Insight into Signal Transduction: Structural Alterations in Transmembrane Helices Probed by Multi-1...

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Insight into Signal Transduction: StructuralAlterations in Transmembrane Helices Probed byMulti-1 ns Molecular Dynamics SimulationsJean-Pierre Duneau a , Norbert Garnier a b & Monique Genest aa Centre de Biophysique Moléculaire, CNRS, Rue Charles Sadronb Université d'Orléans, 45071, Orléans Cedex 2, FranceVersion of record first published: 21 May 2012.

To cite this article: Jean-Pierre Duneau , Norbert Garnier & Monique Genest (1997): Insight into Signal Transduction:Structural Alterations in Transmembrane Helices Probed by Multi-1 ns Molecular Dynamics Simulations, Journal ofBiomolecular Structure and Dynamics, 15:3, 555-572

To link to this article: http://dx.doi.org/10.1080/07391102.1997.10508966

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Insight into Signal Transduction: Structural Alterations in Transmembrane Helices

Probed by Multi-1 ns Molecular Dynamics Simulations.

http://www.albany.edu/chemistry/sarma/jbsd.html

Abstract

The hypothesis of structural alteration in transmembrane helices for signal transduction process is viewed by molecular dynamics simulation techniques. For the c-erbB-2 transmembrane domain involved in oncogenicity, the occurrence of conformational changes has been previously described as transition from the a to 1t helix. This dynamical feature is thoroughly analyzed for the wild phenotype and oncogenic sequences from a series of 18 simulations carried out on one nanosecond time scale. We show that these structural events do not depend upon the conditions of simulations like force field or starting helix coordinates. We demonstrate that the oncogenic mutations Val659 Glu, Gin and Asp do not prevent the transition. Furthermore, we show that ~ branched residues, in conjunction with Gly residues in the c-erbB-2 sequence, act as destabilizers for the a helix structure. 1t deformations are tightly related to other local structural motifs found in soluble and membrane proteins. These structural alterations are discussed in term of structure-activity relationships for the c-erbB-2 activating mechanism mediated by transmembrane domain dimerization.

Introduction

The neu/c-erbB-2 proto-oncogene encodes a 185 kDa tyrosine kinase receptor (TKR) exhibiting a high homology with epidermal growth factor receptor (I, 2). These receptors, which are part of the erbB family, are involved in signal transduction across cell membrane. They all possess a large glycosilated extracellular domain with two cysteine rich subdomains to which a ligand binds, a single hydrophobic transmembrane domain (TMD) and a cytoplasmic domain housing the tyrosine kinase activity. Ligand binding to the extracellular domain triggers the activation of the kinase domain (3). An oncogenic point mutation Val664 Glu was identified in the rat neu TMD (4) but was not found to be associated with human neoplasia for which the human protein c-erbB-2 is overexpressed (5). Nevertheless, directed mutagenesis experiments have shown that the c-erbB-2 proto-oncogene can be also transformed in its oncogenic form by the analogous amino-acid substitution Val659 Glu. This site, Val659 for c-erbB-2 or Val664 for nue, embedded in a specific intramembrane sequence (6) is probably involved in wild receptor dimerization that mediates activation. Also, at this site, only substitutions by Glu, Gin or Asp were found to induce the transforming phenotype whereas substitutions by Gly, Lys, His or Tyr lead to the wild phenotype (7, 8). These mutation experiments tested on neu and c-erbB-2 show that both the position and the nature of the substituting residue are critical for receptor activation.

The high specificity of the mutation in neu/c-erbB-2 TMD has retained much attention to explain the role of the presumed a helical sequence in receptor activation and dimerization. Several hypotheses have been suggested to explain the critical function of amino-acids 664/659 in the neu/c-erbB-2 activation. One hypothesis is based on the structural alteration of the TMD a helix. From molecular mechanics

Journal of Biomolecular Structure & Dynamics, ISSN 0739-1102 Volume 15, Issue Number 3, ( 1997) ©Adenine Press ( 1997)

Jean-Pierre Duneaul*, Norbert Garnier1,2 and Monique Genestl* ICentre de Biophysique Moleculaire,

CNRS, Rue Charles Sadron,

2Universite d' Orleans,

45071 Orleans Cedex 2 France

*Authors to whom correspondence should be addressed. Phone: 33 2 38 25 76 68; Fax: 33 2 38 63 15 17; E-mail: duneau @cnrs-orleans.fr; geneste@cnrs-orleans.fr 555

Duneau et a/.

studies, Brandt-Rauf and coworkers (9-11) proposed that a sharp bent found in non transforming TMD could hinder the dimerization process and that the whole a helical structure, preferentially displayed by the transforming mutated sequence, is needed for driving the dimerization process. Besides, in our laboratory, molecular dynamics (MD) simulation methods have been used to determine whether mutations could influence the dynamical behavior of this c-erbB-2 transmembrane domain. On a 100 ps time scale, a flexible behavior was observed for the transmembrane domain of the wild receptor whereas a more rigid a helix was found for the Glu transforming mutant (12). Another hypothesis, based on stereochemical modelling of TMD association, postulates that dimers constituted of a helical peptides are stabilized by additional interhelical hydrogen bonds (HB) involving the protonated Glu side chains (13). This later hypothesis has been recently supported by Cl3 NMR experiments (14) and theoretical investigations of coiled-coil structures of c-erbB-2 TMD (15). Both hypotheses are not necessarily opposed and this last study also shows that intramolecular and intermolecular processes may act cooperatively.

The TMD dynamics was studied by performing further MD simulations on the Val659 Gly non transforming mutant of c-erbB-2 showing that the flexible behavior results from events that induce a cooperative transition from a well defined a helix to a helix containing several successive 1t type (i~ i+5) HB, both at the mutation site and in its middle ( 16). We were the first to thoroughly describe this behavior for transmembrane helices and later this has been also reported for soluble peptides (17). Such a conformational transition observed on a short time scale (100 ps) and characterized as a rare event was not sufficient to establish relationships between the dynamical behavior of the peptides and the phenotype of the whole receptor.

The first aim of this work is to firmly establish the existence of the a ~ 1t transition in c-erbB-2 TMD. A series of 1 ns MD runs was undertaken for the Val659 Gly mutant under different conditions to examine possible artifacts of simulations. Several parameters have been tested, the most important being the force field used for calculating interatomic interactions. We therefore compare simulations carried out with two force fields namely GROMOS (18) and CHARMM (19). We have also checked that constraining bond lengths with the SHAKE algorithm (20) does not modify the results of the simulations. Since they are performed on TMD, a small protein portion, we have compared the results obtained on free helices that ignore the rest of the protein and those obtained on helices constrained at their extremities that approximate its presence. Finally, in order to get a better sampling of the phase space, different sets of coordinates and velocities have been used. None of these parameters inhibits the formation of the a ~ 1t transition.

Evidence of 1t HB patterns in helical structure led us to compare, in a second point, the dynamical behavior of 4 other TMD sequences differing only by one residue at the mutation site in c-erbB-2. One is the wild sequence (Val659) and the three others are mutated sequences Val659 Glu, Asp or Gin conferring the oncogenic phenotype of the whole receptor. Several runs performed under different conditions also show the appearance of the a ~ 1t transition possibly modulated by the mutation.

Additionally to the biological interest of studying models for signal transduction mechanisms, the study of TMD sequences also raised the question of the stability and accommodation of helix breaker and ~ strand former residues in membrane environment. Among the 22 hydrophobic residues of the c-erbB-2 TMD, 12 ~ branched residues, known as inducers of extended conformation, are often found in successive arrangement and associated with Gly residues. A structure prediction using the statistical methods of Chou and Fasman (21) gives this sequence predominantly as an extended structure. This prediction can be related to experimental studies that indicate that both lie and Val residues have a helix destabilizing effect in aqueous solution (22-29). However, by circular dichroism experiments,

Deber and coworkers have shown that in micelle and membrane environment, these residues may accommodate within helical structure although the ellipticity is sequence-dependent (30-33). In particular, Gly residues can obviously affect peptide helicity (34 ).

As a third point of this paper, the role of the ~ branched residues has been examined relatively to their possible role in the a~ 1t transition. The c-erbB-2 TMD a helix stability has been analyzed from two I ns MD simulations of the modified Val659 Gly c-erbB-2 TMD in which the 6 remaining Val residues were substituted by 6 Ala residues.

Methods

The TMD of the c-erbB-2 receptor comprises 29 amino-acids including residues 651 to 679 of the whole protein. The sequence of the model peptide is:

ACE-Leu L Thr-Ser-lle-lle-Ser-Ala-Val-Xaa9-GJy-lie-Leu-Leu-Val-Val-Val- LeuGly-Val-Val-Phe-Gly-lie-Leu-lie-Lys-Arg-Arg-GJn29_ NME

Acetyl (ACE) and N-methyl amide (NME) groups are added to theN- and C-terminus respectively and ensure uncharged ends. Xaa denotes the mutation position 659 of the whole protein and corresponds to position 9 in the peptide model.

5 peptides have been studied with Xaa=Val (wild transmembrane sequence), Gly, Glu, Asp and Gin respectively. Glu and Asp are considered in the protonated state. A last peptide designed to study the role of the ~ branched residues was built by replacing the 6 remaining Val residues of the Gly mutant by 6 Ala residues. This peptide will be referred to as 6V-6A.

The a helical structure for each peptide was built using the SYBYL program (35) with the 4> and \jf backbone dihedral angles set to -57° and -47° respectively. The X side chain dihedral angles are those of the SYBYL library. This gave a first initial coordinate set termed CO. A second set of initial helical coordinates (termed Cl) was built with the Xl dihedral angle values taken from the backbone independent rotamer library of Dunbrack and Karplus (36, 37). These values are reported in Table I.

Table I xi side chain dihedral angles (in degrees) in CO and C1 initial coordinate sets.

Leu Thu'Ser Ile Val Phe Lys Arg Gin Asp Glu co 180 45 60 60 70 60 60 60 70 60 C1 -60 60 -60 -180 -60 -60 -60 -60 -60 -60

Each initial structure was submitted to 200 steps of steepest descent energy minimization for removing steric contacts. Minimization and MD were performed using the GROMOS software (18) or the X-PLOR software (CHARMM force field (38)), considering explicit polar hydrogen atoms but aliphatic hydrogen as united atom. In vacuo calculations were carried out with a relative dielectric constant of 1. No cutoff distance was applied for van der Waals and electrostatic interactions. Basic residues in the C-terminal juxtamembrane portion were considered without net charges owing to the absence of explicit water at the membrane surface but their hydrogen bonding ability is maintained. The peptides are globally uncharged.

For all MD simulations using GROMOS, the same heating-equilibrium protocol has been applied. From the minimized structure, the temperature was increased up to 300 K by 50 K steps, each step during 4 ps with atomic velocities reassigned every 0.2 ps. This was followed by a 150 ps equilibration period using a strong coupling to a thermal bath for the first 50 ps (relaxation time equal to 0.01 ps) and a weak coupling (relaxation time equal to 0.1 ps) for the remaining 100 ps. During

Insight into Signal Transduction

Duneau et a/.

the heating-equilibration period, end fraying was reduced by constraining the first turn of the helix and the last turn to keep close to the a structure, but helix bent and length changes are allowed. The structural constraints approximate the structure of the transmembrane helix extremities within the whole protein which obviously does not exhibit free ends. The peptide dynamics has been examined by performing simulations either with or without these terminal constraints. Pairs of atoms involved in the applied distance restraints are given in Table II. The force constant value of 5000 kJ.moJ-I.nm-2 used leads to terminal interatomic distance fluctuations magnitude equal to those observed in the inner helix.

Table II Pairs of atoms involved in distance restrained potential applied to maintain the structure of the first a helix tum and the last a helix tum along the MD simulations.

Helix end Atom I Atom 2 Distance (nm)

N-terminal CaThr2 C(CO)ACE 0.44

CaSer3 C(CO)ACE 0.47

CaLeul CaSer3 0.57

O(CO)ACE H(NH)Ile4 0.20

C-terminal CaArg27 CaGln29 0.57

CaArg27 N(NH)NME 0.52

CaArg28 CaGln29 0.46

0 (CO) Ile25 H(NH)Gln29 0.20

Simulations performed with X-PLOR were carried out following the same heatingequilibrium procedure using CHARMM force field parameters (explicit polar hydrogen). However the equilibration period has been reduced to 50 ps using a strong coupling to the thermal bath (friction parameter set to 100 ps-1). The same distance constraints were imposed at helix extremities using a square well potential function.

With both softwares production periods ranging from 500 to 1500 ps were carried out with coordinates and energy values stored every 0.4 ps. Tumbling of the whole molecule was removed at the first production step and removed again every 30 ps. A weak coupling to the thermal bath was applied with a coupling constant value of 0.1 ps when using GROMOS and a friction parameter equal 10 ps-I when using X-PLOR.

Several trajectories were produced starting either from initial coordinate sets CO or C 1, varying initial velocities, maintaining or not distance restraints at helix ends or applying or not the SHAKE algorithm. The time increment was 0.002 ps when SHAKE was used and 0.0005 ps otherwise. Five simulations were produced for the Gly mutant referred to as MDG1,2,3,4,5. Two simulations were produced for the wild sequence, noted MDV6,7 and three for each of the Glu, Gin and Asp mutants noted MDE8,9, 10, MDQ11, 12,13 and MDDJ4, 15,16 respectively.

Dynamics and structure analyses are based on energy time series, average dihedral angle values and a detailed examination of hydrogen bond formation. The criteria for the HB formation correspond to a donor (D) -acceptor (A) distance smaller than 0.35 nm and a minimum value of 130° for the DHA angle. These large limits allow to detect weak HB including bifurcated HB formed between one acceptor (CO) and two donors (NH). Residue numbers i- j will denote a COi-NHi HB and residue numbers j - i - (j+ 1) will denote a bifurcated NHrCOi-NHi+ 1 HB in the following text.

In order to emphasize amplitude variations in energy and HB changes, a smoothing technique has been applied for the plots of the corresponding time series. Each point of a time series is replaced by the average calculated over a window of 2n+ 1 values cen-

tered on this point. All calculations were perfonned on Silicon Graphics workstations. 559

Results Insight into Signal Table III summarizes the results of all 1 ns simulations perfonned on the different sequences along with the simulations conditions.

Table III Condition of simulations, n; hydrogen bond pattern and energy changes for the wild sequence (MDV6,7) and the different Val659 mutants of c-erbB-2 transmembrane domain (the residue at the mutation-site appears in one letter code as the upper index and the number gives the simulation order (see text)). +or- : terminal constraints are applied or not. i-j numbers denote backbone hydrogen bond (HB) between the atom accep-tor of residue i and the atom donor of residue j.1[ :simulation performed with the coordinate set Cl (see table 1). ± :SHAKE algorithm not applied. 6E : potential energy difference between the two states observed before and after the conformational transition.

Force Field Simulation Terminal Initiation HB nb of n; first n; constraints time (ps) initiation site HB HB

GROMOS MD0 1 500 5-10 16 2-7

MDV6 500 17-22 X> 3-8

MDE8 200 17-22 16 2-7

MD"14 30 23-28 21 3-8

GROMOS MD0 2 + 16 17-22 Z2 3-8

MDG31[ + 700 17-22 7 11-16

MD0 4± + 900 14-19 15 3-8

MDV7 + 100 7-12 4 5-10

MDEg + 0

MlflO + 550 17-22 15 3-8

MD"15 + 0

MD"16 + 350 24-29 Z2 3-8

CHARMM MD0 5 300 17-22 14 5-10

MD0 11 0

CHARMM MD0 12 + 50 13-18 14 4-9

MD0 13 + 800 13-18 4 10-15

Gly Mutant

Free Dynamics (MDC 1) Figure 1 shows the time evolution of the potential energy of the Val659 Gly mutated TMD from a free MD simulation.

Over the first 500 ps, the system fluctuates around an average energy value of -1170 kJ.moJ-1 with no significant drift showing a correctly equilibrated system. An increase

in potential energy of 50 kJ.moJ-1 occurs between 600 and 700 ps followed by the relaxation of the system towards a state with a potential energy 50 kJ.moJ·IJower than that of the initial state. Such a profile, closely related to that mentioned in our previous study (16), strongly suggests the occurrence of a confonnational transition.

Figure 2 shows the time series of both i~i+4 and i~i+5 HB occurrence all along

last n; Llli HB (kJ.mol" 1

17-22 -50

22-27 -70

17-22 -30

24-29 -100

24-29 -80

17-22 0

17-22 -30

8-13 0

17-22 ..()()

24-29 -90

17-22 0

17-22 -30

13-18 0

Transduction

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Figure 1: Time evolution of the potential energy of the Val659 Gly mutant of the c-erbB2 transmembrane domain (MDGI ). A smoothing technique has l~cn applied to the curves with a window of 21 elenlcnts to discard short time energy fluctuations (see Methods).

0 E -1200 -~

- 1 400 o~---'----=2=-=o=-=o=--..____-=-4-=o-=o----'-s~o~o=--.....~...-.....,a:-'oL:o=---'---1~ooo

time (ps) the simulation (no other HB type is detected at a meaningful levtel). It can be observed that the HB network stays in the standard i~i+4 a helical pattern during the first half of the simulation. Then, from 500 to 700 ps, a cooperative mechanism leads to the successive replacement of i~i+4 HB by the related i~i+5 HB from 2-7 until 9-I4 with the CO GlyiO excluded from the HB network. This pattern includes 8 successive i~i+5 HB and covers the mutation site. It is related to a local distortion termed 1t bulge. The C-terminal boundary of this deformation is strongly similar to a structural motif observed in protein databases called a aneurism (39). In both motifs, a Gly residue is part of the interface between the i~i+5 and the i~i+4 HB network and corresponds to GlyiO in MDGJ. The free CO Gly/10 state lasts 100 ps until 775 ps before it incorporates into a i~i+5 HB (I5-20). This provides the i~i+5 HB propagation towards the C-terminus in a few ps until the exclusion of the Glyi8 CO group from the HB network. Figure 3 gives snapshots of the helix structure along the simulation showing in particular the 1t bulge/a aneurism deformation at 570 and 700 ps. The conformational changes give rise to a large 1t helix including I61t HB as illustrated by the last snapshot in Figure 3. The structural characteristics of the conformation averaged over the last 200 ps and minimized are given in Table IV. The mean values of the parameters have been calculated for the residues involved in the 1t HB pattern both with their amide and carbonyl groups (residues 7 to I7).

It is important to underline that the mean values of <j> and 'If given in Table IV for MDGI are not those of a canonical1t helix structure. Indeed, the helix built with these dihedral angles gives rise to a 1t structure with a pitch reduced by I A per turn, totally inconsistent with proper van der Waals interactions. These different 1t structures come from the deviations in the <j> and 'If dihedral angles with respect to the mean values (10 degrees for Gly and 5 degrees for other residues).

The lower rise of the MDG I 1t portion results in a decrease of the length of the whole helix from 4.5 to 4.0 nm and an increase of the helix diameter of 0.05 nm.

Table IV Structural parameters for 1t and a helices. «!>> and <\jl>: mean values of backbone dihedral angles; q : unit twist (in degrees); h : unit height (in A); p : pitch (in A); n : residue number per tum.

Helix type «I» <\jl> 8 h p n

1t helix 60 -57.1 -69.6 82 1.15 5.060 4.4

MD0 I (n portion) -64.5 -65.7 ID 1.15 5.175 45

a helix 61 -57 -47 100 1.5 5.5 3.6

a helix in globular protein 62 -62 -41 5.4 3.54

In this structure, the residues i and i+8 are exactly separated by two helix turns so that their side chains are eclipsed when the helix is observed along its axis.

The PROCHECK ( 40) Ramachandran plot displayed in Figure 4 shows that this 1t

type helix stands in the most favoured area of the diagram except for residues Val8 and Valli which are located at the boundary with the additional allowed region. The two Gly residues (Gly9 and GlylO) are slightly shifted from the zone where the other residues converge. As observed by Creighton ( 41 ), the cavity measured in the interior of the helix as the excluded volume between van der Waals spheres is larger in the 1t structure than in the a one. The en tropic cost of this feature is unfa-

Figure 2: Time series of the existence of .main chain HB between the donor of a residue i and acceptors of residue i+4 (a HB type) and i+5 (7t HB type) in the MDGJ simu· lation. In ordinate, the value I characterizes the existence of the i -7 i+4 HB and the value -I characterizes the existence of the i -7 i+5 HB. For both HB types the ordinate value of 0 characterizes the absence of HB. Intermediate values exist owing to the application of a smoothing technique on all time series (window of II elements).

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vorable in aqueous environment but not in a membrane medium.

Influence of Constrained Extremities (MDG2) MDG2 was produced from the same initial coordinate set as MDG1 (CO) but with distance constraints applied at helix extremities (see Methods section). Two distinct periods are observed in the time series of the potential energy plot over 1 ns (not shown). One covers the first 350 ps and is characterized by a mean energy value of -1150 kJ.moi-1 and the other covers the rest of the simulation and corresponds to a potential energy state 80 kJ.moi-1 lower.

Examination of the HB formation (data not shown) evidences that an a~ 1t helix transition occurs. It starts very early (near 16 ps) and leads to the formation of a 1t

bulge from 13-18 to 17-22 during the first 75 ps. Then, a rearrangement in the HB pattern leads to a displacement of the 1t bulge from 10-15 to 13-18. Near 250 ps, the deformation extends towards the C-terminus without stopping at the Gly 18 position and leads to the 24-29 1t HB in the last 200 ps. Similarly, at step 350, the 1t deformation extends towards the N-terminus until the 3-8 1t HB. So, the transition has covered almost the whole helix giving rise to the most important stretch of 1t helix we have never encountered in our simulations.

Influence of Initial Conformation (MDGJ) Using the same conditions as for MDG2, we have performed another 1 ns simulation with a new set of initial coordinates (C1) with different side chain rotamers (see Table 1). A structural transition occurs at 700 ps and leads to a it bulge from 11-16 to 17-22, the CO group ofGly18 being free. No change in the potential energy is associated with this transition. This state prevails until the end of the simulation.

Influence of Bond Length Restraints (MDG4) A fourth simulation has been carried out using the set CO as initial coordinates but without applying the SHAKE algorithm. Under such conditions, the a ~ 1t helix transition occurs at about 900 ps, giving rise very quickly to a 1t deformation from 3-8 to 17-22. At the transition time, the potential energy exhibits a peak of 50 kJ.mol-1. To evaluate the mean energy after the transition the simulation has been extended until 1500 ps. We observed that the helix lies at an energy level30 kJ.mol-1 lower than that of the whole a helix conformation.

Influence of the Force Field (MDG5) Although the GROMOS force field has been extensively used by many authors to characterize the structure and the dynamics of proteins we verified that the spontaneous transition from a to 1t helix on large peptide portions is not an artifact of the force field. Therefore a 1 ns MD simulation was produced with the CHARMM force field used in X-PLOR starting from the initial coordinates set CO (see Table 1).

An a ~ 1t helix transition is still observed and is initiated at 300 ps. It obeys to the same mechanism as already described for the GROMOS simulations. The initiation of the transition takes place at the Gly 18 site with the 18-22 HB disruption followed by an elongation towards the N-terminus until the 5-10 HB formation. No significant energy variations are measured during the process.

Wild type and Oncogenic Sequences

Multiple MD simulations have been undertaken on the wild sequence, and on the Glu, Gin and Asp mutants. All the simulations have been carried out with the same initial coordinate set CO and SHAKE was applied. The other conditions of the simulations along with the results are summarized in Table III.

Wild Peptide 2 simulations were performed, one on the free helix and the second on the end con-

strained helix. For the free MD, we observe the initiation of the transition a few steps before 500 ps. 20 ps are sufficient to lead to a 1t helix which extends from 3-8 to 17-22. Then, a further propagation occurs near 675 ps towards the C-terminal side until the 22-27 HB. For the constrained simulation a 1t bulge appears at 100 ps ( 4 1t HB from 5-10 to 8-13) but fails to elongate.

Glu Mutant Concerning the Glu protonated transmembrane mutant, a 500 ps unconstrained MD (MDE8) was sufficient for the appearance of a 1t helix from 2-7 to 17-22.. The initiation step occurs at 200 ps and the 1t elongation lasts 50 ps. No further elongation have been encountered towards the C-terminus after the Glyl8 residue but the simulation has been stopped owing to a failure of the SHAKE algorithm caused by its inefficiency to compensate an unconstrained step perpendicular to the plane of a planar group at the C-terminal extremity ( 42). Another MD run with constrained helix extremities (MDE9) does not give rise to the transition. The a helix remains stable all along the production period. Nevertheless, in a third simulation starting from a different set of initial atomic velocities (MDE I 0), a short 1t helix appears from 11-16 to 17-22 after 525 ps. 230 ps later, it elongates towards theN-terminus in less than I 0 ps until the 3-8 1t HB formation.

Gin Mutant X-PLOR was used for this mutant. The Gin side chain is structurally and fonctionnally very close to that of the Glu protonated side chain and the use of CHARMM force field for this mutant allows usefull comparisons with GROMOS.

Three simulations have been performed, one considering helix extremities free of any constraints (MDQll), the two others with end constraints using two different values of the restraining force constant. In the unconstrained simulation, the a helix remains stable except for a short time from 550 to 575 ps during which a transient 5 HB 1t bulge appears from 14-19 to 18-23. When weak constraints are applied (MDQ12), leading to a constrained potential energy of 3-4 kJ.mol·I, the system undergoes a transition which starts at 50 ps leading to a 1t stretch from 4-9 HB to 17-22 HB (MDQ9) 150 ps later. When constraints are reinforced by a factor 4 (MDQ13) an a~ 1t transition is recorded at time 800 ps corresponding to 4 1t HB between 1 0-15 and 13-18.

Asp Mutant The unconstrained simulation produced with the GROMOS force field on the Asp mutated peptide (MDDI4) results nearly in a whole 1t helix structure as earlier as 120 ps. The initiation of the phenomenon occurs in the first 30 ps at an unusual site corresponding to the C-terminal25-29 HB. Then the elongation occurs very quickly as a zip from 24-29 to 3-8. Two other I ns simulations have been carried out with constraints. In one case (MDDI5) i~i+4 HB are disrupted at the C-terminus but no transition is observed. This result shows that the distance restrained potential is sufficiently weak to allow deviations from the a helix conformational space. The structural change at this extremity is accompanied by a decrease of 40 kJ.mol· I in potential energy. In the other case (MDDI6), i~i+4 HB disruptions arise during the first 100 ps but i~i+5 HB appear only 250 ps later starting from 24-29 and progressing towards the N-terminus until 3-8.

Transition and Stability

Energy The potential energy variation associated to the conformational changes were analyzed and two major observations can be made: (i) From Table III we remark that when 1t

helices cover more than 15 HB, the potential energy state is lower than that of the entire a helix. This difference reaches -100 kJ.moJ-I for MDDJ4. When less than 14 HB are formed no difference is detected. This is illustrated in Figure 5. (ii) The transition period is not necessarily associated to an energy barrier as observed for MDG I.

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Figure 6 shows the details of electrostatic and Lennard-Jones energy variations along the simulations that give rise to large 1t helices. The plots correspond to simulations performed for several mutants either with the GROMOS or the CHARMM force field. It emerges that energy changes are correlated with the conformational transition. In Figure 6a, energy plots relative to MDG5 (CHARMM) show that the electrostatic energy increase by 40 kJ.mol-1 but this change is over compensated by a gain in Lennard-Jones energy (-70 kJ.mol-1). In the case of MDV6 (Figure 6bGROMOS) both electrostatic and Lennard-Jones energy variations contribute to the conformational stabilization. In the case of MDD16 (Figure 6c - GROMOS), the change in Lennard-Jones energy is strongly stabilizing (-100 kJ.mol-1) while electrostatic energy does not evidence large variations. Finally, another example concerns MDEIO (Figure 6d) for which electrostatic energy fluctuations are large but do not lead to a distinct energy level. The decrease in Lennard-Jones energy occurring at 700 psis about 70 kJ.mol-1.

With the GROMOS force field, the Lennard-Jones interactions are the strong stabilizing component and the electrostatic potential does not change during the transition. For the simulations carried out with X-PLOR the Lennard-Jones energy stabilization is still important but the electrostatic energy displays unfavourable variations.

Role of {3 Branched Residues: 6V-6A Peptide A first 1 ns simulation on the Val659 Gly TMD in which all the Val residues have been substituted by Ala was performed under the same conditions as MDG2. During this simulation, the potential energy fluctuates around -1150 kJ.mol-1 and no drift is observed. As expected, the systematic substitution of the Val ~ branched residues results in a stabilization of the a helical conformation along the simulation. A detailed analysis of i~i+4 and i~i+5 HB formation confirms the a helix stability. However, the occurrence of a transitory 1t deformation that results in two 1t HB (3-8 and 4-9) is observed between 475 and 500 ps. The observation of this reversible 1t bulge was newly exemplified in a second simulation carried out under the same conditions but with a different set of initial atomic velocities. In that case, the reversible deformation appears four times at 150 ps, 715 ps, 830 ps and 960 ps. It can be related to the presence of two successive ~ branched Ile residues at position 4 and 5 in the sequence shown in Figure 7. Discussion

The aim of the present work was to assert that 1t HB type exist in the transmembrane domain of c-erbB-2. This structural feature in a helices was previously reported from 100 ps molecular dynamics simulations for wild phenotype sequences (wild sequence and Val659 Gly mutant), but not for the oncogenic Val659 Glu mutant (12, 16). Clearly, starting from a helical structures, a local deformation called 1t bulge was sometimes observed. However, this a ~ 1t transition was reported as a rare event, so that no conclusions could be drawn from these first observations. Then, it was therefore of interest to perform other simulations on a longer time scale by varying initial conditions to complete a best statistics. This led us to carry on 16 MD runs on the nanosecond range on five different mutants.

For 13 runs over 16 the a~ 1t transition is detected showing that on 1 ns time scale this structural phenomenon is rather frequent. In some cases 1t HB patterns may cover nearly all the peptide, but more generally it extends from 3-8 to 17-22 with the Glyl8 CO group excluded from the HB network. 1t helices result from a deformation often initiated in the central helix part, or more sparsely at the level of the mutation point. For this latter case we verified, from HB time series, that the transition is not triggerred by hydrogen bonds between the hydrophilic side chains and the backbone. Hydrogen bonds of this type do not exist at a significant percentage ( <1% ). The mechanisms for the initiation and the elongation of the deformation are

Figure 3: Snapshots of instantaneous confonnations taken from the MDG I simulation. a helix portions are colored in cyan and 1t helix portion is colored in yellow. The location of the Phe ring is chosen as an example to illustrate the changes of residues along the helix faces induced by the 1t defonnations.

Figure 4: PROCHECK Ramachandran plot (40) for residues involved in the 1t helix portion during the MOO I simulation (structure averaged over the last 200 ps of the trajectory). Area A, B, L correspond to the most favoured regions, a, b, I, p are the additional allowed regions and -a, -b, -1, -p are the generously allowed regions. Filled triangles represent the Gly residues and filled squares represent the other residues.

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Number of 1t hydrogen bonds completely similar to those previously reported (16).

1t helix formation is not related to possible simulation artifacts. Indeed, among the different parameters that have been tested (force field (GROMOS and CHARMM), SHAKE algorithm, initial coordinates and velocities, structural constraints at helix extremities) any of them prevent from the occurrence of the a~ 1t transition. We show that it is rather sequence-dependent and particularly induced by the ~ branched amino acids.

Another important point is the vacuo approximation used to examine the dynamics of transmembrane peptides. As known, the core of the membrane is a low dielectric constant and a weak viscosity medium (43) and thus, vacuo approximation, also applied by other authors ( 44-45), seems correct. The lipidic environment, essentially that of the hydrocarbon chains, result on dumped motions owing to van der Waals interactions without electrostatic contributions. But at the interface with head groups of lipids and water molecules, hydrogen bond interactions with helix extremities may interfer with the intrinsic dynamics of the peptide particularly when hydrophilic side chains are present. Undoubtly electrostatic contributions and the potentiality of hydrogen bond formation in this polar environment is not well approximated by vacuum conditions. Consequently, it remains difficult to speculate on the significance of the length of the 1t deformation especially for large 1t helices initiated at the level of the polar interfacial residues. However, 1t HB observed for the different sequences are frequently initiated in the middle of the helix where electrostatic is negligible when it is embedded in the membrane. This is in line with the first results of simulations of the wild type sequence in explicit membrane (work in progress).

Stability of the Deformed Helices Our analysis shows that larger is the 1t deformation, stronger is the van der Waals stabilization of the system. However, this may result either from a continuous van der Waals energy gain with the length of the 1t bulge or from a sudden release of internal constraints proper to the a helix. This last hypothesis requires a minimum number of 1t HB before to lead to a complete stabilization. This is observed in the MDE9 simulation for which 7 1t HB are formed during 150 ps without affecting the

Figure 5: Potential energy difference between the a helix in the initial state and the structure that prevails after the transition versus the number of i ~ i+S HB in the 1t portion. Data are given for the 16 simulations of the wild and mutated peptides performed with the GROMOS (18)or the CHARMM (19) force fields.

568 Figure 6: Smoothed time series (window of 21 elements) of electrostatic and van der Waals energies for simulations leading to the formation of large 1t helix portions (more than 14 i ~ i+5 HB).

Duneau et a/.

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1----------------1 van der Waals energy. A strong stabilization arises when 8 additional 1t HB are formed. As a general fact, short 1t helix portions do not change the energy of the peptide. Unfavourable interactions at the a/1t junctions probably need to be readjusted before this gain in stability.

~ branched residues are reported to destabilize a helical structures (26) owing to steric hindrance or en tropic considerations (22, 23, 28). As shown in Figure 8, these residues, inducers of extended structure, are present in successive arrangements in the c-erbB-2 sequence (Ile4-Ile5, Val8-Val9, Vall4-Val15-Vall6, Vall9-Val20) and are flanked by Gly residues at positions 10, 18 and 22 in the wild sequence. Nevertheless in low dielectric media such as membranes, a backbone HB network has to be fully formed to ensure an optimal stability balancing the destabilizing effect of ~ branched residues. The 1t helix, with larger turns than in a helix, could be a good compromise to account for both effects. This hypothesis is supported by the results obtained on the Val659 Gly sequence where all Val residues have been replaced by Ala residues. In that case, no stable 1t deformation is observed from two I ns simulations except a short bulge, temporarily detected at the level of the remaining~ branched residues Ile4 and Ile5.

7r Helix in Literature 1t helix formation has been seldom mentioned in molecular dynamics studies of polypeptides. We have reported the existence of such 1t HB in the study of the cerbB-2 TMD (12) and these patterns have been characterized in a recent work (16). Kovacs et at. ( 46) also described an a ~ 1t helix transition on the highly hydrophobic sequence from the non-palmitoylated pulmonary surfactant lipoprotein (SP-C) in agreement with circular dichroism and NMR data (47, 48) that suggest a less helical structure than the native one. This transmembrane peptide shows some similarities with c-erbB-2 since it contains successive arrangements of Val residues interrupted by Gly residues. Recently, lyer and Vishveshwara (49) performed molecular dynamics simulations on isolated helices of Bacteriorhodopsin using the AMBER 4.0 force field. Helices of this membrane protein also contain numerous ~ branched and Gly residues. The authors do not explicitly give details on the structures, but they present helix snapshots that undoubtly include a significant amount of 1t turns. These observations confirm that 1t HB pattern is not force field dependent. Very recently, results of MD simulations on the calmodulin central helix show 1t HB near a site involved in the helix flexibility (50).

Figure 7: Central position of lie residues in the transitory 1t pattern observed in the 6V-6A peptide. The dotted lines figure out the two 1t HB.

Figure 8: Helical network (a) and helical wheel (b) representations for the wild c-erbB2 transmembrane sequence from Ser 3 to Leu 25. The ~ branched residues are circled.

Duneau et a/.

Although few high resolution structures of membrane proteins are available, several cases of 1t deformations have been reported in helical TMD. In the Photosynthesis Reaction Center (chain M and chain C) 16 1t turns are present in the crystal structure (51). Another case concerns the X-ray structure of the 13-subunits oxidized Cytochrome c Oxidase (52) which reveals 1t hydrogen bonding in the helices located at the dimer interface (Figure I B in ref 52 and personal communication). As a last example, the NMR structure of the TMD of subunit C from the H+-transporting FIFO ATP synthase (53) (entry name IATY in the Brookhaven Protein Data Bank (54)) shows significant amount of 1t HB.

Small 1t helices have also been experimentally observed for soluble proteins such as streptococcal nuclease (55) and soybean lipoxygenase-1 (56) or cytochrome P450 (57). Other structures related to 1t deformation and termed looping-out (58) or <X aneurism (39) can be found in protein structure databases. A compilation of a representative set of protein structures made by Rajashankar and Ramakumar (51) reveals a great number of 7t-turns in proteins. Some of them occur as distortion in the middle of <X helices. Thus, it emerges that 1t helix hydrogen bonding, often considered as a hypothetical structure, is in fact present in many proteins.

Biological Implications A small1t HB pattern may have important biological consequences for the function of the whole receptor. First, the properties of the helix faces are considerably modified once the <X ~ 1t transition is established. Thus, interactions between transmembrane sequences mediated by dimerization can be strikingly different for full <X helices and for helices containing 1t deformations. Second, a protein domain attached to one extremity of the intramembrane sequence (as the kinase domain in TKR) will be rotated relatively to the other domain attached to the opposite extremity with an angle depending upon the 1t helix length. In a dimeric complex such a rotation may also change intermolecular interactions between extramembrane domains. Finally, the lower rise of the 1t helix compared to that of the a helix reduces the length of the TMD by 0.35 A per residue. Each of these structural changes could account for an efficient mechanism for the control of tyrosine kinase activity.

The existence of such a 1t bulge has also been observed at the mutation site in cerbB-2 dimer models (15). Similarly, the helices that exhibit the 1t deformation in the 13-subunits oxidized Cytochrome c Oxidase (subunits VIa of the complex) are the main components of the dimer interface. From these two examples it could be suggested that the flexibility brought by 1t deformations could help for optimizing transmembrane packing at the dimer interface.

Regarding the difference between the dynamics behaviors of the V659 mutants, relationships between oncogenicity and dynamics effects can not be clearly established. Nevertheless, it is worth to note that among 16 simulations, 3 do not exhibit the 1t structural deformation and correspond to the three oncogenic sequences Glu, Asp and Gin, although it is obvious that these sequences may undergo the <X ~ 1t transition. However, the statistic is not sufficient to validate the tendency for oncogenic phenotype sequences to prevent for a helix deformation.

Monitoring Transition by MD Finally, the present study newly exemplifies the ability of molecular dynamics simulation to monitor structural transition phenomena that otherwise would be ignored with conventional experimental techniques. Indeed, the fact that 1t helices could arise in specific transmembrane sequences allows to design specific experiments for testing this finding. For example, Electron Spin Resonance experiments have been recently and successfully associated to MD to follow 3-10 /a helix equilibrium in model peptides (59).

Acknowledgment

We thank D. Genest for helpful discussions. We also thank Eiki Yamashita for the data on the helix deformation in the Cytochrome c oxidase structure and the CITU from University of Orleans for computational facilities.

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Date Received: October 10, 1997

Communicated by the Editor Rick Ornstein

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