Molecular Dynamics Simulations of the Interactions of Kinin Peptides

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Molecular Dynamics Simulations of the Interactions of Kinin Peptides with an Anionic POPG Bilayer Moutusi Manna and Chaitali Mukhopadhyay* Department of Chemistry, University of Calcutta, 92, A. P. C. Road, Kolkata - 700 009, India

bS Supporting Information ABSTRACT: We have performed molecular dynamics simulations of peptide hormone bradykinin (BK) and its fragment desArg9-BK in the presence of an anionic lipid bilayer, with an aim toward delineating the mechanism of action related to their bioactivity. Starting from the initial aqueous environment, both of the peptides are quickly adsorbed and stabilized on the cell surface. Whereas BK exhibits a stronger interaction with the membrane and prefers to stay on the interface, des-Arg9-BK, with the loss of C-terminal Arg, penetrates further. The heterogeneous lipid-water interface induces β-turn-like structure in the otherwise inherently flexible peptides. In the membrane-bound state, we observed C-terminal β-turn formation in BK, whereas for des-Arg9-BK, with the deletion of Arg9, turn formation occurred in the middle of the peptide. The basic Arg residues anchor the peptide to the bilayer by strong electrostatic interactions with charged lipid headgroups. Simulations with different starting orientations of the peptides with respect to the bilayer surface lead to the same observations, namely, the relative positioning of the peptides on the membrane surface, deeper penetration of the des-Arg9-BK, and the formation of turn structures. The lipid headgroups adjacent to the bound peptides become substantially tilted, causing bilayer thinning near the peptide contact region and increase the degree of disorder in nearby lipids. Again, because of hydrogen bonding with the peptide, the neighboring lipid’s polar heads exhibit considerably reduced flexibility. Corroborating findings from earlier experiments, our results provide important information about how the lipid environment promotes peptide orientation/conformation and how the peptide adapts to the environment.

’ INTRODUCTION A central area of research in membrane biophysics is the detailed investigation of peptide/protein-lipid interplay, which is crucial for many cellular processes, including membrane trafficking, transport, and signal transduction.1,2 The intracellular communication generally consists of interactions between the peptide messengers with their cell-surface receptors, often catalyzed by the biological membrane.3 According to the membrane compartments theory,4 the inherently flexible endogenous peptide ligands first interact with the membrane, where they adopt a preferred conformation/orientation for interaction with the receptor.4-6 Then, through a lateral 2D diffusion on the membrane surface, the ligand interacts with the receptor, leading to binding and activation.4 Moreover, the peptide binding can modify the lipid packing of the host membrane,7-9 which in turn may affect the conformation and functionality of the membrane-embedded receptor.10-12 Bradykinin (BK, Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8Arg9) is a cationic neuropeptide hormone produced by the action of kallikrein on high-molecular-weight precursor protein kininogen upon the occurrence of tissue injury or trauma.13,14 It r 2011 American Chemical Society

is naturally present in human body fluids, exhibiting a broad spectrum of physiological activity. BK is one of the most potent vasodilators and can increase vascular permeability.15 It is active in the central nervous system, initiating pain stimuli, and may be associated with the symptoms of the common cold and other inflammatory disorders.16 BK elicits the contraction of smooth muscles of the respiratory and gastrointestinal tracts and also of the uterus.17 The activity of this kinin peptide is mediated by a G-protein coupled receptor (GPCR) (B2), expressed in nearly all cells.18-20 Whereas B2 receptors mediate most of the BK actions, B1, opposite to B2, recognizes and binds mostly to BK fragment des-Arg9-BK, a natural kinin metabolite.13-21 Previous experiments reveal that the kinin receptor (B1/B2) selectivity is largely dependent on the presence of charged terminal peptide residues.22-24 The linear short oligopeptides (up to about 20 residues) generally do not have definite secondary structure in isotropic Received: October 8, 2010 Revised: January 24, 2011 Published: February 28, 2011 3713

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Langmuir media, and they exist in a random coil conformation in aqueous solution25 but can undergo a conformational transition when complexed to its receptor18-20,26,27 or in the presence of a lipidic environment.28-35 The extent of the peptide-lipid interaction largely depends on the surface charge density of the host lipid matrix,28-30,36-39 and it was found that BK does not interact with lipids bearing no net charge but can easily interact with acidic lipids.7,8,37 In the presence of anionic GM1 (ganglioside monosialytated type 1) micelles, we had earlier reported a turn formation in the BK backbone upon membrane binding.28,29 The receptor-bound structure of BK contains a well-defined turn comprising residue S6P7F8R9,18,19 and the B1-specific antagonist (B-9858) has a type II β-turn involving residues 2-5.40 For the B1-selective des-Arg9 analogue, the absence of a C-terminal β-turn, which is the main structural feature of BK, is not unexpected. The deletion of Arg9 prevents such turn formation and alters the conformational features of the C-terminus.22 The B2-receptor affinity is shown to be drastically reduced by the removal of Arg9, and des-Arg9 analogues exhibit a high affinity for the B1-type receptor.22 The greater affinity of BK to the B2-type receptor is associated with regions of the protein with negative charge, whereas for des-Arg9 analogues the loss of the Arg9 residue reduces the electrostatic attraction, thus favoring the interaction with hydrophobic regions of the B1-type receptor.8,18,22,24 To gain better insight into the possible bioactive conformation and to develop a structure-activity relationship, the conformational analysis of BK has garnered potential interest in recent years.26,27,30,33 Despite intense research, there is still a gap in relating the peptide conformational alteration as well as the change in macroscopic membrane properties to the peptide-membrane interaction. One of the grand challenges amenable to molecular modeling is to provide an atomistic-level insight into such biologically important complex phenomenon, which is difficult to achieve by the conventional experimental techniques.41 Few earlier molecular dynamics (MD) simulation studies on kinins have been performed in biphasic membrane-mimetic (H2O/ CCl4) solvent,34,42 but such a two-phase box sacrifices the detailed description of the water-lipid interface. In the present work, we have performed an MD simulation study of BK and its fragment des-Arg9-BK in the presence of an explicit POPG bilayer in order to illustrate the peptide’s conformational modification as well as its reciprocal effects on bilayer structure and dynamics. We also present a detailed description of the peptidelipid interplay, highlighting the important role of specific amino acid residues in modulating such interactions. Our result depicts the spontaneous adsorption (Figure 1) of both of the peptides on the bilayer surface, followed by the adoption of backbone β-turnlike structure with a considerable modification of bilayer fluidity, which further supports the active role of the biological membrane as a promoter of the ligand-receptor interaction.

’ COMPUTATIONAL METHODOLOGY System Setup and Parameters. POPG lipids are frequently used to model the anionic lipid bilayer.43-48 Our model lipid bilayer consists of 128 anionic POPGs, 5470 water molecules, and a total of 128 Naþ counterions to ensure electroneutrality. The bilayer is a racemic mixture of equal numbers of D-POPG and L-POPG (Figure S1 in the Supporting Information).43 The coordinates (final configuration of 150 ns MD simulation) and force field parameters (popg.itp) for POPG were downloaded from http://www.softsimu.org/downloads.shtml and the

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Figure 1. Snapshots showing the adsorption and stabilization of peptide hormone BK (left panel, bk_traj1) and its fragment des-Arg9-BK (right panel, des9_traj1) on the water-lipid interface. The lipids are shown with hydrophobic tails in cyan and phosphorus atoms as spheres. The peptide is represented as a blue cartoon, with Arg and Phe residues highlighted in red and green, respectively. The image rendering is done with VMD.76 lipid.itp was taken from the home page of Dr. P. Tieleman (http:// moose.bio.ucalgary.ca/). All simulations were performed using the GROMACS 3.3.1 software package49,50 and the GROMOS87 force field.51 The ffgmx force field was used for the peptide in conjunction with the Berger lipids.52 The SPC model was used for water molecules.53 The bilayer system was subjected to energy minimization, followed by 25-ns-long equilibration (further equilibration was not done as we use 150 ns equilibrated coordinate as our starting structure43), and the structural properties (Figure S2 and Table S1 in the Supporting Information) of the equilibrated bilayer are in good agreement with previous studies on the POPG lipid.43,44 Now the peptide hormone bradykinin (BK) and its fragment des-Arg9-BK were placed at a distance of approximately 15 Å from the bilayer surface. We used three different starting orientations for both of the peptides with respect to the membrane surface (Figure S3). In the first case (traj1), the aromatic Phe residues of the peptides were oriented toward the membrane surface, whereas basic Arg residues were pointing away from the interface (Figure S3). In this orientation, we have conducted two independent simulations for each peptide, initialized with different starting velocities (traj1 and traj2). In the second case (traj3), peptides were rotated by 90, and in the third case (traj4), peptides were rotated by 180 around their axis parallel to the membrane surface (Figure S3). 3714

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Table 1. Systems under MD Simulation Study systems with conditionsa

abbreviations

length of MD (ns)

(1) POPG128/water5470/Naþ143/Cl-15

PG_traj

(2) BK/POPG128/water5438/Naþ143/Cl-17

bk_traj1

50


bk_traj2b

50


bk_traj3c

25

(5) BK/POPG128/water5438/Naþ143/Cl-17 (6) des-Arg9-BK/POPG128/water5438/Naþ143/Cl-16 (7) des-Arg9-BK/POPG128/water5438/Naþ143/Cl-16 (8) des-Arg9-BK/POPG128/water5438/Naþ143/Cl-16 (9) des-Arg9-BK/POPG128/water5438/Naþ143/Cl-16 (10) BK/water1249/Cl-2 (11) des-Arg9-BK/water1340/Cl-1

bk_traj4d

25

25

des9_traj1

50

des9_traj2b

50

des9_traj3c des9_traj4d

25 25

bk_sol

10

des9_sol

10

BK (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) with þ2 charge and des-Arg9-BK (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8) with a þ1 charge at neutral pH. The subscripts correspond to the number of each component in the system. b Traj2 having the same initial peptide orientations as traj1, but initialized with different starting velocities. c In traj3, peptides were rotated 90 around their axis. d In traj4, peptides were rotated 180 around their axis. a

In all cases, counterions were added to maintain the electroneutrality of the systems and additional NaCl salt was added to achieve a physiological salt concentration of 150 mM. The systems were then subjected to energy minimization. The equilibration of the combined system was achieved by performing a 5 ns molecular dynamic (MD) run with position restraint on peptide heavy atoms, followed by unrestrained MD simulations. A list of the simulations performed is summarized in Table 1. Simulation Protocols. All MD simulations were carried out in the isobaric-isothermal (NPT) ensemble with a time step of 2 fs and imposed 3D periodic boundary conditions. The Berendsen thermostat,54 with a coupling constant of τT = 0.1 ps, was employed to keep the temperature constant (300 K). This temperature was selected to maintain bilayer fluidity because the main phase-transition temperature (Tm) of POPG is close to 0 C46-48 and also to maintain a physiologically relevant temperature. To keep the pressure constant (1 bar), the pressure coupling was applied semi-isotropically with a coupling constant of τP = 1.0 ps using the Berendsen algorithm.54 Lennard-Jones interactions were truncated at a cutoff distance of 1.2 nm. The long-range electrostatics was handled by the particle mesh Ewald (PME) method with a real-space cutoff of 1.2 nm.55 The LINCS algorithm was used to constrain all bond lengths.56 Analyses were performed with GROMACS analysis tools. The results of traj1 are presented in the main text, and the results of trajectories 2-4 are given in the Supporting Information (Figures S4-S7, S10-S15, and S20b,d).

’ RESULTS AND DISCUSSION For the peptide/lipid systems, the time evolution of the area per lipid molecule has been calculated along with the system potential energy as a function of time (Figures 2 and S4).43-45 The area per lipid is the most widely used parameter for characterizing lipid bilayer systems because it is related to various other properties of membranes, such as the lateral diffusion, membrane elasticity, and permeation.43-45 Figures 2 and S4 indicate that the systems were stable and well-equilibrated during the simulation time span. Peptide Adsorption and Orientation at the Water-Lipid Interface. The membrane binding of peptide hormones is crucial

to their bioactivity. To monitor the binding process, we have presented here the peptide insertion depth57 and number of lipid-peptide contacts as a function of time (Figure 3a,b). At the beginning of the simulations, both peptides quickly came into

Figure 2. Time evolution of the area per lipid molecule in bk_traj1 (black) and des9_traj1 (gray). The inset represents the potential energy of the system as a function of time.

close contact with the anionic lipid headgroups and remained at the surface for the rest of the time (Figures 1 and 3a,b). Similar observations were obtained for the other three trajectories (Figures S5 and S10 for traj2, Figures S6 and S12 for traj3, and Figures S7 and S14 for traj4). The adsorption of basic peptides on negatively charged cell surface is well documented in the literature.58-60 In our case, the increasing number of lipidpeptide contacts correlates well with the spontaneous adsorption of peptide at the interface. Electrostatic forces are believed to aid the initial approach of cationic peptides toward the negatively charged membrane interface.58-61 Because of the cooperative electrostatic attraction by two terminal Arg residues, BK prefers to stay on the anionic bilayer surface. However, des-Arg9-BK, with loss of one terminal Arg, moves deeper into the interface and forms more contacts with lipids compared to BK. Starting with different initial orientations of the peptides, we observe that a similar orientation toward the membrane surface was achieved by the peptides within the first 15 ns of the simulations (Figures S6, S7, S12, and S14). The total number of contacts between lipid non-hydrogen atoms within 6 Å of any peptide non-hydrogen atom is ∼700 for BK and ∼850 for des-Arg9-BK (Figure 3a,b). Turchiello et al. had earlier examined the 3715


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Figure 3. (a, b) Time dependence of the distance between the peptide center of mass and the average plane of phosphorus atoms in the contact monolayer (black). The total number of contacts between lipid non-hydrogen atoms within 6 Å of any peptide non-hydrogen atom is plotted as a function of time (gray). (c, d) Depth of insertion of peptide residues into the bilayer. The time-average (last 5 ns) distance between the peptide side chain center of mass from the water-lipid interface is plotted as a function of the residue number. The dotted line corresponds to the headgroup phosphorus atom. The left panel is for BK (bk_traj1), and the right panel is for des-Arg9-BK (des9_traj1). Error bars are the estimated standard deviations.

interaction and partial penetration of fluorescently labeled BK and its fragments with an anionic DMPG vesicle.7,8 After correcting for electrostatic interactions, they obtained a higher affinity of BK fragments for the hydrophobic phase of the bilayer.8 The peptide adopts a favorable topological orientation after accommodating itself on the bilayer surface.58 Figure 3c,d depicts the vertical positions of the side chains of different peptide residues.58 BK penetrates through its N-terminal part, whereas the central part (residues 2-5) lies near the interface. In this case, N-terminal Arg1 inserts below the plane defined by lipid phosphate atoms and C-terminal Arg9 stays near the surface, in strong association with the charged headgroup. In accordance with our results, a recent NMR spectroscopic study has revealed a similar orientation of BK in an anionic DOPC/DOPA/ DOPE vesicle, where Arg1 forms tight contacts with the lipid headgroup and the central part (2-5) anchors the bilayer environment.30 The membrane binding of BK was reported previously.28,29,32,35,37 For des-Arg9-BK, the peptide penetrates

the membrane via both of its termini. Here again, Arg1 acts as a tethering point for the peptide to the membrane surface. The aromatic amino acid residues (phe5 and phe8) of des-Arg9-BK insert below the average phosphate plane. An almost similar topological arrangement is observed for the B1-selective desArg9 analogue of Lys-BK in the presence of lipid micelles.22 Peptide Conformation. The earlier experiments have revealed the conformational modification of BK upon membrane binding.28-30,32-35 To gauge the influence of the lipid matrix on the structure of bound peptide, here we have calculated the time profile of the peptide’s secondary structure (using VMD, as in refs 2 and 62) of BK both in aqueous solution (Figure S8a) and in the presence of the lipid bilayer (Figure S9a). BK is mostly unstructured in the aqueous medium (Figure S8a), in accordance with previous experiments.25,28 However, in the membranebound state (Figure S9a), BK spends a considerable amount of time in the β-turn ensemble. As depicted in Figure S9a, the formation of the β-turn at the C-terminus of BK, with an unstructured N-terminal part is evident. A similar conformation 3716


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Figure 4. Superposition of peptide configurations (using Pymol77) in the membrane-bound states (last 1 ns) of (a) BK (bk_traj1) and (b) des-Arg9-BK (des9_traj1). The snapshots are separated by 100 ps.

of BK was also obtained from other trajectories (traj3 and traj4 with different starting orientations, Figures S11a, S13a, and S15a). The C-terminal β-turn formation of BK agrees with our previous experiments (NMR, CD, and fluorescence) in the GM1 micelle28,29 and also with the solid-state NMR spectroscopic study by Lopez et al.19 in the presence of dodecyl maltoside (DDM) micelles. The C-terminal β-turn formation is a prerequisite of B2 receptor binding18-20 and has been successfully utilized in the design of agonists and antagonists.26,27,40,63 On the basis of solid-state NMR spectroscopy, Lopez et al.19 recently proposed the structure of BK bound to human B2 GPCR, where BK has a C-terminal β-turn (S6P7F8R9) with an N-terminal R-helical bend (R1P2P3G4).19 Using homology modeling and docking simulation, Kyle et al.18 have proposed a twisted S-shaped model of BK bound to rat B2 receptor, where “C-terminal β-turn is buried in the receptor just below the extracellular boundary of the cell membrane and the N-terminus is interacting with negatively charged residues in extracellular loop 3 of the receptor (most notably Asp268 and Asp286)”. These models are supported by the higher structural order we find at the C-terminus and less-well-defined N-terminus.19 Figure 4a represents the superimposed structures of BK (from the last 1 ns of the present simulation) that converge with an average backbone root-mean-square deviation (rmsd) of 0.50 ( 0.17 Å. The average backbone dihedral angles are listed in Table S2 (Supporting Information) and compared with the experimental results by our group28 and by Lopez et al.19 and also with the homology model of BK by Kyle et al.18 Thus, our result agrees with the overall description of the lipid/receptorinduced turn formation of BK. Moreover, the C-terminal β-turn is stabilized by several inter-residue hydrogen bonds, as listed in Table S3 (Supporting Information). The geometric criteria that we chose for H-bonding are as follows: the acceptor-hydrogen distance dAH < 0.25 nm and the donor-hydrogen-acceptor angle θDHA > 90.2,43 We observe hydrogen bonding between the backbone amide NH of Arg9 with -OdC of Pro7 and the backbone amide NH of Phe8 with -OdC of Ser6 (Figure S16a).

Hydrogen bonding between side chains of Arg9 with side-chain -OH of Ser6 (Figure S16b) is also present. These results are in nice correlation with the NMR study of BK,28 where NOE cross peaks between Ser6HR-Phe8HN and Pro7HR-Arg9HN, which are the type of HRi-HNiþ2, characteristic of a β-turn and also between Ser6RH with a side chain of Arg9, were observed. Such interactions further confirm the folded BK conformation at the C-terminus. Regarding the structural requirements for binding to the B1 receptor, much less data is available.22,40,64 For des-Arg9-BK, all four trajectories exhibit turn formation occurring in the middle of peptide (Figures S9b, S11b, S13b, and S15b). Figure 4b represents the superimposed structures of des-Arg9-BK, converging with an average backbone rmsd of 0.60 ( 0.16 Å. The backbone dihedral angles are plotted in Figure S18 (Supporting Information). The turn structure is stabilized by several interresidue hydrogen bonds (Table S4 in the Supporting Information). Hydrogen bonding between Phe5-NH with -OdC of Pro3, between Ser6-NH with -OdC of Gly4 and, between side-chain -OH of Ser6 with -OdC of Phe8 (Figure S19) are present. Our result agrees with the overall description of the B1-specific antagonist (B-9858) having a β-turn in the center of the peptide. For B1-selective analogues such as des-Arg9-BK or des-Arg9-Lys-BK (H-Lys-Arg-Ado-Ser-Pro-Phe-OH),22 the presence of a β-turn at the C-terminus has not been ascertained and would require a shift in the sequence because of the lack of the C-terminal Arg, which is the fourth residue in the turn observed for B2-selective BK analogues.22 Altogether, the membrane-induced conformational modifications of kinin peptides are suitable for their recognition by the corresponding cell-surface receptors. The decrease in the solvent-accessible surface area (ΔSASA) of peptides (Table S5 of the Supporting Information) on going from the water to lipid environment can also contribute favorably to the solvation free energy of the peptides. Lipid-Peptide Interactions. To shed light on the factors that confer the stability and activity of the kinin peptides on the 3717


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Figure 5. Probability distribution of the interaction energy of BK (bk_traj1, upper panel) and des-Arg9-BK (des9_traj1, lower panel) with the POPG lipid bilayer. The total interaction energy is partitioned into the electrostatic and vdW terms.

anionic bilayer surface, we have calculated the total lipidpeptide interaction energies of BK and des-Arg9-BK with the surrounding lipid matrix and also its partition into electrostatic and van der Waals (vdW) terms (Figure 5).2,60 As shown in Figure 5, BK exhibits a stronger interaction (∼-590 KJ/mol) with the membrane than does its fragment (∼-530 KJ/mol), in accordance with a previous report.7 Although the vdW contributions of the two peptides are comparable, a stronger BK interaction originates from its higher electrostatic contribution. In the distribution curve, the peak for the electrostatic contribution per BK is located at ∼-425 KJ/mol, whereas that of des-Arg9-BK, with the loss of one terminal Arg, is shifted to ∼-370 KJ/mol. Thus, though the lipid-peptide interaction energy is the outcome of both electrostatic and van der Waals contributions, in the presence of an anionic lipid, electrostatic interactions may play the dominant role. To elucidate the source of such Coulombic interactions, a careful study of hydrogen bonds (H-bonds) formed and broken along the MD trajectory is carried out (Figure 6).11 We also calculate the average number of H-bonds shared by peptide residues with different functional groups (Figure S1) of the lipid head (inset in Figure 6). As depicted in Figure 6, the major H-bonding contribution arises from charged Arg residues. For both peptides, Arg residues are involved in H-bonding during more than half of each trajectory, thus acting as “hooks” for the membrane, as observed for BK in ref 30. Although the side-chain functional group of Arg exhibits a high affinity for the anionic phosphate group, additional H-bonds with glycol or ester oxygen are also formed. The role of basic Arg/Lys residues in penetrating the cell membrane is well documented in the literature.2,58-61,65 As far as Ser6 is concerned, it is mostly solvated by water molecules. The Gly and Phe residues contribute to H-bonding via their backbone amide nitrogen. Such interactions facilitate the stabilization of peptides on the membrane interface and provide the favorable orientation of the peptides, thus aiding their recognition by a suitable cell-surface receptor.22,58 Differential Effects of Peptide Binding on Bilayer Properties. In the preceding section, we have discussed the important roles of lipid in inducing the conformation/orientation of peptides at the bilayer interface. Because the lipid bilayer makes up the

Figure 6. Residues of (a) BK (bk_traj1) and (b) des-Arg9-BK (des9_tarj1) involved in lipid-protein hydrogen bonding interactions. The total bar length indicates the percentage of snapshots where the interaction exists. The inset plots represent the average number of H-bonds formed between peptide residues and lipid headgroups. The last 5 ns of data has been used.

platform where membrane-associated signaling molecules interact and function, the earlier studies revealed that any change in the bilayer properties can have a strong effect on signaling dynamics.10-12 A detailed understanding of peptide-induced changes in the membrane properties is thus necessary to gain better insight into their mode of membrane interaction, and such studies have drawn significant attention recently.2,58,66,67 In this particular section, we will emphasize the effects of a peptide on the bilayer structural properties (conformational ordering, headgroup orientation, and bilayer thickness) as well as on the dynamic (flexibility of lipid heads) reorganization at the lipidwater interface. Lipid Tail Order. The modulation of bilayer fluidity by membrane-active peptides/proteins, hormones, cholesterol, and ions is well documented in the literature.2,60,66-72 The ordering of lipid acyl chains can be characterized by the molecular order parameter, Smol46 Smol ¼

1

3 cos2 θn - 1 2

ð1Þ

where θn is the instantaneous angle between the nth segmental vector (i.e., the (Cn - 1, Cnþ1) vector connecting (n - 1) and 3718


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Figure 7. Molecular order parameters, Smol, for the palmitoyl (left column) and oleoyl (right column) chains for the different lipid categories of a lipid bilayer containing BK (bk_traj1, upper panel) or des-Arg9-BK (des9_traj1, lower panel) and that of the pure POPG bilayer (without peptide). The last 5 ns of data has been used.

(n þ 1) carbon atoms in the lipid hydrocarbon chain and the bilayer normal (Z axis). The angular brackets Ææ denote the average over time and the ensemble. To simplify the analysis, here we have divided the lipids into two categories: local (those with a lipid non-hydrogen atom within 10 Å of a peptide nonhydrogen atom) and bulk (all others) lipids.2,66 Figure 7 depicts a significant drop in the order parameter values of local lipids as compared to the pure bilayer (without peptide). This indicates a localized bilayer perturbation around the peptide contact regions. The bulk lipids, however, become more ordered than the pure bilayer.66 The order parameter value of the pure POPG bilayer agrees well with the previous findings.43,44 The effect of BK and its fragments (des-Arg9-BK, des-Arg1-BK, and Arg-ProPro-Gly-Phe or BK1-5) on the fluidity of the DMPG vesicle was examined previously by Turchiello et al.7 Headgroup Orientation. To characterize the packing of lipid polar heads around the surface-embedded peptide, we have calculated the angle (θi) between the outward membrane normal and a vector defined by the phosphorus and glycol center carbon atom (P-C12, Figure S1) of the PG headgroup.43 The average θi values were then plotted as a function of the average in-plane position of lipid phosphorus atoms in the upper bilayer leaflet (Figure 8, first row).58 For both peptides, adjacent lipids become parallel to the membrane surface. The effect is more pronounced at the termini of the peptides. For rest of the lipids, the headgroup orientation angle is between ∼60 and 80, which compares well with a recent theoretical study.43 Such an effect arises because of the ability of the peptide to orient the lipid heads to itself through strong electrostatic and H-bonding interactions (Figure 6).

Other groups have recently reported such a reorientation of the lipid head, caused by peptide insertion.58,67,73 Membrane Landscape. The local disorder of the membrane upon peptide insertion, together with the tilting of the lipid head, can cause bilayer thinning near the peptide contact region.2 The membrane landscape is characterized by the in-plane distribution of ΔZi= Zi - ÆZæ, where Zi represents the Z coordinates of the ith lipid in the contact monolayer and ÆZæ is the average Z value of this surface.58 Figure 8 (second row) exhibits roughness on the bilayer surface. For both BK and des-Arg9-BK, the peptide depresses the bilayer in its immediate vicinity by 0.7-0.8 nm, forming distinct grooves or dents beneath the peptide. The effect is very localized, confined to the peptide-lipid contact region, especially at the two peptide termini. Similar behavior is also reported for other cell-penetrating peptides.58,60,67 Dynamics of the Lipid-Water Interface. It was previously reported that lipids adjacent to peptide/protein (enriched in basic (Lys, Arg) and aromatic (Trp, Tyr) amino acid residues) exhibit lower diffusion than the rest of the bilayer.74,75 The dynamics at the bilayer interface can be characterized in terms of the flexibility of the lipid polar head, Δrmsfi = rmsfi - max(rmsfipure),58 where rmsfi is the root-mean-square fluctuation (i.e., the standard deviation) of the polar head of the ith lipid relative to its equilibrium position (calculated using the g_rmsf program of GROMACS). The term max(rmsfipure) is the maximum of the distribution of rmsfi in the pure POPG bilayer (Figure S21). The 2D map (Figure 8, third row) illustrates the inplane distribution of the flexibility of lipid heads and differentiates distinct regions on the bilayer surface, with lipids having different overall mobility. As shown in Figure 8 (third row), the 3719


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Figure 8. Disturbance at the lipid-water interface induced by the insertion of BK (bk_traj1, left column) and des-Arg9-BK (des9_traj1, right column). The data are averaged over the last 5 ns of the simulation trajectory. The first row shows the in-plane distribution of the lipid head orientation. The second row represents the surface distribution of the change in bilayer thickness around the embedded peptides. The third row shows the in-plane distributions of dynamic fractions of lipids having different headgroup flexibilities. The average positions of peptide CR atoms are highlighted as yellow points.

headgroup flexibility of the neighboring lipids was reduced significantly from that of the rest of the membrane. Such a freezing or arresting effect is attributed to the trapping of lipid polar heads through electrostatic and H-bonding interactions by the charged/aromatic residues of the peptide. Our results reveal peptide-induced local bilayer perturbation in terms of lipid tail order, membrane thinning, headgroup orientation, and flexibility of the lipid head. We have measured the properties of the bilayer (area per lipid and order parameter) for each leaflet separately (Table S6). For both systems, the upper bilayer leaflet is slightly more disordered and has a marginally greater area per lipid molecule than does the lower leaflet. For example, with BK the values are 0.521 ( 0.003 and

0.509 ( 0.002 nm2 whereas for des-Arg9-BK the values are 0.529 ( 0.002 and 0.515 ( 0.002 nm2 for the upper and lower leaflets, respectively. Such an effect arises from the asymmetric interactions of peptides with respect to the two bilayer leaflets. Because peptides bind at the lipid-water interface of the upper monolayer, the area per lipid increases to make room for the peptide. Moreover, because of the interaction with the peptide, the adjacent lipids (located mostly in the upper leaflet) become more disordered, causing local bilayer thinning. According to membrane elasticity theories,78 the local perturbation is propagated into the bilayer and the distant lipids change in a manner that maintains membrane integrity. Similar results are reported in many other MD simulations,60,66,67 but like other MD 3720


Langmuir studies,60,66,67,72 the present work is also limited by the system size, with a fixed lipid member in each leaflet, and thus the ordering of distant lipids may be influenced by the simulation artifact. Recently, Martinez-Seara et al.79 showed that the description of a double bond80 in a lipid hydrocarbon chain is a subtle matter in the atomistic simulation of lipid bilayers, which have a measurable influence on system properties. However, using the GROMOS87/Berger force field (as in our case), many simulations81,82 were reported to reproduce the overall shape of the order parameter profile of the unsaturated oleoyl tail. As obtained from the NMR experiment,83 the disordered region in the middle of the hydrocarbon chain arises from the unsaturated carbon atoms. To allow comparison with experimental data, we have calculated the average area per lipid and order parameter profile over the whole membrane (Table S6). The overall peptide-induced changes in membrane fluidity compared well with a previous experiment on BK and its fragments in a DMPG vesicle.7 Recently, an AFM study revealed that a peptide located at the lipid hydrophilic/hydrophobic interface “does not induce uniform membrane thinning, but instead leads to the formation of distinct domains in the lipid bilayer”.84 The result is in nice correlation with our simulation finding (Figure 8, second row).

’ CONCLUSIONS The kinin peptides have received significant attention as potential therapeutic agents and exert their bioactivity through binding to specific cell-surface receptors. Partitioning or binding to the lipid bilayer is likely to favor or facilitate receptor binding by the peptides. To explore the influence of lipids on the conformation and dynamics of peptides and vice versa, we have simulated neuropeptide hormone BK and its fragment des-Arg9BK, a natural kinin metabolite, in the presence of the anionic POPG bilayer. Although BK with two terminal Arg’s, prefers to stay at the interface, des-Arg9-BK with the loss of the charged group penetrates further. For kinins, the presence of charged terminal residues is related to their receptor selectivity. As explained in earlier experimental studies,8 the greater affinity of the B2-type receptor for BK can be associated with regions of the protein with a negative charge. For des-Arg9-BK, the loss of the Arg9 residue favors the interaction with hydrophobic regions of the B1-type receptor.8 Again, on binding at the surface, both BK and des-Arg9-BK adopt a β-turn-like structure, which is crucial to their receptor binding. Simulations with different starting orientations of the peptides with respect to the bilayer surface lead to the same observations, namely, similar membrane-bound states, relative positioning of the residues on the membrane surface, deeper penetration of des-Arg9-BK, and formation of the turn structures at the C-terminal of BK and in the middle for desArg9-BK. Moreover, the peptide binding has reciprocal effects on membrane structure and dynamics. The lipid headgroups adjacent to the peptides become substantially tilted, causing the lipid tail to spread outward, which leads to bilayer thinning near the peptide contact region. The hydrocarbon tails of the neighboring lipids become more disordered, whereas those of the distant lipids become stretched and ordered. Again, both peptides are capable of trapping lipid headgroups through electrostatic and H-bonding interactions, thereby considerably reducing the overall flexibility of lipid polar heads in their vicinity. Thus, peptide binding can affect the structure, dynamics, and organization of the lipid membrane. Altogether, our simulations provide

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molecular-level insight into the details of lipid-peptide interactions, highlighting the roles of different peptide residues, peptide conformation, and orientation in surface binding and peptideinduced membrane structural disturbances and the dynamical reorganization of the lipid-water interface.

’ ASSOCIATED CONTENT

bS

Supporting Information. Data regarding the equilibration of POPG bilayer, results of the control simulations, SASA values of the peptides, phi-psi dihedral angles, Ramachandran plots, the list of inter-residue hydrogen bonds of peptides, and the schematic representation of POPG molecules. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected], [email protected].

’ ACKNOWLEDGMENT We are thankful to the UPE project of the Department of Chemistry, University of Calcutta, Kolkata, India, for the computational facilities and to the RCAMOS of the Indian Association for the Cultivation of Science, Kolkata, India, for providing access to the High Performance Computing Facility. This work is supported by a fellowship through SRF CSIR-NET to M.M. by the Government of India. ’ REFERENCES (1) Cho, W.; Stahelin, R. V. Annu. Rev. Biophys. Biomol. Struct. 2005, 34, 119–151. (2) Manna, M.; Mukhopadhyay, C. Langmuir 2009, 25, 12235– 12242. (3) Moroder, L.; Romano, R.; Guba, W.; Mierke, D. F.; Kessler, H.; Delporte, C.; Winand, J.; Christophe, J. Biochemistry 1993, 32, 13551– 13559. (4) Schwyzer, R. Biopolymers 1995, 37, 5–16. (5) Wymore, T.; Wong, T. C. Biophys. J. 1999, 76, 1199–1212. lvarez, R. M. S. J. Phys. Chem. B (6) Petruk, A. A.; Marti, M. A.; A 2009, 113, 13357–13364. (7) Turchiello, R. F.; Juliano, L.; Ito, A. S.; Lamy-Freund, M. T. Biopolymers 2000, 54, 211–221. (8) Turchiello, R. F.; Lamy-Freund, M. T.; Hirata, I. Y.; Juliano, L.; Ito, A. S. Biopolymers 2002, 65, 336–346. (9) Brailoiu, E.; Margineanu, A.; Miyamoto, M. D. IUBMB Life 1998, 44, 203–209. (10) Chachisvilis, M; Zhang, Y.-L.; Frangos, J. A. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 15463–15468. (11) Cordomi, A.; Perez, J. J. J. Phys. Chem. B 2007, 111, 7052–7063. (12) Lee, A. G. Biochim. Biophys. Acta, Biomembr. 2004, 1666, 62–87. (13) Farmer, S. G.; Burch, R. M. In Bradykinin Antagonists: Basic and Clinical Research; Burch, R. M., Ed.; Dekker: New York, 1990; pp 1-31. (14) Moreau, M. E.; Garbacki, N.; Molinaro, G.; Brown, N. J.; Marceau, F.; Adam, A. J. Pharm. Sci. 2005, 99, 6–38. (15) Carbonell, L. F.; Carretero, O. A.; Stewart, J. M.; Scicli, A. G. Hypertension 1988, 11, 239–243. (16) Dray, A.; Perkins, M. Trends Neurosci. 1993, 16, 99–104. (17) Regoli, D.; Barabe, J. C. Pharmacol. Rev. 1980, 32, 1–46. (18) Kyle, D. J.; Chakravarty, S.; Sinsko, J. A.; Stormann, T. M. J. Med. Chem. 1994, 37, 1347–1354. (19) Lopez, J. J.; Shukla, A. K.; Reinhart, C.; Schwalbe, H.; Michel, H.; Glaubitz, C. Angew. Chem., Int. Ed. 2008, 120, 1692–1695. 3721


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Molecular Dynamics Simulations of the Interactions of Kinin Peptides

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