This journal is c the Owner Societies 2013 Phys. Chem. Chem. Phys., 2013, 15, 19031--19041 19031

Cite this: Phys. Chem.Chem.Phys.,2013,15, 19031

Sequence dependent lipid-mediated effects modulatethe dimerization of ErbB2 and its associative mutants†

Xavier Prasanna,z P. J. Praveenz and Durba Sengupta*

The association of transmembrane helices is an important event in several biological processes, but the

factors governing association, especially the non-specific environmental effects, have still not been

elucidated. Here, we use coarse-grain molecular dynamics simulations to study the association of ErbB2

transmembrane helices and three ‘‘oncogenic mutants.’’ Self-assembly simulations and the dimerization

free-energy profiles confirm an energetically-favorable dimerized state for both the wildtype and the

mutants. The dissociation free energy of all three mutants is calculated to be larger than the wildtype

peptide. Along with favourable protein–protein interactions, non-specific environmental effects

are observed to contribute to the association. In particular, local bilayer thinning along with membrane

perturbations are seen around the mutants. The membrane perturbations are reduced upon helix asso-

ciation, suggesting that lipid chain packing is an important driving force for helix dimerization. Our

results highlight the importance of both specific as well as non-specific driving forces in the association

of transmembrane helices.

1 Introduction

The association of a-helical membrane proteins is a key eventin several cellular processes including cellular signaling andtrafficking.1–3 Consequently, the altered association profiles of thesemembrane proteins are linked with various disease conditions.4–6

The association processes of these large complexes can bemodulated or sometimes even dictated by the assembly of theirtransmembrane domains,7,8 as evidenced by pathogenic mutations9

and the use of short transmembrane peptides as novel inhibitors ofmembrane protein function.10,11 Recent advances in microscopytechniques have allowed us to probe these associations withincreased resolution, challenging several established concepts inmembrane structural biology. One aspect of this change is reflectedin experimental evidence that suggests a need to evolve from simpledimerization sequence motifs to understanding the more complexnature of transmembrane helix association.12 An even moreimportant consequence of state of art microscopy experimentsis the change from a ligand-induced dimerization paradigm tothat of pre-formed dimers for several membrane receptors.13,14

Thus, the need arises to re-evaluate the association profiles of thepermanent and transient associations within membranes.

Helix–helix interactions have been shown to be characterizedby specific motifs such as the GxxxG motif15–23 and the leucinezipper motif.24,25 The presence of polar residues at dimerinterfaces22,26 and the surface complementarity of the inter-acting transmembrane helices have also been extensively char-acterized.27–30 Several studies probing helix–helix interactionsfocussed on glycophorin A as a model system, but the qualitativeconsensus in interface residue identities was in contrast with theincreased association of certain double mutants predicted to bedisruptive, pointing towards more complex pathways and associatedstates.31 The sensitivity of the mutation analysis to lipid compositionhas also been demonstrated.32 It is therefore becoming clear thatresidue based determinants do not always explain the entireassociation and the need arises to systematically analyse othercontributions to transmembrane helix association.

In contrast to the specific helix–helix contacts that stabiliseassociated states, the non-specific contributions from the lipidenvironment have been characterised less.33 In glycophorin A,measurements of helix association in bilayers have revealedthat dimer stability in palmitoyloleoylphosphatidylcholine(POPC) bilayers varies substantially compared to detergent,32

contrary to previous speculations on the relative stabilities.34

Recent computational studies have shown that the effect of themembrane is larger than previously estimated, and lipidophobiceffects may be an important driving force in helix association.35–37

Experimental studies have also suggested that residues withpoor lipid–protein interactions could be a driving force for helixassociation.38 Interestingly, membrane fluidity has also been

CSIR-National Chemical Laboratory, Dr Homi Bhaba, Road, Pune-411008, India.

E-mail: d.sengupta@ncl.res.in; Fax: +91-20-2490-2636; Tel: +91-20-2490-2408

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3cp52447g‡ These authors contributed equally to this work.

Received 13th June 2013,Accepted 14th September 2013

DOI: 10.1039/c3cp52447g

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shown to play a role in tuning associations within the bilayer.39,40

In complex membranes, the varying lipid composition modulatestransmembrane helix interactions,41 and the need arises to system-atically assess the different contributions to helix association.

ErbB2 is a member of the epidermal growth factor receptor(EGFR/ErbB) family belonging to the receptor tyrosine kinasesuperfamily.42 The ErbB family of proteins have been the focusof several studies on helix–helix association due to their centralrole in cellular signalling and as model systems for other single-span transmembrane helices.43 A high resolution structure of theErbB2 dimer has been proposed based on NMR and moleculardynamics simulations.44 Interestingly, ErbB2 represents thefirst system where the transmembrane domain was implicatedin an oncogenic mutation. The V - E mutation in the trans-membrane domain in ErbB2 (also called Neu) was identified inoffsprings of rats9,45 and increased association was proposed tolead to receptor over-activation and oncogenesis.46 However,experimental measurements of the association of the mutantshowed it to be destabilising,47–49 with the exception of onemeasurement.50 Alternative hypotheses have been proposed toexplain the over-activation of the mutant, including structuralchanges, differences in membrane insertion, molecular switchor rotational coupling and multiple modes of interaction.48,51

The solvent effects contributing to the dimerization energeticshave been largely neglected, although a recent study hasattributed differences in association of the mutant to thejuxtamembrane positioning in the presence of charged lipids.52

Here, we use the coarse-grain MARTINI force-field53,54 tounderstand the association of human ErbB2 and three mutantswithin a dipalmitoylphosphatidylcholine (DPPC) membrane.The mutants chosen are V659E (the human mutant corre-sponding to the Neu oncogenic mutant in rats47 and shownto cause cancer in transgenic mice55), V664E (corresponding toa site of increased transmembrane association selected froma high-throughput selection study56) and the correspondingdouble mutant (V659EV664E). Starting from a dissociated state,we observe the spontaneous association of the helices, bothwildtype and mutants. We also calculate the dimerization profiles(potential of mean force) of the four peptides to justify theexperimentally measured differences in association. We furtherelucidate the role of the lipid-mediated interactions and othersolvent effects as opposed to the specific protein–protein inter-actions in driving the association of these transmembranehelices. The work has significance in understanding the roleof the membrane environment in driving the association ofhelical receptors and ErbB2 in particular.

2 Methods2.1 Self-assembly simulations

To study the association of ErbB2 and its mutants in lipidbilayers, coarse-grained molecular dynamics simulations wereperformed. The MARTINI force-field53,54 was used to describethe peptides, lipids and water. The MARTINI force-field hasbeen previously demonstrated to reproduce several aspectsof membrane–protein interactions accurately,57–61 including

protein–protein interactions within the membrane.35,62–64 Thecurrent simulation system consisted of two ErbB2 (or itsmutant) transmembrane helices embedded in a DPPC bilayerof 246 lipids solvated by 4000 coarse-grained water beads.Simulations were performed with the wildtype ErbB2, theV659E and V664E single mutants and the V659EV664E doublemutant. The two single mutants, V659E and V664E, correspondto the human equivalent of the Neu oncogenic mutant in rats47

and a site of increased transmembrane association selectedfrom a high-throughput selection study,56 respectively. Thecoarse-grained structure of the monomeric wildtype ErbB2was mapped from the atomistic structure of the dimer (PDBcode: 2JWA44). The three mutants were modelled from ErbB2 byaddition of side-chain beads followed by a minimisation. All fourpeptides were modelled as ideal helices, using dihedral potentialsto maintain the helicity of the peptides. The terminal residueswere modelled as coil with no secondary structural restraints.The sequences corresponding to the four systems are:

wildtype: PLTSIISAVVGILLVVVLGVVFGILIKRRQV659E: PLTSIISAVEGILLVVVLGVVFGILIKRRQV664E: PLTSIISAVVGILLVEVLGVVFGILIKRRQV659EV644E: PLTSIISAVEGILLVEVLGVVFGILIKRRQMolecular dynamics simulations were performed using the

GROMACS software package, version 4.5.5,65 with the schemedeveloped for the MARTINI model.53,54 The temperature wascoupled (coupling time 0.1 ps) to a thermostat at T = 325 Kusing a Berendsen algorithm.66 The pressure was coupled(coupling time 1.0 ps, compressibility 5 � 10�5 bar�1) using asemi-isotropic coupling scheme, in which the lateral andperpendicular pressures are coupled independently at 1 bar.66

The non-bonded interactions were treated with a switch functionfrom 0.0 to 1.2 nm for the Coulomb interactions and 0.9 to1.2 nm for the Lennard Jones interactions (pair-list updatefrequency every 10 steps). A time step of 20 fs was used. Thetimes reported in the manuscript are simulation times, i.e. whenmultiplied by a factor of four, approximately accounts for thespeed-up of coarse-grained dynamics resulting from the neglectof friction associated with the atomistic degrees of freedom.53,54

Initially, two copies of the monomeric peptide were introducedin a pre-equilibrated DPPC bilayer at a distance of about 6 nmfrom each other. Simulations of 10 ms were performed for each ofthe three peptides, sufficiently long to observe the self-assemblyof the transmembrane helices.

2.2 Potential of mean force

The associated dimers from the self-assembly simulations wereused as the starting structures for the potential of mean force(PMF) calculations. The potential of mean force was calculatedusing the umbrella sampling technique.67 The umbrellapotential acts on the center of mass of the backbone beads ofthe peptides with a force constant of 2000 kJ mol�1 nm�2. Thevalue was optimised to achieve an adequate overlap betweenhistograms. For each system, windows were simulated corre-sponding to a 0.1 nm shift of the monomer per simulationstarting from 0.5 nm to 4 nm. The starting structures for eachwindow were created by pulling the two peptides from their

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associated state, taken from the self-assembly simulations, totheir window location using the umbrella potentials with aforce constant of 2000 kJ mol�1 nm�2 and with a pull rate of0.5 nm ns�1. Each window was then simulated for up to 6 ms. TheWHAM method68 was used to unbias the umbrella potentials. Forthe wildtype, each window was further simulated for up to 16 ms,corresponding to a total of about 2.2 ms of effective time, to testthe convergence of the dimerization profiles.

2.3 Analysis

Interaction energy. The interaction energies: protein–protein,lipid–lipid and water–water were calculated from the sum of theLennard-Jones and electrostatic contributions, as a function ofthe helix–helix distance.

Dimer characteristics. The inter-helical distance was definedto be the distance between the centres of masses of the back-bone of the two helices. The distance between the N-terminiwas calculated as the distance between the backbone bead ofthe residue 7 of both helices and the C-termini as the distancebetween the backbone bead of the residue 24 of both helices.The inter-helical angle (cross-over angle) was calculated as theangle between the two helical axes defined by the backbonebeads of the residues 4 and 24 of both helices. This simplemethod does not accurately describe the helix axis but isadequate for calculating the changes in cross-over angle incoarse-grain models, especially in the presence of secondarystructural constraints.

Dimer interface. The residues at the dimer interfaces weredetermined via a distance criteria with a cut-off of 0.5 nm. Thenormalized probability of its presence in the dimer interfacewas calculated for 2 ms of the trajectory. A value of 1.0 indicatesthat a residue is always present at the interface, and a value of0.0 indicates that it is never present in the interface.

Membrane parameters: thickness and order parameters.The membrane thickness was calculated as the average distancebetween the two phosphate beads (PO4) in the head-group ofDPPC using analysis tools that have been previously developed.69

The lipid chain order parameter, P2 = 1/2(3hcos2y � 1i), wascomputed for consecutive bonds in the coarse-grain lipids,where y denotes the angle between the bond vector and thebilayer normal. Perfect alignment with the bilayer normal isindicated by P2 = 1, perfect anti-alignment by P2 = �0.5, and arandom orientation by P2 = 0. Order parameters were calculatedby averaging over the last 5 ms of the trajectory. To calculate themembrane thickness and order parameters, the translationalmotion of the peptides was removed (i.e., the position of thecentre of mass of both peptides was constant).

3 Results3.1 ErbB2 and its associative mutants self-assemble withinthe membrane

To study the association of ErbB2 and its mutants within themembrane, self-assembly simulations were performed. TwoErbB2 a-helical monomers were inserted in a parallel orienta-tion into a pre-equilibrated DPPC bilayer with the helices

separated at a distance of approximately 6 nm. During thesimulation, the transmembrane helices diffused through themembrane and associated within 1 ms. The representative self-assembled wildtype dimer was close packed in a right-handedstructure with a negative cross-over angle. The time course ofthe inter-helical distance, the distance between the N- andC-terminal residues and the inter-helical angle is shown inFig. 1A. The average inter-helical distance in the dimerizedstate was 0.8 nm. Upon extended sampling, two distinct dimerconformations were observed that differed from each other inthe helix–helix packing. The largest population was that of aC-terminal dimer, characterised by a closer packing of theC-terminal residues and corresponding to an inter-helical angleof �301. The second population observed had a lower inter-helical angle of �201 and a comparative closer-packing ofthe N-terminal dimers. Representative conformations of theN- and C-terminal dimers are depicted in the inset in Fig. 1.An analysis of the residues involved in the dimer interface duringthe simulation is shown in Fig. 2. A cut-off value of 0.5 nmhas been used to determine the interface residues. A value of1.0 indicates that the residue was always present at the dimerinterface, and a value of 0.0 indicates that it was never present.The N-terminal dimer interface was mainly characterised by thepresence of contacts within residues Ser656, Ala657, Gly660,Ile661 and Leu663 and the C-terminal with residues Leu677,Gly678 and Gly682. The helix face for both the N- and C-terminaldimers is similar to residues from the N- and C-terminal side,respectively, being involved at the contact interface. The dimerinterface is flexible and rotation around the dimer interfaceleads to the involvement of the adjacent residues. A fewconformations where the dimer interface is asymmetrical arealso observed. Control simulations starting with differentinitial velocities show that the formation and packing of thedimer is reproducible. No dissociation event was seen in any ofthe simulations.

Self-association simulations were also carried out for themutants under similar conditions and we observed the trans-membrane helices to associate rapidly within 1 ms. All threemutants self-assembled to a right handed dimer. The inter-helical distance of the V659E dimer was on an average 0.9 nm(see Fig. 1B). Again two distinct dimer populations were observed.The N-terminal dimer was similar to the wildtype and had aninter-helical angle of �251. The C-terminal dimer had a muchlarger inter-helical angle of �601. The dimer interface was similarto that of the wildtype, although fewer residues were observed atthe interface. The dimer interface appeared to be ‘‘tighter’’ and norotations around the dimer interface were observed (Fig. 2). Twodistinct conformations were also seen in the right handed dimerstructure of the V664E single mutant (Fig. S1A, ESI†). TheN-terminal dimer had an inter-helical angle of �251 but theC-terminal dimer had a large dynamics with an average inter-helical angle of �551. The inter-helical distance also differed: theN-terminal dimer had an inter-helical distance of 0.9 nm, whilethe C-terminal dimer was further apart at 1.1 nm. The inter-helicalangle of the self-assembled V659EV664E dimers was �431.The average inter-helical distance was 1 nm, though a second

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population with an inter-helical distance of 1.5 nm was alsoseen. The N-terminal dimer could not be distinguished evenon extended sampling (Fig. S1B, ESI†). The absence of theN-terminal dimer is also reflected in the residues involved inthe dimer interface (Fig. 2). In both these mutants, the bilayerwas highly perturbed and several water molecules were seen inthe vicinity of the protein.

3.2 The dimerization profile of ErbB2

To analyse the energetics of dimerization, we performedumbrella sampling and calculated the potential of mean force(PMF) as a function of the inter-helical distance for the humanErbB2 transmembrane domain (shown in Fig. 3, black). Thefree-energy of the two well separated monomers is assumed tobe zero. Consistent with the self-assembly simulations andprevious experimental studies, the dimerized state is observedto be the most favourable. For the wildtype ErbB2, the dimer-ization profile shows only one main minimum and a negligiblysmall barrier (1 kJ mol�1) to association. The minimum of theassociation profile is located at 0.8 nm. The value correspondswell to the inter-helical distance observed in the self-assemblysimulations. Both the N-terminal and C-terminal dimersobserved in the self-assembly simulations are sampled.A population distribution of the inter-helical angle is shownin Fig. S2 (ESI†). The free-energy difference between the fully-separated state and the dimerized state is �34 kJ mol�1.A second minimum, less deep than the first at E1.1 nm, is alsoseen. To test the convergence, we calculated the profiles takinginto account only the first 2 ms, 2–4 ms, 2–6 ms and 2–16 ms(Fig. S3, ESI†). The global shape of the profile remains thesame during the different sampling times. The most strikingdifference is the appearance of a second minimum, whichbecomes apparent only after prolonged sampling. Extendingthe sampling in each window to 16 ms leads to a convergence inthe PMF profile. The apparent dissociation free energy DGdis

can be obtained by integrating the PMF profile GPMF(r) toan appropriate separation, which delineates the limit of associa-tion. In cylindrical coordinates, the association constant can be

written as:70 Ka ¼ pÐ lmax

0l exp �bGPMFðlÞ

� �dl where, lmax denotes

the cylindrical radius separating associated and dissociatedstates of the two a-helices and is taken to be 4 nm. The integral

Fig. 1 Dimer characteristics of the transmembrane helix dimer of (A) the wildtypeand (B) the V659E mutant of ErbB2, as a function of time. The top panel (in bothsub-figures) shows the inter-helical distance, the middle panel shows the distancebetween N- and C-terminal residues in black and red, respectively, and the bottompanel shows the inter-helical angle. The two conformations, an N-terminal and aC-terminal dimer, that are sampled during the simulation are shown in the insetwith the two transmembrane helices coloured in red and blue. The time duringwhich the N-terminal dimer is sampled is marked by dotted lines.

Fig. 2 The time averaged residue–residue contacts at the dimer interface. A cut-off distance of 0.5 nm has been used to determine the contact residues. A value of1.0 indicates that the residue was always present at the dimer interface and a value of 0.0 indicates that it was never present.

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reaches a plateau beyond 2.5 nm and the value of Ka is notsensitive to that of lmax. The apparent dissociation free energyis then given by DGdis = �RTln(Ka). Numerical integration of theequation, using the PMF profile, results in DGdis = 34.7 kJ mol�1

for the wildtype ErbB2.The PMF for the association of the V659E mutant is shown

in Fig. 3 (red). A single minimum was observed, again with asmall barrier (1 kJ mol�1) to association. The minimum in thePMF was shifted further at 0.9 nm. The value of the inter-helicaldistance at the minimum is consistent with the largest populationobserved in the self-assembled dimers. The difference in freeenergy between the fully-separated state and the dimerized statesis about 38 kJ mol�1. The value of DGdis (as calculated above) is39.4 kJ mol�1, about 4.7 kJ mol�1 higher than the wildtype. Thesecond minimum that was observed in the wildtype at 1.1 nmcould not be discerned at these timescales. Similar to the wildtypeassociation profile, the free-energy levels off and reaches a plateauat distances of greater than 2.4 nm.

The association profiles of the V664E single mutant (Fig. 3,green) and the double mutant (Fig. 3, blue) were strikinglydifferent from the wildtype. For V664E, two minima could bediscerned at 0.85 nm and 1.2 nm. The values correspond well tothe inter-helical distances of the two populations observed inthe self-assembly simulations. The depth of both minima was�50 kJ mol�1, and DGdis was calculated to be 54.7 kJ mol�1. Forthe double mutant, the main minimum was observed at aninter-helical distance of 1 nm. The average value of free-energydifference between the fully-separated state and the dimerizedstate is 52 kJ mol�1 (DGdis = 57.9 kJ mol�1). A second minimum,higher in energy than the first is also seen at 1.4 nm. Incomparison to the wildtype, the minimum is broader for thesetwo mutants. The free-energy levels off and reaches a plateau ata distance of about 3 nm.

3.3 Contribution of the solvent effect to dimer association

To analyse the driving forces for the association of ErbB2 andits oncogenic mutants, we calculated the different contributions

to the association profiles, such as the protein–protein, lipid–lipid, and water–water interaction terms (Fig. S4, ESI†). Asthe peptides approach, protein–protein interaction energybecomes more favourable and as expected, the associated stateis stabilised by helix–helix interactions. The decrease in theprotein–protein interaction term as a function of helix separa-tion showed a similar trend for all four peptides. For thewildtype protein, the global minimum in the protein–proteininteraction energy is around 0.6 nm, a distance that is slightlylower than the global minimum in the PMF. Interestingly, asecond minimum is also observed at 1.1 nm that correspondsdirectly to the second minimum in the PMF. For V659E and theV664E mutant, the interaction energy follows a similar trend,but the value of the minimum is higher than the wildtype. Theprotein–protein interaction energy is the most favourable forthe double mutant with a minimum at 0.5 nm, whereas theminimum in the PMF lies at 1 nm. Interestingly, at largeseparations (between 2–3.2 nm), the energy decreases morerapidly for the wildtype and V659E than the V664E and thedouble mutant, consistent with the relative decrease in thePMF. Although the helix interaction energies can explaincertain features of the PMF, it is clear that they cannot fullyaccount for the differences seen in the dimerization freeenergy. The lipid–lipid interaction term also decreases as afunction of helix separation and becomes more favourable asthe helices associate. In the monomer regime, the lipid–lipidinteraction energy is the lowest for the wildtype and the highestfor the double mutant. However, as the helices associate, alarge decrease is seen in the double mutant and only a marginaldecrease in the wildtype. Since the relative decrease in thelipid–lipid interaction energy during dimerization is more forthe double mutant compared to the wildtype, it contributessubstantially towards helix association. The largest contributionfrom the lipid–lipid interaction energy was observed for theV664E mutant. Although the lipid packing energetics appearedto explain the much larger association energy for the V664E andV659EV664E mutants as compared to the wildtype and V659Emutant, they did not explain all aspects of the PMF.

Since none of the interaction energy terms individuallyshowed the same trend as the PMFs, we added their contribu-tions and normalised them to the value at large helical separa-tions (Fig. 4). Interestingly, the decrease in the interactionenergy (summed up contributions) as a function of inter-helicaldistance was maximum for the double mutant, implying that itfavoured association the most. The total interaction energyprofile for the V664E mutant was also highly favourable forassociation. The summed up contributions for the wildtype andthe V659E mutant also decreased upon association, but thedecrease was less than that of the above two mutants. Atthe dimerized state, the interaction energies were the leastfavourable for the wildtype, the most favourable for the doublemutant and intermediate for the V659E and V664E mutants,consistent with the dimerization propensities seen in theassociation profile. None of the interaction energy termscontributed to the increase seen in the PMF at low helixseparations and could arise due to entropic contributions.69

Fig. 3 The potential of mean force (PMF) of dimerization of wildtype ErbB2transmembrane helices (black) and three mutants V659E (red), V664E (green)and V659EV664E (blue). The error bars shown are estimated from bootstrapanalysis.

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We would like to point out that the trend in the total energiesshould be considered instead of the exact values, since theentropy–enthalpy balance that gives rise to the correct freeenergy value differs in a coarse-grain force-field as comparedto the more accurate atomistic force-fields. Taken together,

it thus appears that dimerization, at least in the case of ErbB2and its oncogenic mutants, is a complex process driven by theinteraction energies of the protein, lipid and water.

3.4 Membrane packing effects in helix association

A visual inspection of the trajectories revealed large membraneperturbations around the mutant dimers. To characterise thelocal membrane perturbations, we calculated the differences inthe membrane packing around the four peptides in theirdimerized states and in the monomer regime. The bilayerthickness was calculated for the entire bilayer by binning intogrids and is shown in Fig. 5. Large variations were observed inthe bilayer thickness of the annular lipids around each of thefour proteins. Around the wildtype dimer structure, a marginaldecrease in the bilayer thickness was observed, followed by anincrease in the bilayer thickness that persisted for about 2 nm.In the monomeric regime, i.e. when the helices were separatedby a distance of 3 nm, the variation in the bilayer thickness waseven less. For the V659E mutant, the trend was the same as thewildtype, but the differences in membrane thickness were morepronounced. The decrease in the membrane thickness aroundthe receptors was substantial also in the monomer regime.The local decrease in membrane thickness was associatedwith perturbations of the phosphate bead and increased water

Fig. 4 The summed contributions from the protein–protein, lipid–lipid andwater–water interaction energies as a function of inter-helical distance, normal-ized to the value at large helical separations for wildtype ErbB2 (black) and itsV659E (red), V664E (green) and V659EV664E (blue) mutants. The error barsshown denote the standard deviations calculated within the trajectory.

Fig. 5 Local membrane thickness calculated over the entire membrane by binning into grids. The two columns show the membrane thickness around the dimer(left column) and the two monomers (right column) of the wildtype ErbB2 (A, B), V659E (C, D), V664E (E, F) and V659EV664E (G, H) mutants, respectively.

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penetration into the membrane. In the case of the V664Emutant and the double mutant, a substantial decrease inmembrane thickness was seen around the dimer structure.The subsequent increase in the bilayer thickness was similarto that of the wildtype dimer. The extent of perturbation aroundthe monomers was observed to be larger than in the dimer. Tocharacterise the membrane perturbations, we also calculatedthe water partial density along the membrane normal, againfor the dimer and monomer regimes (Fig. S5, ESI†). In the caseof the wildtype no perturbations were seen, and the waterdensity decreased uniformly at the head-group region. In themutants, an increasing perturbation was seen near the peptidesin both the dimerized and the monomeric states. The bulgeobserved in the water densities near the head group region wasmaximum in the double mutant.

Lipid chain order parameters (defined in Methods) were alsocalculated and averaged over either only the first lipid shellaround the protein (annular lipids) or the lipids much further

away (bulk lipids). The order parameters (averaged over thedimer regime) for the annular and bulk lipids are shown inFig. 6A and B, respectively. The order parameters of the annularlipids around the helix dimers were highest for the wildtype(black) and lowest for the double mutant (blue). Thus, thedisorder is highest around the V659EV664E dimer, followed bythe V664E and the V659E mutant dimers, and least around thewildtype ErbB2 dimer. The disorder (and perturbations) appearto be local in nature since no differences were seen in the orderparameters of the bulk lipids. Comparison of the order para-meters for the annular and bulk lipids for each peptide (Fig. S6,ESI†) clearly shows that the annular lipids are perturbed and adecrease in the order parameters is seen in the annular lipidsas compared to the bulk lipids. The perturbations are seenaround the peptides in both the dimerized state and in themonomer regime. The disorder in the lipids chains is negligiblefor the wildtype and highest for the double mutant. The decreasein the membrane order around the four peptides is consistentwith the decrease in membrane thickness. Averaging the value ofthe order parameters over all bonds and binning over the entiremembrane (Fig. 7) showed that although the annular lipidsare equally perturbed, the extent of disorder decreased upondimerization. It therefore appears that dimerization of themutant transmembrane helices reduces the membrane perturba-tions, increasing membrane chain packing.

4 Discussion

The association of the receptor ErbB2 that belongs to the EGFRfamily of receptors has been probed extensively due its centralrole in cellular signalling, and its implication in neurogenetivediseases, development defects as well malignancy in tumours.In particular, an oncogenic mutation in the correspondingNeu receptor in rats (V664E), has been shown to enhancereceptor activation, although it is still debated whether it arisesfrom an increased receptor association or conformationalswitching.47–49,52 We have used coarse-grain simulations andumbrella sampling simulations to probe the association betweenhuman ErbB2 receptors and the corresponding oncogenicmutant V659E. We have also looked at another putative asso-ciative mutant V664E and the corresponding double mutant.Our results show an increased association for all three mutantscompared to the wildtype, albeit with different values. Theresults highlight the importance of factors other than directsequence based metrics in driving helix association withinmembranes.

The first interesting feature of our results, is the marginalincrease of association of the V659E mutant, compared with thewildtype, consistent with experiments on rat ErbB2 in bilayers,50

but in contrast with association studies in micelles andmembrane mimetics.48 It is not clear whether the contradictoryresults arise from the differences in the membrane system(bilayer vs. vesicles) or from differences in measurements. Theassociation free energy calculated here for the wildtype is about�34.7 kJ mol�1, that corresponds well to previous estimates.48

It has been earlier shown that the corresponding Neu mutant is

Fig. 6 The average membrane order parameters, P2, of the (A) annular lipidsand (B) the bulk lipids around the wildtype ErbB2 dimer (black) and its V659E(red), V664E (green) and V659EV664E (blue) mutants. The values of P2 overlapfor the bulk lipids and cannot be distinguished.

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more tilted with the glutamic acid hydrogen bonded.71 The dimerconformation of the mutant in our simulations is also more tilted.Due to membrane deformations, water is also seen in the vicinityof the residue, which would presumably be hydrogen-bonded inan atomistic description. A transverse repositioning toward themembrane surface has also been attributed to the mutant, basedon fluorescence quenching,72 which perhaps could be attributedto the membrane deformation and increased water accessibility ofthe glutamic acid residue seen in our simulations. Previousatomistic simulations with the modelled structure of the V664Emutant have also reported increased water penetration andhydrogen bonding with lipid head groups.73,74 The membraneperturbations allow the screening of the side chain charges inboth coarse-grain and atomistic representations75 and the PMFprofile of a glutamate residue traversing the membrane calculatedfrom MARTINI simulations matches the atomistic profileclosely.54,76 The glutamate mutant also retains its a-helical char-acter in molecular dynamics simulations of the modelled ErbB2mutants (rat or human).44,73,74,77 Circular dichroism (CD)72 andNMR studies78 have also confirmed a high helical content of thetransmembrane domain of the glutamate mutant.

We also see in our simulations, two distinct dimer confor-mations that we term as the N-terminal and the C-terminal

dimer. No distinction could be made between the wildtype andV659E mutant based on the relative populations of the twodimer conformations. It has been suggested before for bothErbB2 and other members of the family that their C-terminaldimer is the inactive conformation while the N-terminal dimeris the active state.42,48 Recent microsecond timescale atomisticmolecular dynamics simulations of a related protein, EGFR,have also shown that the N-terminal dimer is more stable thanthe C-terminal dimer and corresponds to the active form.79 Inthe rat V664E mutant, it has been suggested that the C-terminalassociation is blocked leading to a constitutive activation.48

Although no conformational restriction was observed in oursimulations, the importance of the extra-membrane domainsin restricting certain conformations cannot be ruled out. Thedimer interfaces of the wildtype ErbB2 dimer and its threemutants observed in this study are similar, and are character-ized mainly by inter-helical residue contacts via the glycineresidues. The V659E dimer however appears to be less flexiblethan the wildtype and no rotation is observed around the dimerinterface. The lower flexibility of the V659E dimer interfaceis also reflected in the fewer residues that characterise thedimer interface. The NMR structure of the human ErbB2 trans-membrane dimer is that of the N-terminal dimer, characterized

Fig. 7 Local average membrane chain order parameter, P2, calculated over the entire membrane by binning into grids. The two columns show the average orderparameter around the dimer (left column) and the two monomers (right column) of the wildtype ErbB2 (A, B), V659E (C, D), V664E (E, F) and V659EV664E (G, H)mutants, respectively.

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by the GG4-like motif Thr652-X3-Ser656-X3-Gly660.44 In thestudy, the authors suggest that the local conformation of thedimerization interface is asymmetrical; although, in the courseof molecular dynamics relaxation, hydrogen bond switchingoccurs and a symmetrical dimer is obtained. They have alsoperformed molecular dynamics simulations with the model ofthe mutated V659E protein and observed the direct interactionof the Glu659 in the dimer interface. Although we observe severalconformers with a direct contact of Glu659, it is not presentthroughout the simulation and the contact metrics of Gly660 ishigher. In contrast, NMR studies on rat ErbB2 were not consistentwith hydrogen bonding between the glutamate residues, butsuggested the packing of the subsequent glycine residue in thedimer interface.78 Previous atomistic molecular dynamics simula-tions of ErbB2 models have also predicted a right handeddimer73,77,80 characterized by the GG4 motif, although a few lefthanded dimers have also been predicted.77,80,81

An increased association is also seen in the V664E mutant(which is distinct from the rat oncogenic V664E mutant) andthe corresponding double mutant. Although the associationenergy is substantially more favourable compared to the wild-type the inter-helical distances are larger, suggesting a less wellpacked structure. An increased inter-helical distance has alsobeen previously predicted for a highly associative mutant in theEpo receptor.82 Yet another aspect brought out in our work isthe importance of the sequence context to association. A highenergetic penalty is associated with the membrane insertionof charged residues such as glutamic acid83 and it has beensuggested that inter-residue hydrogen bonding can providestability and specificity to helix association.28 However, althoughwe see an increase in association upon introduction of aglutamic acid residue, it is based on the position of the residueand it is sequence/position specific instead of residue specific.Furthermore, there was no systematic change in the helix–helixinteraction energy, and it is a combined difference in theenvironment (membrane and water) along with the helix–helixinteractions that lead to the increased association of the mutantscontaining a glutamate residue.

In conclusion, we have carried out microsecond time scalesimulations of ErbB2 and three associative mutants. Two dis-tinct conformations were observed, an N-terminal and aC-terminal conformation, characterised by different cross-overangles. Umbrella sampling calculations were also carried outto determine the dimerization profiles of these peptideswithin the membrane. The dimerization energy of ErbB2 isabout �34.7 kJ mol�1. The V659E mutant dimer is stabilised incomparison to the wildtype by 4.3 kJ mol�1. The dimerizedstates of the V664E mutant and the V659EV664E double mutantare stabilised further by 20.0 and 23.2 kJ mol�1, respectively.Along with the protein specific interactions, lipid chain packingappears to be an important driving force for dimerization. Ourresults highlight the importance of non-specific driving forcesin membrane protein association and the need to re-evaluateprotein–environment interactions. Both sequence and solventbased criteria need to be considered to fully understand theprocess of membrane protein association.

Acknowledgements

D.S. gratefully acknowledges the support of the RamalingaswamiFellowship from the Department of Biotechnology (D.B.T), and theFast Track grant from D.S.T., Govt. of India. X.P. gratefully acknowl-edges the research fellowship from University Grants Commission(UGC), Govt. of India and P.J.P. the CSIR JRF fellowship. Theauthors thank the Center of Excellence in Scientific Computing(CoESC) and Center of Excellence in Polymers (SPIRIT) at CSIR-NCLfor computational time. D.S. and X.P. also acknowledge helpfuldiscussions with Srinivasa M. Gopal.

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