophobic N- and C-terminal domains of LRAT is still unknown. Measurements of the membrane binding properties of tLRAT and of its N- and C-terminal peptides should therefore be very useful to improve our understanding of the membrane topology of LRAT which is still poorly understood. Langmuir monolayers have been extensively used as a model membrane to study lipid−protein interactions.12−18 This method allows to control the lipid density at the interface and the subphase composition and has also been extensively used to study the activity of lipolytic enzymes (for reviews, see refs 10, 13, 14, 16, and 19−22). Different approaches have been used to determine the extent of protein and peptide binding to lipid monolayers. The determination of the “maximum insertion pressure” (MIP) and of the “synergy factor a” were shown to be very useful to compare the extent of protein binding to different lipid monolayers.23,24 Moreover, polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) as well as IRRAS allow the observation of infrared absorption bands of phospholipids and proteins in monolayers and to estimate the orientation and secondary structure of peptides and proteins (for reviews, see refs 25 and 26). In the present study, the measurement of the MIP and synergy factor a have been used to determine the extent of binding of tLRAT onto different phospholipid monolayers.

INTRODUCTION Lecithin:retinol acyltransferase (LRAT) is a membrane-bound protein that plays an essential function in the visual cycle. It catalyzes the esterification of retinol into retinyl esters in the retinal pigment epithelium (RPE) as well as in other tissues including testis, liver, and intestine.1−3 The retinyl esters are then either stored in the RPE or further metabolized to produce the chromophore of rhodopsin, 11-cis-retinal.4 The nucleotide sequence of LRAT indicates an open reading frame of 690 bp encoding a 230 amino acid protein with a calculated mass of 25.3 kDa.5 The primary sequence of LRAT is novel5 and does not show any homology to enzymes that catalyze similar reactions, such as lecithin:cholesterol acyltransferase.6,7 A hydropathy analysis of the primary sequence of LRAT allowed to suggest that its N- and C-terminal segments could include hydrophobic transmembrane domains located at positions 9−31 and 195−222, respectively.5 The deletion of these two putative hydrophobic domains was shown to produce a fully active and soluble truncated form of LRAT (tLRAT) (amino acids 31−196).8,9 tLRAT includes the three essential residues for its activity (C161, Y154, and H60) which are conserved in the LRAT family of enzymes.8 This protein was shown to bind and hydrolyze phospholipid monolayers,10 thus suggesting that the absence of its N- and C-terminal hydrophobic domains does not prevent its membrane hydrolysis activity. This was surprising since these N- and Cterminal peptides were postulated to allow membrane binding of LRAT.5 However, it was recently reported that only the Cterminal extension of LRAT is essential for its membrane © 2012 American Chemical Society

Received: October 4, 2011 Revised: January 19, 2012 Published: January 19, 2012 3516

dx.doi.org/10.1021/la203896n | Langmuir 2012, 28, 3516−3523

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of the incident beam.31,32 To remove the isotropic contributions from bulk water and water vapor and experimental drifts and to get rid of the dependence on the Bessel function, the spectrum of the peptide in the presence or absence of the phospholipid monolayer was divided by that of the pure buffer to produce the resulting normalized PM-IRRAS spectrum. Each PM-IRRAS spectrum was the result of the coaddition of 600 scans at a resolution of 8 cm−1. Consequently, a typical PMIRRAS spectrum was obtained after ∼9 min of acquisition. In these experiments, a buffer containing 50 mM Tris at pH 7 and 150 mM NaCl was poured in a 4.5 mL trough made of Delrin. Phospholipids were slowly spread at the surface of the buffer until the desired surface pressure was reached (varying between 17 and 23 mN/m). After an equilibrium period of 15 min, 10 μg of peptide in HFIP was injected into the subphase (final concentration of 1.2 μM), and surface pressure was monitored with a NIMA tensiometer during the measurement of the infrared spectra. Membrane Topology Model of LRAT Using I-TASSER. Prediction of 3-dimensional protein structures from amino acid sequences represents one of the most important problems in computational structural biology. This task is even more challenging in the case of LRAT because, as mentioned above, its primary sequence is novel,5 and it does not show any homology to enzymes that catalyze similar reactions. The comparison of I-TASSER designed models with several X-ray determined structures allows to appreciate the accuracy of this prediction tool.33,34 A 3D model of LRAT was thus built based on multiple-threading alignments by LOMETS and iterative TASSER simulations; function insights are then derived by matching the predicted models with protein function databases.35 LRAT full sequence was uploaded to the I-TASSER online server, and this algorithm allowed to create five ab initio structures. One structure was selected because of its consistency with the data.

Moreover, the secondary structure, monolayer kinetics of binding, and orientation of the N- and C-terminal hydrophobic peptides of LRAT were determined by circular dichroism and PM-IRRAS. Then, on the basis of these data, a topology model is proposed for the membrane organization of LRAT.

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MATERIALS AND METHODS

Materials. The deionized water used for the preparation of all buffer solutions was highly purified with a NANOpure purification apparatus (Barnstead). This water had a resistivity of no less than 18.2 MΩ·cm and a surface tension of 72 ± 0.1 mN/m at room temperature. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were from Avanti Polar Lipids (Alabaster, AL) and used as received. NaH2PO4 and Na2HPO4 were from Merck (Darmstadt, Germany). Chloroform and Tris were from Laboratoire MAT (Quebec, Canada). Sodium chloride was from Ultrapure Bioreagent (Phillipsburg, NJ). Econo-Pac 10DG columns were from Bio-Rad (Hercules, CA). His-Trap columns were from Amersham Pharmacia (Piscataway, NJ). The N-terminal (MKNPMLEVVSLLLEKLLLISNFTLFSSGAA; purity >90%; molecular weight 3280 g/mol) and C-terminal (LGLASIVCTGLVSYTTLPAIFIPFFLWMAG; purity >70%; molecular weight 3202 g/mol) peptides of LRAT were purchased from AnaSpec (Fremont, CA). Cloning, Expression, Solubilization, and Purification of tLRAT. tLRAT was produced and purified as previously described.10 After the elution step from the His-Trap column, the elution buffer was exchanged for a phosphate buffer (50 mM, pH 7.0) using an Econo-Pac 10DG column previously equilibrated with this buffer. The molecular weight of tLRAT is 20.9 kDa. Circular Dichroic Spectroscopy of the N- and C-Terminal Peptides of LRAT. Circular dichroic spectra were recorded on a Jasco spectropolarimeter (Model J-710, Jasco, Easton, MD). The spectra have been measured at a peptide concentration of 50 μM in HFIP (hexafluoroisopropanol). For each spectrum, 20 scans were collected from 190 to 260 nm using a 0.1 mm path length cuvette. Binding of tLRAT to Phospholipid Monolayers and Determination of Its MIP. Measurements of tLRAT binding onto Langmuir monolayers were performed using a Kibron DeltaPi-4 research microtensiometer and a multiwell plate (Kibron Inc., Helsinki, Finland). Phospholipids solubilized in chloroform were slowly spread at the surface of a buffer (50 mM phosphate, pH 7) poured in one of the 500 μL glass troughs of the Multiwell plate until the desired surface pressure was reached. The waiting time period for the spreading solvent evaporation and for the film to reach equilibrium varied with the type of lipid, the spreading volume, the initial surface pressure, and the lipid concentration. Then, 5 μg of tLRAT was injected into the subphase (final concentration of 0.5 μM) underneath phospholipid monolayers at different initial surface pressures (Πi) until the equilibrium surface pressure (Πe) was reached. This allowed calculation of the surface pressure increase (ΔΠ = Πe − Πi). This optimal final protein concentration has been determined by performing ΔΠ measurements at different protein concentrations, which allowed to obtain tLRAT surface saturation (0.5 μM). The plot of ΔΠ as a function of Πi allowed determining the MIP by extrapolating the regression of the curve to the x-axis.23 The ΔΠ0 is obtained by extrapolating the regression of the same curve to the y-axis.23 The values of MIP and ΔΠ0 can be used to compare the extent of protein/ lipid interactions.23,24,27,28 The calculation of the uncertainty of the MIP measurement and of the slopes and the statistical analysis were performed as previously described.23,24 PM-IRRAS Measurements of the N- and C-Terminal Peptides of LRAT. PM-IRRAS spectra were measured as previously described.29,30 Briefly, PM-IRRAS combines Fourier transform midIR reflection spectroscopy (FT-IR) with rapid polarization modulation

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RESULTS

Extent of tLRAT Binding onto Different Phospholipid Monolayers. The extent of tLRAT binding (ΔΠ) to different phospholipid monolayers was measured at different initial surface pressures (Πi) to determine its MIP. The phospholipids used were selected to mimic the lipid content of the RPE.36 Indeed, the RPE contains approximately 44% phosphatidylcholine, 32% phosphatidylethanolamine, and 7% phosphatidylserine. Moreover, the fatty acyl chain composition of the phospholipids of the RPE include approximately 31% palmitoyl (C16:0), 19% stearoyl (C18:0), and 30% oleoyl (C18:1).36 Therefore, the phospholipids DMPC, DOPC, DOPE, DOPS, DPPC, DPPE, DPPS, and DSPC have been selected to study the monolayer binding of tLRAT. An example of the determination of the MIP of tLRAT is given in Figure 1. Typical kinetics of tLRAT binding onto a DPPC monolayer are presented in Figure 1A. The extent of the surface pressure increase varies with the initial surface pressure. Indeed, for example, at a low initial surface pressure of 6 mN/m, a large and quick increase in surface pressure is observed after tLRAT injection into the monolayer subphase followed a slow decrease until a stable surface pressure is reached. For larger initial surface pressures, the quick initial increase is followed by a slower increase in surface pressure until a final stable surface pressure is obtained within 25−30 min. Moreover, it can be seen that the larger is the initial surface pressure, the smaller is the increase in surface pressure (ΔΠ). Indeed, ΔΠ of 24.8, 20.2, 18.6, and 13.6 mN/m have been measured for Πi of 6, 18, 23, and 30 mN/m, respectively. A negative slope is thus obtained when plotting these values of ΔΠ as a function of Πi (Figure 1B), which allowed to calculate a MIP of 53 ± 2.4 mN/m for tLRAT in the presence of a DPPC monolayer. The values of MIP of tLRAT obtained with different phospholipids 3517


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Figure 2. Histogram presenting the MIP values of tLRAT obtained in the presence of different phospholipid monolayers. These MIP values have been determined as described in Figure 1. Statistical analysis of the data allowed to determine the values which are significantly different (**: p = 0.05).

(p = 0.05) from that measured with DOPE. Interestingly, the unsaturation of the phospholipid fatty acyl chains leads to a significant decrease of the MIP of tLRAT compared to the data obtained with the saturated phospholipids except for DOPE (Figure 2). However, most important is that all of these MIP values are larger than the estimated lateral pressure of membranes, which lies in the range of 30−35 mN/m.37−43 This suggests that tLRAT is able to strongly bind membranes, despite the absence of its N- and C-terminal hydrophobic domains. A new approach for the study of protein binding to lipid monolayers was recently described by Calvez et al.24 Briefly, it has been shown that the plot of the equilibrium surface pressure, Πe (ΔΠ + Πi), as a function of the initial monolayer surface pressure, Πi, follows a linear regression and that the decrease of ΔΠ with the increase of Πi (Figure 1B) are related by the parameter a, called synergy factor, which modulates protein adsorption.24 In fact, this previously derived parameter was shown to demonstrate the preferencial binding of proteins for specific phospholipids.24 A typical plot of Πe as a function of Πi resulting from the binding of tLRAT onto a DPPC monolayer can be seen in Figure 3. It allows to determine the

Figure 1. (A) Typical example of the kinetics of binding of tLRAT in the presence and absence of a phospholipid monolayer. Plot of the surface pressure increase (ΔΠ) as a function of time after the injection of tLRAT underneath a DPPC monolayer at different initial surface pressures (Πi). The final concentration of tLRAT in the subphase is 0.5 μM. Only the results obtained at Πi of 6, 18, 23, and 30 mN/m are shown for clarity. (B) The ΔΠ at equilibrium (Πe) obtained in “A” is then plotted as a function of Πi. Extrapolating the regression of the plot to the x-axis allows determining the MIP. The ΔΠ0 is obtained by extrapolating the regression of the plot to the y-axis. Subphase contains 50 mM phosphate buffer pH 7.0.

are easier to compare when plotted as histograms as shown in Figure 2. Values of MIPs of 47.7 ± 3.8, 44.9 ± 4.5, 53 ± 2.4, 48.3 ± 3.5, and 47.6 ± 3.9 mN/m have been obtained for tLRAT in the presence of the saturated phospholipids DMPC, DSPC, DPPC, DPPE, and DPPS, respectively (Figure 2). It can be seen that only the MIP value obtained with DPPC is significantly different (p = 0.05) from the other saturated phospholipids. Therefore, the MIP values obtained with DMPC, DSPC, DPPE, and DPPS are not significantly different. This suggests that tLRAT does not show any preference for phospholipids with shorter fatty acyl chains (DMPC, C14:0) or longer fatty acyl chains (DSPC, C18:0) and, conversely, that the physical state of the phospholipid has no influence on tLRAT binding (DMPC, liquid-expanded state; DSPC, solidcondensed state). Moreover, these data also suggest that the type of polar headgroup has no effect on tLRAT binding. In addition, values of MIPs of 40.6 ± 5.6, 49 ± 4.5, and 37.1 ± 5.1 mN/m have been obtained for tLRAT in the presence of the monounsaturated phospholipids DOPC, DOPE, and DOPS, respectively. The values obtained with DOPC and DOPS are not different from each other, but they are significantly different

Figure 3. Plot of Πe as a function of Πi. The slope of the plot allows to determine the synergy factor a. The other parameters are the same as in Figure 1.

value of the synergy factor a which corresponds to the slope of this linear regression. The values of the synergy factor a for all 3518


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phospholipids studied are shown in Table 1. It can be seen that all values obtained lie between 0.31 and 0.48. Values of a > 0 Table 1. MIP, Synergy Factor a, and ΔΠ0 of tLRAT in the Presence of Different Phospholipid Monolayers phospholipid DMPC DOPC DOPE DOPS DPPC DPPE DPPS DSPC

MIP (mN/m) 47.7 40.6 49 37.1 53 48.3 47.6 44.9

± ± ± ± ± ± ± ±

3.8 5.6 4.5 5.1 2.4 3.5 3.9 4.5

synergy factor a 0.48 0.35 0.31 0.45 0.42 0.31 0.34 0.43

± ± ± ± ± ± ± ±

0.06 0.16 0.09 0.14 0.05 0.09 0.08 0.10

ΔΠ0 (mN/m) 24.8 26.6 33.1 20.5 30.6 33.1 31.3 25.5

± ± ± ± ± ± ± ±

1.6 3.5 2.4 3.3 1.4 2.4 2.0 2.9

correspond to the most favorable conditions for protein monolayer binding.24 Moreover, the value of ΔΠ0 is also interesting to analyze.24 It has been postulated that this value, which corresponds to ΔΠ at Πi = 0 mN/m, could be equivalent to the surface tension of the protein in the absence of a lipid monolayer (ΔΠ0). This value of surface tension is 24 mN/m for tLRAT as can be seen in Figure 1A. It has been suggested that, when it is larger than the surface tension of the protein, ΔΠ0 could be ascribed as the trend of the phospholipid monolayer to modify the surface activity of the protein.24 As can be seen in Table 1, the values of ΔΠ0 in the presence of DPPC, DPPE, DPPS, and DOPE are larger than the surface tension of tLRAT, whereas those obtained with DMPC, DSPC, DOPC, and DOPS are similar to that of the surface tension of tLRAT when taking the experimental error into account. Secondary Structure of the N- and C-Terminal Peptides of LRAT in Solution and in Monolayers. The circular dichroic spectra of the N- and C-terminal peptides solubilized in HFIP are presented in Figure 4A. Both peptides show the characteristic positive and negative bands located respectively at 190−195 and 208−222 nm, which are typical of α-helical structures.44−49 Moreover, Figure 4B shows the PMIRRAS spectra obtained after spreading the individual N- and C-terminal peptides at the air−water interface in the absence of a phospholipid monolayer. It can be seen that the amide I band of the N- and C-terminal peptides is centered at 1653 and 1650 cm−1, respectively, which is typical of α-helices.30,50 However, the amide I band of the C-terminal peptide also comprises a strong component at 1632 cm−1, thus suggesting that this peptide contains β-sheets in addition to α-helices.51 Moreover, it has been previously demonstrated that information can be obtained on the orientation of peptides with an α-helical structure from the amide I/amide II ratio of PM-IRRAS spectra.13,52 A large variation of the amide I/amide II ratio can indeed be seen when an α-helical peptide is oriented at 40 (ratio of ∼1.1), 50 (ratio of ∼2.1), and 60° (ratio of ∼3.2) with respect to the normal of the monolayer. Therefore, on the basis of the amide I/amide II ratio of 1.9 of the N-terminal peptide, its orientation should lie between 45° and 50° (angle θ) with respect to the normal of the monolayer (Figure 4B).53 Although the amide I/amide II ratio of the C-terminal peptide (Figure 4B) is very different from that of the N-terminal peptide, a proper estimation of its orientation can not be performed because it contains β-sheets in addition to α-helices. Binding and Orientation of the N- and C-Terminal Peptides of LRAT in the Presence of Phospholipid

Figure 4. (A) Circular dichroic spectra of the N- and C-terminal peptides of LRAT solubilized in HFIP at a concentration of 50 μM. (B) PM-IRRAS spectra of the N- and C-terminal peptides of LRAT in the absence of a phospholipid monolayer. The intensity of the amide I band of these two spectra is almost exactly the same. They have nevertheless been normalized to facilitate their comparison. An intensity scale bar is also provided. Subphase contains 50 mM Tris at pH 7 and 150 mM NaCl. The final concentration of the peptides in the subphase is 1.2 μM.

Figure 5. Typical kinetics of adsorption of the N- and C-terminal peptides of LRAT after their injection underneath a monolayer of DPPC at 17 and 23 mN/m, respectively. The value of Πe (Πi + ΔΠ) for the N- and C-terminal peptides was ∼30 (Πi of 17 mN/m + ΔΠ of 13 mN/m) and ∼34 mN/m (Πi of 23 mN/m + ΔΠ of 10.7 mN/m), respectively. Subphase contains 50 mM Tris at pH 7 and 150 mM NaCl. The final concentration of the peptides in the subphase is 1.2 μM.

Monolayers. The N- and C-terminal peptides of LRAT were injected into the subphase of the phospholipid monolayers used to mimic the lipid content of the RPE (DMPC, DOPC, DOPE, DOPS, DPPC, DPPE, DPPS, and DSPC). Typical adsorption isotherms of these peptides showing a very fast kinetics of binding are shown in Figure 5. It can be seen that, after the injection of the N- or the C-terminal peptide underneath a 3519


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Very similar PM-IRRAS spectra of the N-terminal peptide have been obtained in the presence of DPPC, DPPE, and DPPS monolayers, thus suggesting that the charge and size of the polar headgroup of these phospholipids has no influence on the secondary structure and orientation of this peptide. The amide I band of all of these spectra is centered at 1653 cm−1, thus showing that this peptide is mainly constituted of α-helices. Moreover, the amide I/II ratio of these spectra varies between 1.1 and 1.5. According to the plot of the amide I/II ratio as a function of the angle θ,53 the orientation of the N-terminal peptide in the presence of these phospholipids should lie between 40° and 45° with respect to the normal of the monolayer. Figure S1 in the Supporting Information compares the PM-IRRAS spectra of the N-terminal peptide in the presence and absence of a phosholipid monolayer. The slightly larger width of the amide I band in the absence of a monolayer suggests that this peptide is more ordered in the presence of a phospholipid monolayer. Moreover, the small difference between the amide I/II ratio suggests a slight difference in the orientation of this peptide in the presence (40°−45°) and absence (45°−50°) of a DPPC monolayer. The PM-IRRAS spectra of the C-terminal peptide in the presence of DPPC, DPPE, and DPPS monolayers can be seen in Figure 6B. These spectra are very similar with an amide I band located at ∼1652 cm−1. This peptide thus also adopts a major α-helical conformation. Furthermore, the amide I/II ratio varies between 1.2 and 1.4, thus suggesting that its orientation should lie between 40° and 45° with respect to the normal, such as the Nterminal peptide. The PM-IRRAS spectra of the C-terminal peptide in the presence and absence of a phosholipid monolayer are compared in Figure S2 of the Supporting Information. A striking difference can be seen between these two spectra. Indeed, the presence of the monolayer modifies the secondary structure of this peptide, leading to the disappearance of the β-sheet structural component. In addition, very similar spectra were obtained with these peptides in the presence of the other phospholipids assayed (DMPC, DOPC, DOPE, DOPS, and DSPC; data not shown). These data thus suggest that the chain length and unsaturation of the fatty acyl chains of the phospholipid monolayer as well as their physical state have no influence on the secondary structure and orientation of these peptides. The spectra of the N- and C-terminal peptides in the presence of a DPPC monolayer are compared in Figure S3 of the Supporting Information. The amide I band of the Cterminal peptide is slightly wider than that of the N-terminal peptide, and their amide I/II ratio is very similar. It can thus be postulated that the conformation and orientation of these two peptides are very similar in the presence of a phospholipid monolayer. Moreover, on the basis of the very small bandwidth of the amide I band of these peptides (Figure S3), the presence of conformations other than α-helices can virtually be excluded. It is also important to stress that the PM-IRRAS spectra of the N- and C-terminal peptides remain unchanged when their subphase concentration was increased from 0.5 up to 12 μM (data not shown), which is much larger than their saturating surface concentration of 1.2 μM. This contrasts with the data of Kerth et al.,54 who observed that the α-helical secondary structure of KLAL model peptides would be converted into a βsheet structure at smaller areas per molecule, and those of Yassine et al.,55 who found that the peptides corresponding to the transmembrane domain of the SNARE proteins VAMP/ synaptobrevin and syntaxin 1 exhibited a transition from an α-

monolayer of DPPC, a large and quick increase in surface pressure can be observed followed by a slow decrease of surface pressure until an equilibrium is reached. The ΔΠ of the two peptides measured at equilibrium is quite similar (10.7 and 13 mN/m). Similar binding isotherms of these peptides have been obtained in the presence of the other phospholipid monolayers. PM-IRRAS spectra of the N- and C-terminal peptides were then measured in the presence of the phospholipid monolayers used to mimic the lipid content of the RPE. Given the acquisition time of 9 min of the PM-IRRAS spectra and the very fast kinetics of adsorption of these peptides (Figure 5), no difference has been observed in the spectra as a function of time. Figures 6A and 6B respectively show typical infrared

Figure 6. (A) PM-IRRAS spectra of the N-terminal peptide of LRAT injected underneath monolayers of DPPC, DPPE, and DPPS at initial surface pressures between 17 and 23 mN/m. The intensity of the amide I band of the spectrum in the presence of DPPE is ∼1.2 times larger than that in the presence of DPPS and DPPC. They have been normalized to facilitate their comparison. (B) PM-IRRAS spectra of the C-terminal peptide of LRAT injected underneath monolayers of DPPC, DPPE, and DPPS. The intensity of the amide I band of the spectrum in the presence of DPPE is respectively ∼1.1 and 1.5 times larger than those in the presence of DPPS amd DPPC. They have been normalized to facilitate their comparison. The intensity scale bar is for the most intense spectrum. Subphase contains 50 mM Tris at pH 7 and 150 mM NaCl. The final concentration of the peptides in the subphase is 1.2 μM.

spectra of the N- and C-terminal peptides in the presence of DPPC, DPPE, and DPPS monolayers. The CO ester band is located at 1735−1737 cm−1 for DPPC and DPPS, whereas it is slightly shifted to 1740−41 cm−1 for DPPE (Figures 6A and 6B). 3520


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helical to a β-sheet structure when increasing the peptide/lipid ratio in monolayer.

but they can be as short as 12 and as long as 34 amino acids.57−59 The average length of transmembrane α-helices typically fits quite well with the thickness of a bilayer. For example, the average length of the 7 transmembrane α-helices of rhodopsin is ∼41 Å (varying between 33 and 47 Å),60 which is very close to the typical thickness of a bilayer (∼40 Å61). However, the longer transmembrane α-helices of rhodopsin are tilted up to 33° with respect to the normal of the bilayer plane presumably because of the hydrophobic mismatch between their length and the thickness of the bilayer.60 It can thus be postulated that the orientation of the N- and C-terminal peptides of LRAT in the presence of a phospholipid monolayer, lying between 40° and 45°, could be due to differences between the thickness of the lipid monolayer and the length of these peptides. A recent study performed with eukaryotic cells proposed that only the C-terminal hydrophobic domain is essential for the activity and the targeting of LRAT to the membrane of the endoplasmic reticulum.11 They also proposed that ΔCterminal-LRAT and tLRAT are inactive and lack interaction with ER membranes. 11 In contrast, the present data demonstrate that tLRAT strongly binds lipid monolayers and that both N- and C-terminal segments of LRAT behave similarly. Indeed, the binding, secondary structure content, and orientation in lipid monolayers of both N- and C-terminal segments are almost identical. These data thus allow to postulate that both N- and C-terminal hydrophobic α-helical peptides could serve to anchor tLRAT to the membrane. This is further supported by preliminary MIP values obtained with these peptides in the presence of a phospholipid monolayer, which are larger than the estimated lateral pressure of membranes. A new topology model has thus been generated using the I-TASSER prediction tool (Figure 7). This structural

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DISCUSSION The full-length sequence of LRAT has not yet been successfully expressed and purified presumably because of the high hydrophobicity of its N- and C-terminal segments. Besides, it has been postulated that both N- and C-terminal hydrophobic segments5 or either only the C-terminal segment11 could allow to anchor and properly orient LRAT with respect to the membrane, such as hydrophobic transmembrane α-helices. However, no information is available regarding the structure of the N- and C-terminal segments of LRAT and the extent of their membrane binding. Moreover, although it was previously shown that purified tLRAT hydrolyzes phospholipid monolayers,10 its extent of membrane binding was still unknown. In the present work, we have thus expressed and purified tLRAT, lacking the N- and C-terminal hydrophobic segments of LRAT, to study its membrane binding. In addition, the N- and Cterminal peptides of this enzyme were synthetized to study their secondary structure and orientation as well as their binding to lipid monolayers. The present data provide evidence for a strong binding of tLRAT to typical phospholipids of the RPE with MIP values much higher than that of the estimated lateral pressure of membranes (for a review, see ref 40). Furthermore, the values of the synergy factor a and ΔΠ0 also argue in favor of a strong binding of tLRAT to phospholipid monolayers, thus suggesting that this protein has a strong affinity for membranes despite the absence of its N- and C-terminal hydrophobic segments. Moroever, it is noteworthy that a significantly larger MIP of tLRAT was obtained with the DPPC substrate which was shown to be preferentially hydrolyzed by LRAT.56 The detailed analysis of the sequence of the N- and Cterminal peptides shows that they are very hydrophobic (see Figure S4 in Supporting Information). Indeed, the N-terminal peptide contains 17 hydrophobic, 9 polar, and 4 charged amino acids whereas the C-terminal peptide includes 21 hydrophobic, 9 polar amino acids, and no charged amino acid. Moreover, the N-terminal peptide contains a short stretch of 7 polar and charged amino acids on the N-terminal side, which also includes 2 hydrophobic amino acids. There are also 2 charged amino acids in the center of this sequence followed by stretches of polar and hydrophobic amino acids. The C-terminal peptide is obviously more hydrophobic than the N-terminal peptide. It includes a stretch of 2 hydrophobic and 2 polar amino acids on the C-terminal side. There is then a long stretch of 10 hydrophobic amino acids, which includes 2 prolines. One of them is located almost exactly in the center of the peptide and shall result in the bending of the peptide structure. The very fast adsorption of these two peptides onto phospholipid monolayers is consistent with the high hydrophobicity of their amino acid sequence. The circular dichroic and infrared spectra of the N- and Cterminal peptides both in solution and in monolayer suggest that they adopt an α-helical conformation. Moreover, the orientation and secondary structure of both peptides are very similar. Therefore, these two putative hydrophobic N- and Cterminal segments of LRAT could serve as transmembrane αhelical peptides. In fact, the length, the sequence and the secondary structure of these two peptides allow to postulate that they could both include a transmembrane segment. In fact, transmembrane α-helices contain 20 amino acids in average,

Figure 7. Topology model of LRAT. The 3D representation of LRAT has been chosen from five ab initio structures constructed with the online tool I-TASSER. The catalytic triad that includes the amino acids H60, Y154, and C161 is displayed prominently.

model of LRAT is consistent with our experimental findings and more consistent with its function than previous models11,62 in that the catalytic triad of LRAT must be in close contact with the membrane to hydrolyze its phospholipid substrate. Indeed, as shown in Figure 7, the three amino acids of the catalytic triad (H60, Y154, and C161) of LRAT are close enough to the 3521


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membrane surface to carry out the hydrolysis of the sn-1 acyl chain of phospholipids. Such a membrane binding of LRAT would be highly favored if both N- and C-terminal α-helical peptides would allow to anchor this protein to the membrane. In contrast, previous models proposed that either only the Cterminal segment of LRAT is membrane bound (the catalytic triad being far away from the membrane)11 or only the N- and C-terminal α-helical transmembrane segments are membrane bound (the central core of LRAT (residues 31−195) being far from the membrane).11,62 In their recent review, Kiser et al.63 have generated a structural model of LRAT using the SWISS-MODEL server64 based on the NMR structure of HRASLS3 (PDB accession code 2KYT).65 This model proposes that LRAT is made of a core of four β-sheets surrounded by three α-helices. It is noteworthy that this model does not include the C-terminal hydrophobic segment of LRAT because only the structure of the N-terminal catalytic domain of human HRASLS3 has been solved. Moreover, there is a significant discrepancy between the number of amino acids of LRAT (230) and that of HRASLS3 (166). As shown in Figure S5 of the Supporting Information, the alignment of LRAT with HRASLS3 shows ∼23% identity between the sequence of these two proteins (with a score of at least 7). On the basis of this sequence identity and the known structure of HRASLS3,65 one can estimate that LRAT contains 10% β-sheets and 10% α-helices. In contrast, we have recently reported by circular dichroism that tLRAT contains approximately 60% α-helices, 8% β-sheets, 10% β-turns, and 22% random structures.66 This large content in α-helices is also in very good agreement with our previous PM-IRRAS experiments showing a strong amide I band located at 1655 cm−1 upon monolayer binding of tLRAT.10 Accordingly, the model proposed in Figure 7 includes 9 α-helices and a small number of β-sheets located in the central core of LRAT, which is consistent with our circular dichroism analysis. Therefore, the topology model presented in Figure 7 is consistent with that of Kiser et al.63 in that the structure of LRAT might contain a central core of β-sheets but inconsistent with their content of αhelices. In conclusion, despite tLRAT lacks its two hydrophobic terminal domains, the present results suggest that it strongly binds membranes, which is consistent with its function involving the hydrolysis of membrane phospholipids. Furthermore, the two peptides corresponding to the putative Nand C-terminal domains of LRAT spontaneously bind to lipid monolayers with an α-helical structure. These data also allowed to design a new topology model for LRAT which is consistent with the membrane binding activity of this enzyme as well as its secondary structure and enzymatic function. This topology model also suggests that the N- and C-terminal hydrophobic peptides could allow to provide LRAT with a proper orientation to efficiently perform its enzymatic phospholipid hydrolysis function.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (418) 682-7569; Fax: (418) 682-8000; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work described here was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). The Banque d’Yeux Nationale is partly supported by the Réseau de Recherche en Santé de la Vision from the FRSQ. S. Bussières is the recipient of the Frederick Banting and Charles Best Graduate Scholarship from the Canadian Institutes of Health Research (CIHR) and a travel fellowship from the Réseau FRSQ de recherche en santé du Québec.

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ABBREVIATIONS DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPE, 1,2-dioleoylsn-glycero-3-phosphoethanolamine; DOPS, 1,2-dioleoyl-snglycero-3-phosphoserine; DPPC, 1,2-dipalmitoyl-sn-glycero-3phosphocholine; DPPE, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine; DPPS, 1,2-dipalmitoyl-sn-glycero-3-phosphoserine; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; HFIP, hexafluoroisopropanol; LRAT, lecithin:retinol acyltransferase; tLRAT, truncated LRAT; PM-IRRAS, polarization modulation infrared reflection absorption spectroscopy; RPE, retinal pigment epithelium; SDS, sodium dodecyl sulfate.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Additional analyses of the PM-IRRAS spectra (Figures S1−S3), the analysis of the primary structure of the N- and C-terminal peptides (Figure S4), and the comparison between the primary structure of HRASLS3 (HREV107) and that of LRAT (Figure S5). This material is available free of charge via the Internet at http://pubs.acs.org. 3522


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Binding of a Truncated Form of Lecithin:Retinol Acyltransferase and Its

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