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The Mechanism by which Luteolin Disrupts the Cytoplasmic Membrane of Methicillin-Resistant Staphylococcus Aureus Tao Zhang, Yunguang Qiu, Qichao Luo, Lifen Zhao, Xin Yan, Qiaoce Ding, Hualiang Jiang, and Huaiyu Yang J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b05766 • Publication Date (Web): 08 Jan 2018 Downloaded from http://pubs.acs.org on January 8, 2018

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The Journal of Physical Chemistry

The Mechanism by Which Luteolin Disrupts the Cytoplasmic Membrane of Methicillin-Resistant Staphylococcus Aureus Tao Zhang†,#, Yunguang Qiu‡,§,#, Qichao Luo‡,§, Lifen Zhao‡, Xin Yanǁ,‡, Qiaoce Ding‡, Hualiang Jiang*,†,‡,§, and Huaiyu Yang*,‡,§ †

School of Pharmacy, East China University of Science and Technology, 130 Meilong

Road, Shanghai 200237, China ‡

Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai

Institute of Materia Medica, Chinese Academy of Science, 555 Zuchongzhi Road, Shanghai 201203, China §

University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049,

China ǁ

School of Life Science and Technology, Shanghai Tech University, 393 Huaxiazhong

Road, Shanghai 201210, China E-mail addresses: [email protected] (H. Jiang) and [email protected] (H. Yang). #

These authors contributed equally to the work.

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ABSTRACT: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most versatile human pathogens. Luteolin (LUT) has anti-MRSA activity by disrupting the MRSA cytoplasmic membrane. However, the mechanism by which luteolin disrupts the membrane remains unclear. Here, we performed differential scanning calorimetry (DSC) and all-atomic molecular dynamics (AA-MD) simulations to investigate the interactions and effects of LUT

on

model

membranes

composed

of

phosphatidylcholine

(PC)

and

phosphatidylglycerol (PG). We detected the transition thermodynamic parameters of dipalmitoylphosphatidylcholine (DPPC) liposomes, dipalmitoylphosphatidylglycerol (DPPG) liposomes, and liposomes composed of both DPPC and DPPG at different LUT concentrations and showed that LUT molecules were located between polar heads and the hydrophobic region of DPPC/DPPG. In the MD trajectories, LUT molecules ranging from five to fifty had different effects on the membranes thickness, fluidity and ordered property

of

lipids,

and

lateral

pressure

of

lipid

bilayers

composed

of

dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG). And most LUT molecules were distributed in the region between the phosphorus atoms and C9 atoms of DOPC and DOPG. Based on the combination of these results, we conclude that the distinct effects of LUT on lipid bilayers composed of PCs and PGs may elucidate the mechanism by which LUT disrupts the cytoplasmic membrane of MRSA.


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1. INTRODUCTION Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most versatile human pathogens and has been commonly detected in communities, clinics and hospitals since the 1960s.1-2 MRSA infects people with relatively weak immune systems and causes many hospitalized patients to be at higher risk of infection, particularly people who recently have undergone surgery.1 According to previous reports, more people in US hospitals have died of MRSA infections than of acquired immune deficiency syndrome and tuberculosis combined since the 1990s.3-4 Thus, MRSA poses a huge threat to Americans’ health and even to global human health. During the past decade, several anti-infective agents, such as vancomycin, linezolid, daptomycin, tigecycline, telavancin and ceftaroline, have provided sick patients with skin and skin structure infections (SSSIs) and pneumonia caused by MRSA with multiple treatment choices.5-6 However, these agents have recently been associated with adverse neuropathic events and nephrotoxicity, emerging resistance problems, and high drug costs. Thus, MRSA remains a major public health problem worldwide and a therapeutic challenge to treat in humans. In addition, because of difficulties in discovering new antibiotics that cure MRSA, different strategies for discovering new agents with activity against MRSA must be identified.2,7 Through the emergence of high-throughput screening and structure-based drug design, natural products from plants have recently attracted renewed interest in drug discovery.8-10 Based on the report by Dae-Ki Joung et al., luteolin (LUT; Figure 1), a



classic flavonoid compound derived from plants, has anti-MRSA activity through its membrane-binding and ATPase-inhibiting activities. Moreover, using transmission electron microscopy, the authors observed that LUT induced the disruption of the MRSA cytoplasmic membrane.11 This research is very meaningful and suggests that LUT represents a potential anti-MRSA drug. However, the mechanism by which LUT disrupts the MRSA cytoplasmic membrane remains to be elucidated before its use in further pharmaceutical applications. Due to the complexity of biological membranes, model membranes prepared from desired membrane components have proven to be an invaluable tool in membrane research, including ion, drug and protein-membrane interactions.12-15 In our case, we constructed two classical model membranes, spherical liposomes and planar lipid bilayers, with phosphatidylcholine (PC) and phosphatidylglycerol (PG) to study the interactions between LUT and the cytoplasmic membrane of MRSA. The molar ratio of PC to PG was 7:3, consistent with the formulations described in other studies.16-17 In biomembrane studies, biophysical techniques, such as differential scanning calorimetry (DSC), can provide valuable information on the phase transition or thermotropic mesomorphism of liposomes.18-22 Both dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) DSC diagrams have two phase transition peaks, one pre-transition peak and one main-transition peak, and the main phase transition temperature (Tm) of both peaks is ~41 °C.23-25 Furthermore, the pre-transition peak is very sensitive to any kind of perturbation, as it might be altered not


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only by compounds that penetrate the lipid bilayer, such as cholesterol,26 but also by cosmotropic substances, such as sucrose.27 Substances that incorporate into the polar-nonpolar interface region of the lipid bilayer, such as formononetin, abolish the DPPC pre-transition peak, even when used at low concentrations.22 Thus, we used DSC to observe the LUT-induced changes in the thermodynamic behaviors of liposomes to determine the location of LUT in liposomes composed of DPPC/DPPG, DPPC and DPPG with varying LUT concentrations. All-atom molecular dynamics (AA-MD) simulations have recently emerged as a powerful tool for investigating ion-lipid, drug-lipid and proteins-lipid interactions.12-13, 28-30

Here, we performed AA-MD simulations to obtain the dynamic and structural

properties of lipid bilayers before and after the insertion of LUT, such as membrane thickness, lipid diffusion coefficients, deuterium order parameter, atom density distributions, lateral pressure profiles, contact lipid numbers, and hydrogen bond numbers, which are not easily assessed using other techniques. These dynamic and structural parameters have been reported to be involved in the biological activity of flavonoids.31 Besides, bacteria can adapt to external stimulus and regulate membrane parameters by altering its composition.32-34 Such an adaptation involves changes in proportion of saturated and unsaturated acyl chains, however, the composition of polar groups does not change.32-33, 35 Thus, the membrane components we used were the same as previous lipid bilayers (dioleoylphosphatidylcholine (DOPC):dioleoylphosphatidylglycerol (DOPG) = 7:3) used in MD simulations that mimic the MRSA cytoplasmic membrane.17 By



combing DSC measurements and MD simulations, this study showed that the distinct effects of LUT on lipid bilayers composed of PCs and PGs may elucidate the mechanism by which LUT disrupts the cytoplasmic membrane of MRSA. 2. METHODOLOGY 2.1 Chemical Reagents

Lyophilized dry powders of LUT and DPPC (> 98%, HPLC grade) were purchased from TCI Development Co., Ltd. (Shanghai, China). HPLC-grade chloroform and methanol were purchased from Ourchem (Shanghai, China). DPPC powders were dissolved in chloroform at a concentration of 25 mg/ml. Highly pure (> 99%) lyophilized dry powders of DPPG were purchased from Corden Pharma (Liestal, Switzerland) and dissolved in chloroform/methanol/water (65:35:8 vol/vol/vol) to a concentration of 25 mg/ml. Both DPPC and DPPG solutions were stored at -20 °C. The structures of DPPC and DPPG are shown in Figure 1. PBS was purchased from Thermo Fisher Scientific (Shanghai, China). All reagents were used without further purification.


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Figure 1. Chemical structures of DPPC, DPPG, DOPC, DOPG and LUT.

2.2 Preparation of Multilamellar Vesicles for DSC

A modified version of a previously reported protocol was employed to prepare the multilamellar vesicles used for DSC measurements.22 LUT was dissolved in CHCl3/CH3OH (2:1 vol/vol) to a concentration of 3.5 mM. Lipid solutions were added in CHCl3/CH3OH (2:1 vol/vol) in a round-bottom flask until the desired composition of DPPC:DPPG lipids (7:3 mol/mol) was obtained, and then the LUT solution was added to obtain molar ratios of lipids to LUT of 1:0.025, 1:0.05, 1:0.1, and 1:0.2. Then, the same experimental procedures were performed to generate LUT-DPPC and LUT-DPPG. The



solvents were evaporated in a gentle gaseous nitrogen stream and then dried under vacuum for approximately four hours to remove the residual solvent. Samples were hydrated with PBS to a final concentration of ~10 mg/ml and vortexed at a temperature (60 °C) greater than the main phase transition of lipids until optical homogeneity of the mixture was observed.

2.3 DSC

DSC measurements were performed using a heat flux instrument (Q2000, TA Instruments, New Castle, Delaware, USA) equipped with a refrigerated cooling system. A nitrogen purge at a flow rate of 60 cm3/min was used to provide an inert gas atmosphere in the DSC cell. The system was calibrated using an indium standard and the samples were run against a hermetic empty reference pan. A scan rate of 5 °C/min and a scan range of 25-70 °C were used for all samples. The total lipid concentration used for DSC was ~10 mg/ml. Data were mathematically analyzed using Thermal Solutions software (TA Instruments, New Castle, Delaware, USA), and the results were expressed as the means ± standard deviations (S.D.) of three determinations.

2.4 Simulation Systems

We constructed a lipid bilayer composed of 140 DOPC molecules and 60 DOPG molecules (DOPC:DOPG = 7:3) to mimic the MRSA cytoplasmic membrane, based on the report by Mitra S. Ganewatta et al.17 The chemical structures of DOPC and DOPG are


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presented in Figure 1. An 82 Å × 82 Å lipid bilayer was generated on the CHARMM-GUI website to establish the system.36 Two systems were constructed for this experiment to study the interactions and influences of LUT on the lipid bilayers. One was a pure membrane system (Pure-DOPC-DOPG (PPCG)), and the other system was a membrane containing LUT (LUT-PCG). As illustrated in Figure 2a, five LUT molecules were initially placed in the bulk water region at a distance of ~0.5 nm from one leaflet of the membrane. When the LUT molecules were observed to have been inserted into the membrane after running the system, we gradually added five more LUT molecules into the system until the fraction of LUT reached 20%, yielding a total of fifty LUT molecules in the final simulation system (shown in Figure 2b). The temperature of each system was maintained at 310 K. Each of the systems was solvated with TIP3P water and 0.15 M NaCl.

Figure 2. (a) Snapshot of the initial lipid bilayers with five LUT molecules. (b) Snapshot of the lipid bilayers in an equilibrium state with fifty LUT molecules.



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2.5 MD Simulation Details MD simulations were performed using the GROMACS 5.1.4 package with an isobaric-isothermal ensemble and periodic boundary conditions.37 The CHARMM 36 force field was used.38 The CHARMM-compatible parameter for LUT was generated using the CGenFF program.39 Energy minimizations were first performed to relieve unfavorable contacts, followed by equilibration steps of 50 ns. The v-rescale method with a coupling time of 0.1 ps40 was used to maintain the temperature of the system, and the Nose-Hoover method41-42 was subsequently used with a coupling time of 0.5 ps in production runs. The pressure was maintained at 1 bar using the Parrinello-Rahman method, with τ = 1.0 ps and a compressibility of 4.5 × 10-5 bar-1.43 The SETTLE constraints44 and LINCS constraints45 were applied to the hydrogen-containing covalent bonds in water molecules and other molecules, respectively. The time step was set to 2 fs, and long-range electrostatic interactions were calculated using the particle mesh Ewald (PME) algorithm.46 A cut-off value of 1.2 nm was used for both electrostatic and van der Waals interaction calculations. The PPCG system was performed for 200 ns until it reached equilibrium. For comparison with DSC measurements, ten simulations of the LUT-PCG system were performed with 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 LUT molecules

(5LUT-PCG,

10LUT-PCG,

15LUT-PCG,

20LUT-PCG,

25LUT-PCG,

30LUT-PCG, 35LUT-PCG, 40LUT-PCG, 45LUT-PCG and 50LUT-PCG), respectively. Each of these systems was run for more than 100 ns until all LUT molecules were inserted into the membrane. The systems comprising DOPC/DOPG lipid bilayers with


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LUT molecules ranging from five to fifty were analyzed and compared with the system comprising pure lipid bilayers. All of them were run for 200 ns. Additionally, Gromacs 4.5.5 program was employed to calculate the lateral pressure profiles according to the methods reported by Juan M. Vanegas et al.47-48. We run 200 ns to produce trajectory files (double precision), containing both positions and velocities, in both two systems. Then the whole set of simulations was rerun in the last 100 ns trajectories with 1000 frames separated by 100 ps using a custom GROMACS version (GROMACS-LS).47-49 GROMACS-LS program computed the local stress based on the Hardy stress definition and the covariant central force decompositions (cCFD) was used to produce the constant pressure for our systems.47 2.6 Calculation of Membrane Properties As shown in Figure S1, the two systems reached equilibrium after 100 ns. The last 100-ns MD trajectories for the PPCG and ten LUT-PCG systems were used to analyze the structural and dynamical properties of membranes. Membrane thickness was calculated using the VMD 1.9.1 MEMBPLUGIN.50 The membrane thickness was defined as the distance between the mass center of the phosphorus atoms of both the top and bottom leaflets along the z-axis (DP-P). The mean square displacements (MSD) and lateral diffusion coefficients (DL) of DOPC, DOPG and water molecules were obtained using the gmx_msd tool. The linear portions of the MSD curves were analyzed to improve the calculation accuracy. Standard errors were estimated by the block averages methods.51-52 For our shorter simulations,



only three blocks were used to determine the standard error.53 DL was calculated using the following equation: lim || − 0|| = 4

→

where is defined as the mass center of phosphorus atom in each lipid. The deuterium order parameter (SCD) is used to determine the structural changes in lipid bilayers by calculating the order of the lipid acyl chains. We calculated the SCD values using the gmx_order program and the following equation: 3 1 S = 〈 cos − 〉 2 2 where is the angle between the vector from Cn - 1 to Cn + 1 and the bilayer normal, and the angular brackets denote a time and ensemble average. The z-axis coordinate of the atom was extracted with the gmx_traj program and then the value of the z-axis coordinate was statistically analyzed to calculate the atom density profiles along the z-axis. To calculate the lateral pressure profiles, the simulation box was divided into slabs by a given grid spacing along the membrane normal. Grid spacing was set to 0.1 nm and the pressure was calculated for every slab along the z dimension. The lateral pressure profile Π ! is defined as55 Π z =

"## ! + "%% ! − "&& ! 2

Where "## !, "%% ! and "&& ! are the diagonal elements of the pressure tensor, which has the opposite sign as the stress tensor, (. Two components of pressure can be reduced from the stress tensor: ") = "## ! + "%% !⁄2 (xy plane of the membrane)


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and "- = "&& ! (normal to the membrane). Furthermore, "## ! and "%% ! would converge to identical values because of the symmetry in the xy plane. We calculated the average of the two to improve the calculation precision. When the bilayer reach to the mechanical equilibrium, "&& ! must be constant for all slabs.55 Due to the symmetry of the lateral pressure profile over the both sides of the membrane, the results only show the one side of the membrane as the previous work.54 The gmx_minidist method was used to calculate the minimum distance between the LUT and DOPC or DOPG molecules. The intermolecular hydrogen bonds between LUT and DOPC or DOPG were analyzed using the gmx_hbond program. All data were statistically analyzed and plotted using Origin 9.0 software (Origin Lab Corporation, Northampton, USA) or GraphPad Prism 5.0 software (GraphPad Prism, San Diego, CA). All statistical data are presented as means ± S.D. or means ± standard errors of the means (S.E.M.). Statistical significance was determined using a two-tailed Student’s t test. Statistical significance was noted as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001. 3. RESULTS 3.1 Differential Scanning Calorimetry Analysis DSC thermograms have been used to detect the transition thermodynamic parameters [(∆H (transition enthalpy), Tm (transition temperature), TOS (temperature at which the transition starts)] of flavonoid/lipid-water systems from the gel phase to the liquid-crystalline phase during an increase in temperature.56 First, we determined the



effects of LUT on the thermotropic behaviors of liposomes composed of DPPC/DPPG (7:3 mol/mol) using DSC. As shown in Figure 3a, the studied LUT molecules alter thermotropic phase behaviors of DPPC/DPPG (7:3 mol/mol) liposomes. For pure lipids, we observed two phase transitions in the range of scanned temperatures (25 °C-70 °C): a pre-transition (Tp = 37.80 ± 0.76 °C) and the main phase transition (Tm = 42.18 ± 0.25 °C). The TOS, and ∆H values for the pre-transition were 36.22 ± 1.19 °C and 1.28 ± 0.77 J/g, respectively, and were 41.78 ± 0.16 °C and 50.64 ± 2.38 J/g, respectively, for the main phase transition. As the concentration of LUT molecules increased, LUT caused the disappearance of the pre-transition phase and a decrease in the TOS, Tm and ∆H values for the main phase transition (Figure 3a and Table 1). We subsequently detected the effects of LUT on the thermotropic behaviors of both DPPC and DPPG liposomes to determine the difference in interactions and effects of LUT with and on DPPC or DPPG in DPPC/DPPG-LUT interactions. As shown in Figure 3b and Table 2, we observed two phase transitions in the pure DPPC liposomes: a pre-transition (Tp = 37.38 ± 1.02 °C) and the main phase transition (Tm = 41.98 ± 0.31 °C). The TOS and ∆H values for the pre-transition and main phase transition of DPPC were 35.73 ± 1.14 °C, 4.09 ± 2.96 J/g, 41.76 ± 0.24 °C and 47.21 ± 2.09 J/g, respectively. However, as the concentration of LUT increased, the pre-transition disappeared and the TOS, Tm and ∆H values of DPPC liposomes decreased. In contrast, pure DPPG liposomes did not show an obvious change in the pre-transition phase and the TOS, Tm and ∆H values showed only a very slight decrease as the concentration of LUT increased (Figure


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3c and Table 3).

Figure 3. (a) DSC diagram of DPPC/DPPG liposomes with LUT at various molar ratios. (b) DSC diagram of DPPC liposomes with LUT at various molar ratios. (c) DSC diagram of DPPG liposomes with LUT at various molar ratios.

Table 1. Calorimetric parameters observed in DPPC/DPPG-LUT mixtures studied at different molar ratios; the values represent the means ± S.D. of three separate measurements. LUT-DPPC/DPPG

TOS a (°C)

Tm b (°C)

∆H c (J/g)

DPPC:DPPG = 7:3

41.78 ± 0.16

42.18 ± 0.25

50.64 ± 2.38

DPPC/DPPG-LUT = 1:0.025

40.96 ± 0.25

41.66 ± 0.21

46.13 ± 1.08


40.09 ± 0.12

41.43 ± 0.37

37.83 ± 2.01


38.95 ± 0.33

40.05 ± 0.27

35.32 ± 2.82


39.86 ± 0.45

41.28 ± 0.27

31.01 ± 1.35

a

TOS, temperature at which the transition starts.

b

Tm, maximum temperature of the

calorimetric peak. c ∆H, transition enthalpy.



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Table 2. Calorimetric parameters observed in DPPC-LUT mixtures studied at different molar ratios; the values represent the means ± S.D. of three separate measurements. TOS a (°C)

Tm b (°C)

∆H c (J/g)

DPPC

41.76 ± 0.24

41.98 ± 0.31

47.21 ± 2.09

DPPC:LUT = 1:0.025

41.10 ± 0.10

41.64 ± 0.19

44.46 ± 1.41

DPPC:LUT = 1:0.05

39.78 ± 0.84

40.82 ± 0.69

38.55 ± 1.09

DPPC:LUT = 1:0.1

37.59 ± 1.11

38.86 ± 1.08

34.66 ± 1.25

DPPC:LUT = 1:0.2

38.03 ± 1.27

39.47 ± 0.95

26.39 ± 0.76

DPPC-LUT

a


b



Table 3. Calorimetric parameters observed in DPPG-LUT mixtures studied at different molar ratios; the values represent the means ± S.D. of three separate measurements. TOS a (°C)

Tm b (°C)

DPPG

40.69 ± 0.24

41.64 ± 0.99

47.54 ± 0.81

DPPG:LUT = 1:0.025

40.20 ± 0.13

41.19 ± 0.47

45.70 ± 2.44

DPPG:LUT = 1:0.05

39.83 ± 0.29

41.17 ± 1.00

43.62 ± 4.35

DPPG:LUT = 1:0.1

39.92 ± 0.43

41.11 ± 0.68

44.52 ± 3.57

DPPG:LUT = 1:0.2

39.88 ± 0.36

41.10 ± 0.47

47.66 ± 7.76

DPPG-LUT

a


b

∆H c (J/g)



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3.2 Molecular Dynamics Simulations In the present study, we performed AA-MD simulations to determine the effects of LUT on the membrane thickness, the lipid diffusion coefficients, the order parameters of lipid chains of DOPC and DOPG, and the location of LUT in the lipid bilayers , and to calculate the lateral pressure profiles, the number of contacts between LUT and lipids in DOPC and DOPG and the probability of the number of hydrogen bonds formed between LUT molecules (5 to 50) with each lipid molecule in DOPC and DOPG, as described below. Membrane Thickness and Lipid Diffusion Coefficients. First, we evaluated the effect of LUT on the lipid bilayers by calculating the thickness of the bilayer before and after the addition of LUT molecules, based on its macroscopic features. As shown in Figure 4, when five LUT molecules were added into lipid bilayers, the average membrane thickness had negligible change compared to pure membrane. By contrast, when additional LUT molecules increased from ten to fifty, the average membrane thickness gradually decreased from 38.47 ± 0.49 Å to 37.71 ± 0.53 Å. In addition, we calculated the lateral diffusion coefficient (DL) value of DOPC and DOPG of the PPCG and LUT-PCG systems to characterize the extent to which the lateral diffusion of lipids in the membrane was affected by LUT molecules of varying concentrations. The calculated diffusion coefficients are exhibited in Figure 5. The DL value of DOPC in our reference



system (PPCG) is 1.08 × 10-7 cm2/s, which is roughly similar with the DL value of DOPC (1.6 × 10-7 cm2/s) reported by Filippov, A. et al..57 The addition of five LUT molecules to membranes had a negligible effect on DL values of DOPC and DOPG compared to those values in PPCG systems. Obviously, an increase of DL values of both DOPC and DOPG lipids can be observed when the added LUT molecules increased from ten to fifty, but not linear increase. It may be caused by the finite size effects and slow diffusion dynamics. To estimate the effects induced by the finite size, we adopted the Bayesian estimates presented by the Venable, Richard M., et al.53 The estimated values were shown in Figure S13-S16.

Figure 4. Lipid bilayers thickness in PPCG system and ten systems ranging from 5LUT-PCG to 50LUT-PCG.


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Figure 5. Diffusion constants of DOPC and DOPG in PPCG system and ten systems ranging from 5LUT-PCG to 50LUT-PCG.

Deuterium Order Parameters. The ordering of the two hydrophobic tails of the lipid is usually characterized by the deuterium order parameter (SCD) that is determined using NMR experiments.58 The order parameter profiles of the four lipid chains (sn-1 and sn-2 of DOPC and sn-1 and sn-2 of DOPG, as shown in Figure 1) for all systems (PPCG, 5LUT-PCG to 50LUT-PCG) are plotted in Figure 6. The profiles of sn-1 and sn-2 chains of both DOPC and DOPG in the nine systems (10LUT-PCG to 50LUT-PCG) are globally less than in the profiles of the PPCG system. By contrast, the order parameter profiles of the four lipid chains of DOPC and DOPG in 5LUT-PCG system are slightly lower than those in the PPCG system. These results indicate an ordering effect of LUT on the phospholipid chains.



Figure 6. Order parameter (SCD) profiles of the sn-1 and sn-2 chains of DOPC (top) and DOPG (bottom) in PPCG and ten systems ranging from 5LUT-PCG to 50LUT-PCG (a, b, c, and d).

Atom Density Profiles. As shown in Figure 7, we determined the depths of fifty LUT molecules in the membrane by calculating their atom density profiles along the z-axis to explore the location of the LUT molecules in the lipid bilayers. The center of the bilayers was defined as 0 Å, and the upper and lower leaflets of the lipid bilayers were defined as negative and positive values, respectively. Consequently, the MD-estimated depths of these LUT molecules in lipid bilayers were mainly at -15 Å and 13 Å. For comparison,


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we also calculated the atom density profiles of the C9, C18 and phosphorus atoms of DOPC and DOPG. The peak values of the atom density profiles for C9 of DOPC and DOPG in the upper leaflet were -9 Å and -8 Å, respectively, and 6 Å and 5 Å for C9 of DOPC and DOPG in the lower leaflet, respectively. The peak values of the phosphorus atom of DOPC and DOPG were -20 Å and 17 Å in the upper and lower leaflet, respectively. Based on these results, LUT molecules are mainly distributed between the polar heads of lipids and C9 of the lipid chains of DOPC and DOPG of lipid bilayers in 50LUT-PCG system. Besides, we also calculated atom density profiles of LUT molecules and lipids along the z-axis (Figure S3-S11) in other nine systems (from 5LUT-PCG to 45LUT-PCG). In 5LUT-PCG system, LUT molecules were mainly distributed between C9 and phosphorus atom of DOPC and DOPG in upper leaflet of lipid bilayers based on atom density profiles of LUT molecules and lipids. For 10LUT-PCG, 15LUT-PCG, and 20LUT-PCG systems, LUT molecules were observed to distribute wider and penetrate gradually deeper into upper leaflet of lipid bilayers than in 5LUT-PCG system and fractional molecules penetrated into lower leaflet of lipid bilayers by comparison of the atom density profiles of LUT and lipids in these four systems. For 25LUT-PCG, 30LUT-PCG, 35LUT-PCG, 40LUT-PCG, and 45LUT-PCG systems, LUT molecules were mainly located between the polar heads of lipids and C9 of the lipid chains of DOPC and DOPG of lipid bilayers seen from Figure S3-S11, which are consistent with the behaviors of LUT molecules in 50LUT-PCG systems.



Figure 7. Plots of the atom density profiles of LUT and the phosphorus, C9, C18 atoms of the sn-1 and sn-2 chains of DOPC and DOPG along the z-axis in the membrane in 50LUT-PCG system.

Lateral Pressure Profiles. Lateral pressure profile has been a fundamental membrane property and its possible changes are of major importance for modulating membrane protein function, and in particular for the behaviors of amphiphilic and polarisable drugs in bilayer.59 Here, we calculated the lateral pressure profiles of lipid bilayers in three systems (PPCG, 5LUT-PCG and 50LUT-PCG) to determine the local effects of LUT on lipid bilayer. The lateral pressure profiles of upper leaflet of lipid bilayer in PPCG and 50LUT-PCG systems are plotted in Figure 8. We defined 0 Å as the bilayer center. The X values in Fig. 8 represent the distance away from the middle of bilayer. It is clear from Figure 8 that two profiles exhibit similar tendency in curves. The pressure profiles exhibit a characteristic trough and two peaks, located at around the X value of 14 Å, 24 Å and 8


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Å, respectively, which are roughly similar to the shapes in profiles reported by Wei Ding et al.60 Obviously, the lateral pressure profile of the 50LUT-PCG system is higher than that in PPCG system in the X range of 12-20 Å around the trough. In contrast, the lateral pressure profile of the 50LUT-PCG system is less than that of PPCG system in the X range of both 6-11 Å and 21-30 Å. These results indicate that LUT molecules at this concentration have significant influence on local region of lipid bilayers. As presented in Figure S12, it can be seen that the lateral pressure has also changed when five LUT molecules were added, especially in the three peaks. Notably, the pressure of the 5LUT-PCG system is slightly higher than that in the PPCG systems at around 14 Å, where LUT molecules were mainly located according to the atom density profiles (Figure S3). By contrast, the pressure of 50LUT-PCG system is much higher than that in the PPCG system in the same region.

Figure 8. Comparison of the lateral pressure profiles across the pure lipid membrane (black curves in PPCG system) with the membranes containing fifty LUT molecules (red curves in 50LUT-PCG system).



Number of Lipids Contacting LUT. We counted the number of DOPC and DOPG contacts with LUT molecules to quantitatively characterize the association of LUT with the lipid bilayers. As depicted in Figure 9a, the lipid was defined as a contact lipid when the distance (D1 and D2) from the LUT molecule to DOPC and DOPG was within 5 Å. As the molar ratio of DOPC to DOPG was 7:3, we calculated the number of neighboring DOPC or DOPG lipids contacting LUT molecules by dividing the total number of DOPC contacts with LUT by 140 and the total number of DOPG contacts with LUT by 60. Based on the results of a statistical hypothesis testing analysis(shown in Figure 9b), the average number of contacts per lipid for DOPC with LUT is significantly larger than the average number of contacts per lipid for DOPG with LUT in systems ranging from 15LUT-PCG to 50LUT-PCG, respectively. The average number of contacts per lipid for DOPC and DOPG with LUT are 0.16 ± 0.02 and 0.15 ± 0.02 in 5LUT-PCG, and 0.31 ± 0.04 for DOPC with LUT and 0.31 ± 0.04 for DOPG with LUT in 10LUT-PCG, respectively. Thus, no obvious difference is observed between average number of contacts per lipid for DOPC with LUT and DOPG with LUT in these two systems.


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Figure 9. (a) Schematic diagram of the definitions of contact lipids of DOPC and DOPG with respect to LUT. (b) Ratio of the number of DOPC and DOPG contacts with LUT.

Hydrogen Bonds. According to a previous report, hydrogen bonds between flavonoids and lipids play an important role in the location of compounds in a membrane.61 Thus, we calculated the hydrogen bond-forming probability of LUT



molecules (5 to 50) with DOPC and DOPG during the last 100 ns of the simulations. Based on our simulations, intermolecular hydrogen bonds formed between the fifty LUT molecules and lipids approximately 80% of the time. We calculated the hydrogen bond-forming probability per lipid of LUT molecules with DOPC and DOPG by dividing the total probability of the formation of hydrogen bonds between all LUT molecules with DOPC by 140 and dividing the probability of the formation of hydrogen bonds between all LUT molecules with DOPG by 60 to determine the difference in the probability of forming intermolecular hydrogen bonds between LUT-DOPC and LUT-DOPG in ten systems ( 5LUT-PCG to 50LUT-PCG) and the results were listed in Table 4 and Table S1-S9. As shown in Figure 10b, the average probability of forming hydrogen bonds between fifty LUT molecules and each DOPC lipid is 0.42 ± 0.02, and that for each DOPG lipid is 0.58 ± 0.05. Furthermore, the oxygen atoms of the hydroxyl, phosphoryl and carbonyl groups in the DOPG heads, as well as the oxygen atoms of phosphoryl and carbonyl groups in the DOPC heads, were involved in intermolecular H-bonds with the phenolic groups of LUT. A snapshot of hydrogen bonds between a DOPG molecule and LUT is shown in Figure 10a. For a more detailed analysis, we grouped the hydrogen bond-forming probabilities of LUT molecules with DOPC and DOPG into three classes, LUT-phosphoryl groups in lipids, LUT-carbonyl groups in lipids and LUT-hydroxyl groups in lipids, and calculated the probability of hydrogen bonds formed between LUT molecules (5 to 50) and each lipid. As presented in Figure 10b and Table 4, the average hydrogen bond-forming probability for fifty LUT molecules with the phosphoryl group


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of DOPC is 0.27 ± 0.02, and that for DOPG is 0.36 ± 0.04. The average hydrogen bond-forming probability for fifty LUT molecules with the acyl chain carbonyl groups of DOPC is 0.21 ± 0.02, and that for DOPG is 0.16 ± 0.02. The average hydrogen bond-forming probability for fifty LUT molecules with the hydroxyl groups of DOPC is 0, and that for DOPG is 0.14 ± 0.02. Thus, the probability that LUT molecules will form intermolecular hydrogen bonds with DOPG is greater than that with DOPC in this system. Hydrogen bond-forming probabilities per lipid of LUT molecules with DOPC and DOPG in nine systems (5LUT-PCG to 45LUT-PCG) were shown in Table S1-S9. The average probability of forming hydrogen bonds between LUT molecules and DOPG is greater than that DOPC in the most systems, except for the 15LUT-PCG and 20LUT-PCG systems.



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Figure 10. (a) A snapshot of intermolecular hydrogen bonds between LUT with DOPG. (b) Ratios of hydrogen bonds per lipid for LUT-total lipids, LUT-phosphoryl groups in lipids, LUT-carbonyl groups in lipids and LUT-hydroxyl groups in lipids of 50LUT-PCG.

Table 4. Average probability forming hydrogen bonds between fifty LUT molecules with DOPC and DOPG in the 50LUT-PCG system. The values represent means ± S.E.M. H-bond

DOPC

DOPG

total

0.42 ± 0.02

0.58 ± 0.05

phosphate

0.27 ± 0.02

0.36 ± 0.04


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carbonyl

0.21 ± 0.02

hydroxyl

0.16 ± 0.02 0.14 ± 0.02

4. DISCUSSION AND CONCLUSIONS Flavonoids are polyphenolic compounds that are delivered to the human body through food and have the ability to prevent neurodegenerative, carcinogenic, cardiovascular, and immune diseases, as well as illness from bacterial infections.62 Luteolin, a classic polyphenolic flavonoid compound, has been shown to exert anti-MRSA activity upon binding the membrane by disrupting the membrane and inhibiting ATPase.11 The membrane-disrupting event is the key function of flavonoids compounds targeting both Gram-positive and Gram-negative bacteria.63 Thus, we performed DSC measurements and AA-MD to study the interactions with and effects of LUT on membranes composed of PC and PG (7:3 mol/mol), which mimic the cytoplasmic membrane of MRSA.16-17 We first investigated the interactions between LUT and liposomes composed of DPPC and DPPG using DSC. When the molar ratio of lipids to LUT was 1:0.025, the pre-transition peak disappeared, the TOS, Tm and ∆H values decreased slightly compared to the values observed for pure liposomes (Figure 3a and Table 1). Based on these results, the LUT molecules seem to be located at polar-nonpolar interface region of the lipid bilayers at low concentrations.22 As the molar LUT:lipid ratio increased, the broaden of main-transition peak imply that LUT penetrated into the hydrophobic part of DPPC/DPPG lipid bilayers and disturbed strong hydrophobic interactions between the



lipid chains, and the decrement of lipid gel-liquid crystalline TOS, Tm and ∆H values demonstrate that LUT molecules primarily distributed with the C1-C9 region of the hydrocarbon chains.64 Furthermore, to determine the discrimination of association between LUT with DPPC and DPPG, we performed separate DSC experiments to study the LUT-DPPC and LUT-DPPG interactions. As shown in Figure 3b and Table 2, as the LUT concentrations increased, LUT caused the disappearance of the pre-transition phase and obviously decreased the TOS, Tm and ∆H values for DPPC liposomes. These results are consistent with the behaviors of other flavonoid compounds in DPPC liposomes and led us to a similar conclusion that LUT was localized in the C1-C9 region of DPPC.22 In contrast, LUT had only a slight effect on the transition thermodynamic parameters of DPPG liposomes (presented in Figure 3c and Table 3). Based on the theory reported by Jorgensen, K. et al.65 that the negligible variation in the ∆H is due to a superficial interaction between drugs and lipids at the polar heads of DPPC, our DSC results indicate that LUT interacts with the lipid heads of DPPG. Therefore, these three DSC results obtained from LUT-DPPC/DPPG, LUT-DPPC and LUT-DPPG liposomes make it seem likely that fractional LUT molecules interact with the lipid heads of DPPG and polar/nonpolar interface region of DPPC, while most LUT molecules associate with the C1-C9 region of DPPC in DPPC/DPPG lipid bilayers. We performed AA-MD simulations to study the interactions between LUT and lipid bilayers composed of DOPC and DOPG and to obtain additional information about the influence of LUT molecules on PC and PG lipids in membranes. Based on our simulation


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results, LUT decreased the membrane thickness by 0.16 - 0.92 Å and increased the diffusion rates of membrane lipids by approximately two to four-fold from 10LUT-PCG to 50LUT-PCG systems, which are roughly consistent with results from other experimental studies showing that quercetin, which has a similar chemical structure to LUT, decreased bilayer thickness of DOPC by 0.8 Å and increased membrane fluidity by 6 mol%;66 while adding five LUT molecules to membrane had negligible effect on the membrane thickness and lipids diffusion rates. Clearly, the insertion of ten to fifty LUT molecules into the membrane increased the disorder of the lipid chains of both DOPC and DOPG, shown in Figure 6. Cholesterol, made up of a hydroxyl headgroup, a fused planar 4-ring assembly and a hydrocarbon tail, has similar chemical structure to LUT. Previous studies determined cholesterol’s hydroxyl group residing near the lipid-water interface with its acyl chain penetrating deep into the bilayer’s hydrocarbon region.67 It is belived that the cholesterol’s ability to increase the lipid hydrocarbon chain order can attribute to its relative little tilt angle in lipid bilayers (~16° in DOPC/Cholesterol at 30% Cholesterol).68 In contrast, the tilt angle of fifty LUT in DOPC/DOPG covers a wide region presented in Figure S2 and the main peak value of tilt angle profile is about 87°. Therefore, LUT disturbs lipids more drastically than cholesterol does. Moreover, due to having a hydrophobic tail and less hydroxyl groups, cholesterol molecules can penetrate deeper into lipid hydrophobic region; and larger planner ring structure make cholesterol molecules pack more with lipid chains than LUT molecules do in lipid bilayers. Based on above two points, cholesterol has



increased-ordering effects, while LUT has decreased-ordering effects on lipid bilayers. According to the atom density analysis of LUT, DOPC and DOPG in lipid bilayers, most LUT molecules were located in the region between the polar heads and C9 of the lipid chains of DOPC and DOPG (Figure 7 and Figure S3-S11), which are consistent with the viewpoint that small molecules located at the polar/nonpolar interface and C1-C9 region of lipid bilayers have significant effect on the packing and ordering properties of lipid hydrophobic chains proposed by Mahendra Kumar Jain et al.64 Also, these depth-estimated results fit well with our DSC results of the LUT-DPPC/DPPG interactions. Lateral pressure profiles were also calculated to determine the local effects of LUT on lipid bilayers. Obviously, it can be seen that the addition of fifty LUT into the membrane increased the pressure in the trough and its nearby region (12-20 Å). The X values of this region approximately correspond to the values of region between C9 and the phosphorus atoms of atom density profiles (Figure 8), demonstrating that fifty LUT molecules have pressure-increased effect on this region of DOPC/DOPG lipid bilayers. By contrast, five LUT molecules have slight pressure-increased effect on this region of DOPC/DOPG lipid bilayers. Thus, we inferred that the lateral pressure profile had a gradual process with concentration of LUT added into the lipid bilayers ranging from five to fifty. Previous studies of interactions of anesthetics with membrane indicated that anesthetics molecules increased the ordering of the lipid tails and the thickness of the membrane might due to their pressure-reduced effect in almost the same region.54 Thus,


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we can make a conclusion that the pressure-increased effect of LUT molecules on lipid bilayers probably caused the decrement of the lipid tail ordering and the thickness of the membrane. In addition, two peaks, located at the about X value of 24 Å and 8 Å, correspond to the lipid heads region and double bonds region of DOPC/DOPG lipids. One peak (at X = 24 Å) attributes to the repulsion interactions of steric, electrostatic and hydration, while the other one peak (at X = 8 Å) may due to double bond effects.47 Figure 8 shows that LUT molecules make these two peaks lower than in pure membrane. For one peak (at 24 Å), LUT’s decreased-effect on the repulsion interactions may be related to interacting with the lipid heads; while for the other peak (at 8 Å), the effect of LUT on dihedral potential that restraints the planar geometry of cis double bond may lead to this pheonomenon.47 One might also consider that intrinsic bilayer curvature is related to the biological functions of bacteria.69 Cardiolipin, which exist in substantial fractions in Gram positive membranes, is negative intrinsic curvature lipid.70 Based on the studies of A.M.Batenburg et al., melittin can induce the curvature change in cardiolipin model membrane by binding of melittin to cardiolipin.71 However, Lihua Yang et al. reported that aphenylene ethynylenes have no influence on the curvature of DOPC/DOPG bilayers, while can change the curvature of DOPG/cardiolipin bilayers.72 In our case, the chemical structure of LUT is smaller than that of aphenylene ethynylenes, so we speculate that LUT may has no effect on the intrinsic curvature of DOPC/DOPG lipid bilayers, but the effects of LUT on that of lipid bilayers containing cardiolipin need to be elucidated in further



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studies. To explore intrinsic reason why LUT molecules prefer to be located at the region between the polar heads and C9 of lipids, the number of contacts per lipid and the ratio of hydrogen bonds between neighboring DOPC and DOPG molecules with the LUT molecules were statistically analyzed in the ten systems (5LUT-PCG to 50LUT-PCG). Figure 9 shows that the average number of contacts per lipid for LUT with DOPC is obviously larger than that with DOPG from 10LUT-PCG to 50LUT-PCG, while the probability that LUT molecules will form intermolecular hydrogen bonds with DOPC is less than with DOPG in the most of studied systems presented in Figure 10 and Table S1-S9. Based on these results, it may be inferred that hydrophobic interaction is the main interaction force between LUT-DOPC interactions, while the intermolecular hydrogen bond

is

the

main

interaction

force

between

LUT-DOPG

interactions

in

LUT-DOPC/DOPG interactions. Therefore, the hydrophobic interactions between LUT and DOPC lipid chains may be one possible factor for the localization of LUT in the region of C1-C9 of lipid bilayers and the intermolecular hydrogen bond between LUT and DOPG may be main factor for the localization of LUT on the lipid heads of lipid bilayers. Here, based on studies of LUT-PC/PG interactions using DSC measurements and AA-MD simulations, we have reached several conclusions. High concentrations of LUT molecules decrease the TOS, Tm and ∆H of DPPC/DPPG liposomes and DPPC liposomes, but only have slight effects on those values for DPPG liposomes. In the AA-MD


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simulation study, although LUT molecules (5 to 50) added in membrane were high concentrations (0.02 to 0.2 M), the different effects on membrane were observed in concentration-dependent manner. LUT at concentration of ten to fifty molecules decreased the membrane thickness, increased the fluidity of the lipids and increased the disorder in the lipid hydrophobic chains. By contrast, LUT at concentration of five molecules had negligible effect on these biophysical parameters. The LUT molecules (5LUT-PCG to 50LUT-PCG) were mainly located in the region of the polar head and C9 of the lipid chains of PCs and PGs, based on the MD- and DSC-estimated depth analyses. The pressure-increased effect of LUT molecules on the C1-C9 region of lipid bilayers caused the decrement of the lipid tail ordering and the thickness of the membrane. Finally, the results of the number of contacts per lipid and the ratio of hydrogen bonds between neighboring DOPC and DOPG molecules with the LUT molecules show that hydrophobic interactions between LUT and DOPC lipid chains may drive most LUT molecules to penetrate into hydrophobic core of lipid bilayers, while intermolecular hydrogen bond-forming of LUT with DOPG or DOPC localize fractional LUT molecules to lipid heads of lipid bilayers. In conclusion, these studies provide indications that the distinct effects of LUT on PC/PG lipid bilayers are related to the membrane-disrupting mechanism of LUT against the cytoplasmic membrane of MRSA. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.



Tables S1-S9, average probability forming hydrogen bond between LUT molecules with DOPC and DOPG; Figure S1, analysis of lipid bilayers thickness; Figure S2, the tilt angle of LUT in lipid bilayers; Figures S3-S11, plots of the atom density profiles; Figure S12, the lateral pressure profiles across bilayers; Figures S13-S16, posterior distributions for the infinite system D.

AUTHOR INFORMATION * Corresponding Authors: E-mail: [email protected]; Tel: +86-21-50800619; Fax: +86-21-50807088 E-mail: [email protected]; Tel: +86-21-50805873; Fax: +86-21-50807088 Notes The authors have no competing financial interests to declare. ACKNOWLEDGEMENTS The work was supported in part by the National Natural Science Foundation of China (21422208), E-Institutes of Shanghai Municipal Education Commission (E09013), Institutes for Drug Discovery and Development, Chinese Academy of Sciences, the Special Program for Applied Research on Super Computation of the NSFC-Guangdong Joint Fund (the second phase) under Grant No.U1501501. We also thank the computer center of East China Normal University for computational resources. ABBREVIATIONS LUT, luteolin; MRSA, methicillin-resistant Staphylococcus aureus; DSC, differential


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scanning calorimetry; AA-MD, all-atomic molecular dynamics; PC, phosphatidylcholine; PG, phosphatidylglycerol; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DPPG, 1,2-dipalmitoyl-sn-glycero-3-phospho-(1’-rac-glycerol);

DOPC,

1,2-dioleoyl-sn-glycero-3-phosphocholine;

DOPG,

1,2-dioleoyl-sn-glycero-3-phospho-(1’-rac-glycerol); DP-P, distance between phosphorus atoms; MSD, mean square displacement; SCD, deuterium order parameter. REFERENCES (1) Gosbell, I. B. Methicillin-Resistant Staphylococcus Aureus: Impact on Dermatology Practice. Am. J. Clin. Dermatol. 2004, 5, 239-259. (2) Jevons MP. “Celbenin”-Resistant Staphylococci. Br. Med. J. 1961; 1: 124-125. (3) Boucher, H. W.; Corey, G. R. Epidemiology of Methicillin-Resistant Staphylococcus Aureus. Clin. Infect. Dis. 2008, 46, S344-S349. (4) Klevens, R. M.; Edwards, J. R.; Tenover, F. C.; McDonald, L. C.; Horan, T.; Gaynes, R.; National Nosocomial Infections Surveillance, S. Changes in the Epidemiology of Methicillin-Resistant Staphylococcus Aureus in Intensive Care Units in US Hospitals, 1992-2003. Clin. Infect. Dis. 2006, 42, 389-391. (5) Rodvold, K. A.; McConeghy, K. W. Methicillin-Resistant Staphylococcus Aureus Therapy: Past, Present, and Future. Clin. Infect. Dis. 2014, 58 Suppl_1, S20-S27. (6) Rehm, S. J.; Tice, A. Staphylococcus Aureus: Methicillin-Susceptible S. Aureus to Methicillin-Resistant S. Aureus and Vancomycin-Resistant S. Aureus. Clin. Infect. Dis. 2010, 51 Suppl_2, S176-S182.



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Negative Intrinsic Curvature Lipids. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 20595-20600.

Table of Contents (TOC) Image



Figure 1 139x88mm (300 x 300 DPI)


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Figure 2 64x24mm (300 x 300 DPI)





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Figure 6 137x110mm (300 x 300 DPI)





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Figure 9 148x277mm (300 x 300 DPI)


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Figure 10 130x212mm (300 x 300 DPI)


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