molecular dynamics simulations of the interactions of kinin peptides with an anionic popg bilayer

Published: February 28, 2011

r 2011 American Chemical Society 3713 dx.doi.org/10.1021/la104046z | Langmuir 2011, 27, 3713–3722

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pubs.acs.org/Langmuir

Molecular Dynamics Simulations of the Interactions of Kinin Peptideswith an Anionic POPG BilayerMoutusi Manna and Chaitali Mukhopadhyay*

Department of Chemistry, University of Calcutta, 92, A. P. C. Road, Kolkata - 700 009, India

bS Supporting Information

’ INTRODUCTION

A central area of research in membrane biophysics is thedetailed investigation of peptide/protein-lipid interplay, whichis crucial for many cellular processes, including membranetrafficking, transport, and signal transduction.1,2 The intracellularcommunication generally consists of interactions between thepeptide messengers with their cell-surface receptors, often cata-lyzed by the biological membrane.3 According to the membranecompartments theory,4 the inherently flexible endogenous pep-tide ligands first interact with the membrane, where they adopt apreferred conformation/orientation for interaction with thereceptor.4-6 Then, through a lateral 2D diffusion on the mem-brane surface, the ligand interacts with the receptor, leading tobinding and activation.4 Moreover, the peptide binding canmodify the lipid packing of the host membrane,7-9 which inturn may affect the conformation and functionality of themembrane-embedded receptor.10-12

Bradykinin (BK, Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) is a cationic neuropeptide hormone produced by theaction of kallikrein on high-molecular-weight precursor proteinkininogen upon the occurrence of tissue injury or trauma.13,14 It

is naturally present in human body fluids, exhibiting a broadspectrum of physiological activity. BK is one of the most potentvasodilators and can increase vascular permeability.15 It is activein the central nervous system, initiating pain stimuli, and may beassociated with the symptoms of the common cold and otherinflammatory disorders.16 BK elicits the contraction of smoothmuscles of the respiratory and gastrointestinal tracts and also ofthe uterus.17 The activity of this kinin peptide is mediated by aG-protein coupled receptor (GPCR) (B2), expressed in nearlyall cells.18-20 Whereas B2 receptors mediate most of the BKactions, B1, opposite to B2, recognizes and binds mostly to BKfragment des-Arg9-BK, a natural kinin metabolite.13-21 Previousexperiments reveal that the kinin receptor (B1/B2) selectivity islargely dependent on the presence of charged terminal peptideresidues.22-24

The linear short oligopeptides (up to about 20 residues)generally do not have definite secondary structure in isotropic

Received: October 8, 2010Revised: January 24, 2011

ABSTRACT:We have performed molecular dynamics simula-tions of peptide hormone bradykinin (BK) and its fragment des-Arg9-BK in the presence of an anionic lipid bilayer, with an aimtoward delineating the mechanism of action related to theirbioactivity. Starting from the initial aqueous environment, bothof the peptides are quickly adsorbed and stabilized on the cellsurface. Whereas BK exhibits a stronger interaction with themembrane and prefers to stay on the interface, des-Arg9-BK,with the loss of C-terminal Arg, penetrates further. Theheterogeneous lipid-water interface induces β-turn-like struc-ture in the otherwise inherently flexible peptides. In themembrane-bound state, we observed C-terminal β-turn forma-tion in BK, whereas for des-Arg9-BK, with the deletion of Arg9,turn formation occurred in the middle of the peptide. The basicArg residues anchor the peptide to the bilayer by strongelectrostatic interactions with charged lipid headgroups. Simu-lations 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 turnstructures. The lipid headgroups adjacent to the bound peptides become substantially tilted, causing bilayer thinning near thepeptide contact region and increase the degree of disorder in nearby lipids. Again, because of hydrogen bonding with the peptide, theneighboring lipid’s polar heads exhibit considerably reduced flexibility. Corroborating findings from earlier experiments, our resultsprovide important information about how the lipid environment promotes peptide orientation/conformation and how the peptideadapts to the environment.

3714 dx.doi.org/10.1021/la104046z |Langmuir 2011, 27, 3713–3722

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media, and they exist in a random coil conformation in aqueoussolution25 but can undergo a conformational transition whencomplexed to its receptor18-20,26,27 or in the presence of a lipidicenvironment.28-35 The extent of the peptide-lipid interactionlargely depends on the surface charge density of the host lipidmatrix,28-30,36-39 and it was found that BK does not interactwith lipids bearing no net charge but can easily interact withacidic lipids.7,8,37 In the presence of anionic GM1 (gangliosidemonosialytated type 1) micelles, we had earlier reported a turnformation in the BK backbone uponmembrane binding.28,29 Thereceptor-bound structure of BK contains a well-defined turncomprising residue S6P7F8R9,

18,19 and the B1-specific antagonist(B-9858) has a type II β-turn involving residues 2-5.40 For theB1-selective des-Arg9 analogue, the absence of a C-terminalβ-turn, which is the main structural feature of BK, is notunexpected. The deletion of Arg9 prevents such turn formationand alters the conformational features of the C-terminus.22 TheB2-receptor affinity is shown to be drastically reduced by theremoval of Arg9, and des-Arg9 analogues exhibit a high affinityfor the B1-type receptor.22 The greater affinity of BK to theB2-type receptor is associated with regions of the protein withnegative charge, whereas for des-Arg9 analogues the loss of theArg9 residue reduces the electrostatic attraction, thus favoringthe interaction with hydrophobic regions of the B1-typereceptor.8,18,22,24 To gain better insight into the possible bioac-tive conformation and to develop a structure-activity relation-ship, the conformational analysis of BK has garnered potentialinterest in recent years.26,27,30,33

Despite intense research, there is still a gap in relating thepeptide conformational alteration as well as the change inmacroscopic membrane properties to the peptide-membraneinteraction. One of the grand challenges amenable to molecularmodeling is to provide an atomistic-level insight into suchbiologically important complex phenomenon, which is difficultto achieve by the conventional experimental techniques.41 Fewearlier molecular dynamics (MD) simulation studies on kininshave been performed in biphasic membrane-mimetic (H2O/CCl4) solvent,34,42 but such a two-phase box sacrifices thedetailed description of the water-lipid interface. In the presentwork, we have performed an MD simulation study of BK and itsfragment des-Arg9-BK in the presence of an explicit POPGbilayer in order to illustrate the peptide’s conformational mod-ification as well as its reciprocal effects on bilayer structure anddynamics. We also present a detailed description of the peptide-lipid interplay, highlighting the important role of specific aminoacid residues in modulating such interactions. Our result depictsthe spontaneous adsorption (Figure 1) of both of the peptides onthe bilayer surface, followed by the adoption of backbone β-turn-like structure with a considerable modification of bilayer fluidity,which further supports the active role of the biological membraneas a promoter of the ligand-receptor interaction.

’COMPUTATIONAL METHODOLOGY

SystemSetup andParameters. POPG lipids are frequently usedto model the anionic lipid bilayer.43-48 Our model lipid bilayer consistsof 128 anionic POPGs, 5470 water molecules, and a total of 128 Naþ

counterions to ensure electroneutrality. The bilayer is a racemic mixtureof equal numbers of D-POPG and L-POPG (Figure S1 in the SupportingInformation).43 The coordinates (final configuration of 150 ns MDsimulation) and force field parameters (popg.itp) for POPG weredownloaded from http://www.softsimu.org/downloads.shtml and the

lipid.itp was taken from the home page of Dr. P. Tieleman (http://moose.bio.ucalgary.ca/). All simulations were performed using theGROMACS 3.3.1 software package49,50 and the GROMOS87 forcefield.51 The ffgmx force field was used for the peptide in conjunctionwith the Berger lipids.52 The SPCmodel was used for water molecules.53

The bilayer system was subjected to energy minimization, followed by25-ns-long equilibration (further equilibration was not done as we use150 ns equilibrated coordinate as our starting structure43), and thestructural properties (Figure S2 and Table S1 in the SupportingInformation) of the equilibrated bilayer are in good agreement withprevious studies on the POPG lipid.43,44 Now the peptide hormonebradykinin (BK) and its fragment des-Arg9-BK were placed at a distanceof approximately 15 Å from the bilayer surface. We used three differentstarting orientations for both of the peptides with respect to themembrane surface (Figure S3). In the first case (traj1), the aromaticPhe residues of the peptides were oriented toward the membranesurface, whereas basic Arg residues were pointing away from theinterface (Figure S3). In this orientation, we have conducted twoindependent simulations for each peptide, initialized with differentstarting velocities (traj1 and traj2). In the second case (traj3), peptideswere rotated by 90�, and in the third case (traj4), peptides were rotatedby 180� around their axis parallel to the membrane surface (Figure S3).

Figure 1. Snapshots showing the adsorption and stabilization of pep-tide hormone BK (left panel, bk_traj1) and its fragment des-Arg9-BK(right panel, des9_traj1) on the water-lipid interface. The lipids areshown with hydrophobic tails in cyan and phosphorus atoms as spheres.The peptide is represented as a blue cartoon, with Arg and Phe residueshighlighted in red and green, respectively. The image rendering is donewith VMD.76


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In all cases, counterions were added to maintain the electroneutralityof the systems and additional NaCl salt was added to achieve aphysiological salt concentration of 150 mM. The systems were thensubjected to energy minimization. The equilibration of the combinedsystem was achieved by performing a 5 ns molecular dynamic (MD) runwith position restraint on peptide heavy atoms, followed by unrestrainedMD simulations. A list of the simulations performed is summarized inTable 1.Simulation Protocols. All MD simulations were carried out in

the isobaric-isothermal (NPT) ensemble with a time step of 2 fsand imposed 3D periodic boundary conditions. The Berendsenthermostat,54 with a coupling constant of τT = 0.1 ps, was employedto keep the temperature constant (300 K). This temperature wasselected to maintain bilayer fluidity because the main phase-transitiontemperature (Tm) of POPG is close to 0 �C46-48 and also to maintain aphysiologically relevant temperature. To keep the pressure constant(1 bar), the pressure coupling was applied semi-isotropically with acoupling 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 LINCSalgorithm was used to constrain all bond lengths.56 Analyses wereperformed with GROMACS analysis tools. The results of traj1 arepresented in themain text, and the results of trajectories 2-4 are given inthe Supporting Information (Figures S4-S7, S10-S15, and S20b,d).

’RESULTS AND DISCUSSION

For the peptide/lipid systems, the time evolution of the areaper lipid molecule has been calculated along with the systempotential energy as a function of time (Figures 2 and S4).43-45

The area per lipid is the most widely used parameter forcharacterizing lipid bilayer systems because it is related to variousother properties of membranes, such as the lateral diffusion,membrane elasticity, and permeation.43-45 Figures 2 and S4indicate that the systems were stable and well-equilibrated duringthe simulation time span.Peptide Adsorption and Orientation at the Water-Lipid

Interface.Themembrane binding of peptide hormones is crucialto their bioactivity. To monitor the binding process, we havepresented here the peptide insertion depth57 and number oflipid-peptide contacts as a function of time (Figure 3a,b). At thebeginning of the simulations, both peptides quickly came into

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

Table 1. Systems under MD Simulation Study

systems with conditionsa abbreviations length of MD (ns)

(1) POPG128/water5470/Naþ143/Cl

-15 PG_traj 25

(2) BK/POPG128/water5438/Naþ143/Cl

-17 bk_traj1 50


-17 bk_traj2b 50


-17 bk_traj3c 25


-17 bk_traj4d 25

(6) des-Arg9-BK/POPG128/water5438/Naþ143/Cl

-16 des9_traj1 50


-16 des9_traj2b 50


-16 des9_traj3c 25


-16 des9_traj4d 25

(10) BK/water1249/Cl-2 bk_sol 10

(11) des-Arg9-BK/water1340/Cl-1 des9_sol 10

aBK (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) withþ2 charge and des-Arg9-BK (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8) with aþ1charge at neutral pH. The subscripts correspond to the number of each component in the system. bTraj2 having the same initial peptide orientations astraj1, but initialized with different starting velocities. c In traj3, peptides were rotated 90� around their axis. d In traj4, peptides were rotated 180� aroundtheir axis.

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


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interaction and partial penetration of fluorescently labeledBK and its fragments with an anionic DMPG vesicle.7,8 Aftercorrecting for electrostatic interactions, they obtained a higheraffinity of BK fragments for the hydrophobic phase of thebilayer.8

The peptide adopts a favorable topological orientation afteraccommodating itself on the bilayer surface.58 Figure 3c,d depictsthe vertical positions of the side chains of different peptideresidues.58 BK penetrates through its N-terminal part, whereasthe central part (residues 2-5) lies near the interface. In thiscase, N-terminal Arg1 inserts below the plane defined by lipidphosphate atoms and C-terminal Arg9 stays near the surface, instrong association with the charged headgroup. In accordancewith our results, a recent NMR spectroscopic study has revealed asimilar orientation of BK in an anionic DOPC/DOPA/DOPE vesicle, where Arg1 forms tight contacts with the lipidheadgroup and the central part (2-5) anchors the bilayerenvironment.30 The membrane binding of BK was reportedpreviously.28,29,32,35,37 For des-Arg9-BK, the peptide penetrates

the membrane via both of its termini. Here again, Arg1 acts as atethering point for the peptide to the membrane surface. Thearomatic amino acid residues (phe5 and phe8) of des-Arg9-BKinsert below the average phosphate plane. An almost similartopological arrangement is observed for the B1-selective des-Arg9 analogue of Lys-BK in the presence of lipid micelles.22

Peptide Conformation. The earlier experiments have re-vealed the conformational modification of BK upon membranebinding.28-30,32-35 To gauge the influence of the lipid matrix onthe structure of bound peptide, here we have calculated the timeprofile of the peptide’s secondary structure (using VMD, as inrefs 2 and 62) of BK both in aqueous solution (Figure S8a) and inthe presence of the lipid bilayer (Figure S9a). BK is mostlyunstructured in the aqueous medium (Figure S8a), in accordancewith previous experiments.25,28 However, in the membrane-bound state (Figure S9a), BK spends a considerable amount oftime in the β-turn ensemble. As depicted in Figure S9a, theformation of the β-turn at the C-terminus of BK, with anunstructured N-terminal part is evident. A similar conformation

Figure 3. (a, b) Time dependence of the distance between the peptide center of mass and the average plane of phosphorus atoms in the contactmonolayer (black). The total number of contacts between lipid non-hydrogen atoms within 6 Å of any peptide non-hydrogen atom is plotted as afunction 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 chaincenter of mass from the water-lipid interface is plotted as a function of the residue number. The dotted line corresponds to the headgroup phosphorusatom. 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.


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of BK was also obtained from other trajectories (traj3 and traj4with different starting orientations, Figures S11a, S13a, andS15a). The C-terminal β-turn formation of BK agrees with ourprevious experiments (NMR, CD, and fluorescence) in the GM1micelle28,29 and also with the solid-state NMR spectroscopicstudy by Lopez et al.19 in the presence of dodecyl maltoside(DDM) micelles. The C-terminal β-turn formation is a prere-quisite of B2 receptor binding18-20 and has been successfullyutilized in the design of agonists and antagonists.26,27,40,63 On thebasis of solid-state NMR spectroscopy, Lopez et al.19 recentlyproposed the structure of BK bound to human B2 GPCR, whereBK has a C-terminal β-turn (S6P7F8R9) with an N-terminalR-helical bend (R1P2P3G4).

19 Using homology modeling anddocking simulation, Kyle et al.18 have proposed a twistedS-shaped model of BK bound to rat B2 receptor, where“C-terminal β-turn is buried in the receptor just below theextracellular boundary of the cell membrane and the N-terminusis interacting with negatively charged residues in extracellularloop 3 of the receptor (most notably Asp268 and Asp286)”.These models are supported by the higher structural order wefind at the C-terminus and less-well-defined N-terminus.19

Figure 4a represents the superimposed structures of BK (fromthe last 1 ns of the present simulation) that converge with anaverage backbone root-mean-square deviation (rmsd) of 0.50(0.17 Å. The average backbone dihedral angles are listed inTable S2 (Supporting Information) and compared with theexperimental results by our group28 and by Lopez et al.19 andalso with the homology model of BK by Kyle et al.18 Thus, ourresult agrees with the overall description of the lipid/receptor-induced turn formation of BK. Moreover, the C-terminal β-turnis stabilized by several inter-residue hydrogen bonds, as listed inTable S3 (Supporting Information). The geometric criteria thatwe chose for H-bonding are as follows: the acceptor-hydrogendistance dAH < 0.25 nm and the donor-hydrogen-acceptor angleθDHA > 90�.2,43 We observe hydrogen bonding between thebackbone amide NH of Arg9 with -OdC of Pro7 and thebackbone amideNHof Phe8 with-OdCof Ser6 (Figure S16a).

Hydrogen bonding between side chains of Arg9 with side-chain-OH of Ser6 (Figure S16b) is also present. These results are innice correlation with the NMR study of BK,28 where NOE crosspeaks between Ser6HR-Phe8HN and Pro7HR-Arg9HN, whichare the type of HR

i-HNiþ2, characteristic of a β-turn and alsobetween Ser6RH with a side chain of Arg9, were observed. Suchinteractions further confirm the folded BK conformation at theC-terminus.Regarding the structural requirements for binding to the B1

receptor, much less data is available.22,40,64 For des-Arg9-BK, allfour trajectories exhibit turn formation occurring in the middle ofpeptide (Figures S9b, S11b, S13b, and S15b). Figure 4b repre-sents the superimposed structures of des-Arg9-BK, convergingwith an average backbone rmsd of 0.60( 0.16 Å. The backbonedihedral angles are plotted in Figure S18 (Supporting In-formation). The turn structure is stabilized by several inter-residue hydrogen bonds (Table S4 in the Supporting Infor-mation). Hydrogen bonding between Phe5-NH with -OdCof Pro3, between Ser6-NH with -OdC of Gly4 and, betweenside-chain -OH of Ser6 with -OdC of Phe8 (Figure S19) arepresent. Our result agrees with the overall description of theB1-specific antagonist (B-9858) having a β-turn in the centerof the peptide. For B1-selective analogues such as des-Arg9-BKor des-Arg9-Lys-BK (H-Lys-Arg-Ado-Ser-Pro-Phe-OH),22 thepresence of a β-turn at the C-terminus has not been ascertainedand would require a shift in the sequence because of the lack ofthe C-terminal Arg, which is the fourth residue in the turnobserved for B2-selective BK analogues.22 Altogether, themembrane-induced conformational modifications of kininpeptides are suitable for their recognition by the correspondingcell-surface receptors. The decrease in the solvent-accessiblesurface area (ΔSASA) of peptides (Table S5 of the SupportingInformation) on going from the water to lipid environment canalso contribute favorably to the solvation free energy of thepeptides.Lipid-Peptide Interactions.To shed light on the factors that

confer the stability and activity of the kinin peptides on the

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


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anionic bilayer surface, we have calculated the total lipid-peptide interaction energies of BK and des-Arg9-BK with thesurrounding lipid matrix and also its partition into electrostaticand van der Waals (vdW) terms (Figure 5).2,60 As shown inFigure 5, BK exhibits a stronger interaction (∼-590 KJ/mol)with the membrane than does its fragment (∼-530 KJ/mol), inaccordance with a previous report.7 Although the vdW contribu-tions of the two peptides are comparable, a stronger BK inter-action originates from its higher electrostatic contribution. In thedistribution curve, the peak for the electrostatic contribution perBK 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 out-come of both electrostatic and van der Waals contributions, inthe presence of an anionic lipid, electrostatic interactions mayplay the dominant role.To elucidate the source of such Coulombic interactions, a

careful study of hydrogen bonds (H-bonds) formed and brokenalong the MD trajectory is carried out (Figure 6).11 We alsocalculate the average number of H-bonds shared by peptideresidues with different functional groups (Figure S1) of the lipidhead (inset in Figure 6). As depicted in Figure 6, the majorH-bonding contribution arises from charged Arg residues. Forboth peptides, Arg residues are involved in H-bonding duringmore than half of each trajectory, thus acting as “hooks” for themembrane, as observed for BK in ref 30. Although the side-chainfunctional group of Arg exhibits a high affinity for the anionicphosphate group, additional H-bonds with glycol or ester oxygenare also formed. The role of basic Arg/Lys residues in penetratingthe cell membrane is well documented in the literature.2,58-61,65

As far as Ser6 is concerned, it is mostly solvated by watermolecules. The Gly and Phe residues contribute to H-bondingvia their backbone amide nitrogen. Such interactions facilitate thestabilization of peptides on the membrane interface and providethe favorable orientation of the peptides, thus aiding theirrecognition by a suitable cell-surface receptor.22,58

Differential Effects of PeptideBindingonBilayer Properties.In the preceding section, we have discussed the important rolesof lipid in inducing the conformation/orientation of peptides atthe bilayer interface. Because the lipid bilayer makes up the

platform where membrane-associated signaling molecules inter-act and function, the earlier studies revealed that any change inthe bilayer properties can have a strong effect on signalingdynamics.10-12 A detailed understanding of peptide-inducedchanges in the membrane properties is thus necessary to gainbetter insight into their mode of membrane interaction, and suchstudies have drawn significant attention recently.2,58,66,67 In thisparticular section, we will emphasize the effects of a peptide onthe bilayer structural properties (conformational ordering, head-group orientation, and bilayer thickness) as well as on thedynamic (flexibility of lipid heads) reorganization at the lipid-water 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 Theordering of lipid acyl chains can be characterized by the molec-ular order parameter, Smol

46

Smol ¼ 12

3 cos2 θn - 1� � ð1Þ

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

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 partitionedinto the electrostatic and vdW terms.

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 theinteraction exists. The inset plots represent the average number ofH-bonds formed between peptide residues and lipid headgroups. Thelast 5 ns of data has been used.


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(n þ 1) carbon atoms in the lipid hydrocarbon chain and thebilayer normal (Z axis). The angular brackets Ææ denote theaverage over time and the ensemble. To simplify the analysis,here we have divided the lipids into two categories: local (thosewith a lipid non-hydrogen atom within 10 Å of a peptide non-hydrogen atom) and bulk (all others) lipids.2,66 Figure 7 depicts asignificant drop in the order parameter values of local lipids ascompared to the pure bilayer (without peptide). This indicates alocalized bilayer perturbation around the peptide contact re-gions. The bulk lipids, however, become more ordered than thepure bilayer.66 The order parameter value of the pure POPGbilayer agrees well with the previous findings.43,44 The effect ofBK and its fragments (des-Arg9-BK, des-Arg1-BK, and Arg-Pro-Pro-Gly-Phe or BK1-5) on the fluidity of the DMPG vesicle wasexamined previously by Turchiello et al.7

Headgroup Orientation. To characterize the packing of lipidpolar heads around the surface-embedded peptide, we havecalculated the angle (θi) between the outwardmembrane normaland a vector defined by the phosphorus and glycol center carbonatom (P-C12, Figure S1) of the PG headgroup.43 The averageθi values were then plotted as a function of the average in-planeposition of lipid phosphorus atoms in the upper bilayer leaflet(Figure 8, first row).58 For both peptides, adjacent lipids becomeparallel to the membrane surface. The effect is more pronouncedat the termini of the peptides. For rest of the lipids, the headgrouporientation angle is between ∼60 and 80�, which compares wellwith a recent theoretical study.43 Such an effect arises because ofthe ability of the peptide to orient the lipid heads to itself throughstrong electrostatic and H-bonding interactions (Figure 6).

Other groups have recently reported such a reorientation ofthe lipid head, caused by peptide insertion.58,67,73

Membrane Landscape. The local disorder of the membraneupon peptide insertion, together with the tilting of the lipid head,can cause bilayer thinning near the peptide contact region.2 Themembrane landscape is characterized by the in-plane distributionofΔZi= Zi- ÆZæ, where Zi represents the Z coordinates of the ithlipid in the contact monolayer and ÆZæ is the average Z value ofthis surface.58 Figure 8 (second row) exhibits roughness on thebilayer surface. For both BK and des-Arg9-BK, the peptidedepresses the bilayer in its immediate vicinity by 0.7-0.8 nm,forming distinct grooves or dents beneath the peptide. The effectis very localized, confined to the peptide-lipid contact region,especially at the two peptide termini. Similar behavior is alsoreported for other cell-penetrating peptides.58,60,67

Dynamics of the Lipid-Water Interface. It was previouslyreported that lipids adjacent to peptide/protein (enriched inbasic (Lys, Arg) and aromatic (Trp, Tyr) amino acid residues)exhibit lower diffusion than the rest of the bilayer.74,75 Thedynamics at the bilayer interface can be characterized in terms ofthe flexibility of the lipid polar head, Δrmsfi = rmsfi - max-(rmsfi

pure),58 where rmsfi is the root-mean-square fluctuation(i.e., the standard deviation) of the polar head of the ith lipidrelative to its equilibrium position (calculated using the g_rmsfprogram of GROMACS). The term max(rmsfi

pure) is the max-imum of the distribution of rmsfi in the pure POPG bilayer(Figure S21). The 2Dmap (Figure 8, third row) illustrates the in-plane distribution of the flexibility of lipid heads and differenti-ates distinct regions on the bilayer surface, with lipids havingdifferent overall mobility. As shown in Figure 8 (third row), the

Figure 7. Molecular order parameters, Smol, for the palmitoyl (left column) and oleoyl (right column) chains for the different lipid categories of a lipidbilayer containing BK (bk_traj1, upper panel) or des-Arg9-BK (des9_traj1, lower panel) and that of the pure POPG bilayer (without peptide). The last5 ns of data has been used.


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headgroup flexibility of the neighboring lipids was reducedsignificantly from that of the rest of the membrane. Such afreezing or arresting effect is attributed to the trapping of lipidpolar heads through electrostatic and H-bonding interactions bythe charged/aromatic residues of the peptide.Our results reveal peptide-induced local bilayer perturbation

in terms of lipid tail order, membrane thinning, headgrouporientation, and flexibility of the lipid head. We have measuredthe properties of the bilayer (area per lipid and order parameter)for each leaflet separately (Table S6). For both systems, theupper bilayer leaflet is slightly more disordered and has amarginally greater area per lipid molecule than does the lowerleaflet. For example, with BK the values are 0.521 ( 0.003 and

0.509 ( 0.002 nm2 whereas for des-Arg9-BK the values are0.529 ( 0.002 and 0.515 ( 0.002 nm2 for the upper and lowerleaflets, respectively. Such an effect arises from the asymmetricinteractions of peptides with respect to the two bilayer leaflets.Because peptides bind at the lipid-water interface of the uppermonolayer, the area per lipid increases to make room for thepeptide. Moreover, because of the interaction with the peptide,the adjacent lipids (located mostly in the upper leaflet) becomemore disordered, causing local bilayer thinning. According tomembrane elasticity theories,78 the local perturbation is propa-gated into the bilayer and the distant lipids change in a mannerthat maintains membrane integrity. Similar results are reportedin many other MD simulations,60,66,67 but like other MD

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. Thesecond row represents the surface distribution of the change in bilayer thickness around the embedded peptides. The third row shows the in-planedistributions of dynamic fractions of lipids having different headgroup flexibilities. The average positions of peptide CR atoms are highlighted as yellowpoints.


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studies,60,66,67,72 the present work is also limited by the systemsize, with a fixed lipid member in each leaflet, and thus theordering of distant lipids may be influenced by the simulationartifact. Recently, Martinez-Seara et al.79 showed that the de-scription of a double bond80 in a lipid hydrocarbon chain is asubtle matter in the atomistic simulation of lipid bilayers, whichhave a measurable influence on system properties. However,using the GROMOS87/Berger force field (as in our case), manysimulations81,82 were reported to reproduce the overall shape ofthe order parameter profile of the unsaturated oleoyl tail. Asobtained from the NMR experiment,83 the disordered region inthe middle of the hydrocarbon chain arises from the unsaturatedcarbon atoms. To allow comparison with experimental data, wehave calculated the average area per lipid and order parameterprofile over the whole membrane (Table S6). The overallpeptide-induced changes in membrane fluidity compared wellwith a previous experiment on BK and its fragments in a DMPGvesicle.7 Recently, an AFM study revealed that a peptide locatedat the lipid hydrophilic/hydrophobic interface “does not induceuniform membrane thinning, but instead leads to the formationof distinct domains in the lipid bilayer”.84 The result is in nicecorrelation with our simulation finding (Figure 8, second row).

’CONCLUSIONS

The kinin peptides have received significant attention aspotential therapeutic agents and exert their bioactivity throughbinding to specific cell-surface receptors. Partitioning or bindingto the lipid bilayer is likely to favor or facilitate receptor bindingby the peptides. To explore the influence of lipids on theconformation and dynamics of peptides and vice versa, we havesimulated neuropeptide hormone BK and its fragment des-Arg9-BK, a natural kinin metabolite, in the presence of the anionicPOPG bilayer. Although BK with two terminal Arg’s, prefers tostay at the interface, des-Arg9-BK with the loss of the chargedgroup penetrates further. For kinins, the presence of chargedterminal residues is related to their receptor selectivity. Asexplained in earlier experimental studies,8 the greater affinity ofthe B2-type receptor for BK can be associated with regions of theprotein with a negative charge. For des-Arg9-BK, the loss of theArg9 residue favors the interaction with hydrophobic regions ofthe B1-type receptor.8 Again, on binding at the surface, both BKand des-Arg9-BK adopt a β-turn-like structure, which is crucial totheir receptor binding. Simulations with different starting orien-tations of the peptides with respect to the bilayer surface lead tothe 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 turnstructures at the C-terminal of BK and in the middle for des-Arg9-BK.

Moreover, the peptide binding has reciprocal effects onmembrane structure and dynamics. The lipid headgroups adja-cent to the peptides become substantially tilted, causing the lipidtail to spread outward, which leads to bilayer thinning near thepeptide contact region. The hydrocarbon tails of the neighboringlipids become more disordered, whereas those of the distantlipids become stretched and ordered. Again, both peptides arecapable of trapping lipid headgroups through electrostatic andH-bonding interactions, thereby considerably reducing the over-all flexibility of lipid polar heads in their vicinity. Thus, peptidebinding can affect the structure, dynamics, and organizationof the lipid membrane. Altogether, our simulations provide

molecular-level insight into the details of lipid-peptide interac-tions, highlighting the roles of different peptide residues, peptideconformation, and orientation in surface binding and peptide-induced membrane structural disturbances and the dynamicalreorganization of the lipid-water interface.

’ASSOCIATED CONTENT

bS Supporting Information. Data regarding the equilibra-tion of POPG bilayer, results of the control simulations, SASAvalues of the peptides, phi-psi dihedral angles, Ramachandranplots, the list of inter-residue hydrogen bonds of peptides, andthe schematic representation of POPG molecules. This materialis 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 ofChemistry, University of Calcutta, Kolkata, India, for the com-putational facilities and to the RCAMOS of the Indian Associa-tion for the Cultivation of Science, Kolkata, India, for providingaccess to the High Performance Computing Facility. This work issupported by a fellowship through SRF CSIR-NET to M.M. bythe Government of India.

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molecular dynamics simulations of the interactions of kinin peptides with an anionic popg bilayer

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