Insights into the Packing Switching of the EphA2 Transmembrane

May 29, 2015 - ... the C-terminal domains, which expressed a mighty binding potency ...... and Stockholm, and the Max Planck Institute for Polymer Res...
0 downloads 0 Views 5MB Size
Download PDF
Article pubs.acs.org/JPCB

Insights into the Packing Switching of the EphA2 Transmembrane Domain by Molecular Dynamic Simulations Fude Sun, Lida Xu, Peng Chen, Peng Wei, Jing Qu, Jialin Chen, and Shi-Zhong Luo* Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China S Supporting Information *

ABSTRACT: Receptor tyrosine kinases play an important role in mediating cell migration and adhesion associated with various biology processes. With a single-span transmembrane domain (TMD), the activities of the receptors are regulated by the definite packing configurations of the TMDs. For the EphA2 receptor, increasing studies have been conducted to investigate the packing domains that induce its switching TMD dimerization. However, the inherent transformation mechanisms including the interrelations among the involved packing domains remain unclear. Herein, we applied multiple simulation methods to explore the underlying packing mechanisms within the EphA2 TMD dimer. Our results demonstrated that the G540xxxG544 contributed to the formation of the right-handed configuration while the heptad repeat L535xxxG539xxA542xxxV546xxL549xxxG553 motif together with the FFxH559 region mediated the parallel mode. Furthermore, the FF557 residues packing mutually as rigid riveting structures were found comparable to the heptad repeat motif in maintaining the parallel configuration. In addition, the H559 residue associated definitely with the lower bilayer leaflet, which was proved to stabilize the parallel mode significantly. The simulations provide a full range of insights into the essential packing motifs or residues involved in the switching TMD dimer configurations, which can enrich our comprehension toward the EphA2 receptor.

■

INTRODUCTION The association between the Eph receptor tyrosine kinases and their ephrin ligands has attracted much attention for its close correlation to cell migration and positioning in human organisms.1,2 As the largest group of the receptor tyrosine kinase (RTK) family, many studies have been reported to elucidate the underlying mechanisms of the signal networks of the Eph family since it was implicated with an increasing number of physiological and pathological processes in various cell types.3−6 Sharing the common structure characters of the RTK family, the Eph receptor consists of a single-span transmembrane domain, an extracellular region with a series of high-affinity ephrin binding sites, and a cytoplasmic tyrosine kinase domain.7,8 It is supposed that binding of ephrin situated on neighboring cells induces the formation of the receptor dimers or oligomers.9 Increasing evidence also suggests that some Eph receptors can assemble together even without the ligands,10 which corresponds to the phenomenon that the receptors are loosely preclustered under inactive conditions.11,12 The ligand-independent clustering is supposed significant since the additional ligand binding can further stimulate the signaling Eph-ephrin cluster formation which contributes to the activity of the Eph receptors. Recent studies proposed that the Eph receptors could adopt an active/inactive configuration to exert their kinase activity regulation, which was associated with the dimerization mode of © 2015 American Chemical Society

the single-span TM domain. There is growing evidence showing that TMDs act as indispensable regulators in conformational switching and signal transduction of the RTKs.13,14 Furthermore, advances in NMR and simulation research have efficiently expanded the investigation of the alternative dimerization of several RTKs, including some Eph receptors and EGFR.15−17 Combining coarse-grained and allatom molecular dynamic simulations, Reddy16 et al. revealed the TMD packing motif and key pathological site which mediated the primary dimerization mode of FGFR3. Chavent18 et al. well estimated two packing states with disparate stabilities of the EphA1 TMDs, which was consistent with the NMR structure and so-called rotation-coupled activation mechanism. As an important member of the Eph receptors, EphA2 has attracted much attention for its important biological roles and ligand-independent clustering characters. Recent studies, including NMR structure resolution and mutational experiments,7,17 elucidated that the TM domain of EphA2 could pack together in a switching manner corresponding to the alternatively active/inactive dimerization mode. Similar to EphA1, the interaction of N-terminal glycine zipper (GxxxG) motifs is considered to participate in the formation of the rightReceived: February 3, 2015 Revised: May 29, 2015 Published: May 29, 2015 7816

DOI: 10.1021/acs.jpcb.5b01116 J. Phys. Chem. B 2015, 119, 7816−7824

Article

The Journal of Physical Chemistry B

type and mutants were constructed by PYMOL32 with defined α-helix secondary structures. The constructed peptides were then transferred into CG models by the martini-1.2.py script. The force parameters that restrain the components adopted the definition of the martini_v2.1.28 All input materials are available on the Martini portal: http://cgmartini.nl/. A mimetic bilayer (size of 7.66781 × 7.66781 × 12.37246 nm3) composed of 128 dipalmitoylphosphatidylcholine (DPPC) lipids and 6400 solvent molecules was applied. The two TM peptides were inserted into the bilayer perpendicular to the membrane surface with an initial distance of 55 Å, and then the system was energy minimized to avoid the irrational atom overlapping. The components including the protein, DPPC, and solvent in the system were separately coupled at 323 K by a Berendsen thermostat33 with a time constant of 1 ps. Semi-isotropic pressure was applied at 1 bar with a time constant of 4 ps and a compressibility of 5 × 10−6 bar−1. The nonbonded Lennard− Jones (LJ) and electrostatic interactions were cutoff at 12 Å using a switching function between 9 and 12 Å for the LJ interactions and 0 and 12 Å for the Coulomb interactions, respectively. Periodic boundary condition was applied, and each simulation step was set as 20 fs. Each system ran for 3.0 μs in duration. Four parallel samples with rescaling initial velocities were carried on for each system. Atomistic Model. All initial protein structures for atomistic simulation were derived from the representative packing dimers stable in the CG simulations. The CG TM structures were transferred into the atomistic model by the Pulchra script.34 Then the AT model peptides were inserted into a preequilibrated lipid bilayer composed of 128 atomistic DPPC molecules. The Inflate GRO35 method was used to set up the feasible system; the water molecules defined with the SPC model were added. Then 1000 steps of steepest descent energy minimization of the system and 1 ns of molecular dynamics (MD) using a weak position restraint (103 kJ·mol−1·nm−2) on the non-hydrogen protein atoms were conducted. All simulations were performed in GROMACS 53a6 force field36 with the force constant of 1000 kJ·mol−1·Å2 to ensure the proper spatial limitation of the peptides. A semi-isotropic pressure of 1 bar with a coupling constant of 2 ps and compressibility of 4.5 × 10−5 bar was applied. The Berendsen thermostat was used to maintain the temperature at 323 K with a coupling constant of 0.1 ps. Electrostatic interactions were modeled at a cutoff distance of 9 Å using a particle mesh Ewald summation, and the van der Waals interactions were cut off at 14 Å. The bond lengths were restricted using the LINCS algorithm. The production simulations were performed in two independent duplicates for 100 ns. All trajectories were analyzed by the tools embedded in the GROMACS 4.5.5 software. Visualization and graphics were performed with VMD.37

handed helix dimer of EphA2, which is verified as the active conformation, while the left-handed dimer is determined by the packing of a so-called heptad repeat motif which has long scale distribution. The switching dimerization modes imply that the association of the EphA2 TM domains is not confined by a predominant interacting motif, and instead, it is the different structural propensity of the separated local motifs that results in the switching of the dimer configuration. For example, during the interconversion process between the active and the inactive states of the EphA1 TMD dimerization,18 a variety of dynamic intermediates and inherent packing regions emerge. As a result, the dynamic interacting mechanisms concerning the switching conformations and the transformation process deserved to be further investigated to better understand the alternative two modes of the EphA2 receptor. There are a number of experimental approaches to investigate the assembling intensity and transformation of the TMD dimerization, including TOXCAT assays,19−21 coimmunoprecipitation, and FRET methods.22 Nowadays, molecular dynamic (MD) simulation has been popularly applied for it enables us to follow and understand the structure and dynamics with extreme detail, literally on the atomistic level. With continuous development of computational power, it has become more practical to explore the associating mechanisms and dynamic structure features hidden in the macromolecule packing processes.23−26 In regard to EphA2 with putative alternative package mode, the simulations can provide valid approaches and microscope insights for us to obtain complementary and detailed structure information that are interrelated with its configuration switching process. The recent studies on FGFR3 and EphA1 TMD dimerization have fully discussed the multiple configurations through simulations and affiliated methods.16,18 Moreover, the simulation technology is innovated rapidly to adapt a definite biology system, such as the Martini force field,27,28 which is excelsior in simulating the longscale assembling process and evaluating the interaction within the proteins near or embedded in the phospholipid environment.29,30 In this study, we explored the underlying TMD interaction mechanism of EphA2 by utilizing multiscale simulation methods. The dimeric conformations were demonstrated by coarse-grained (CG) and atomistic methods, exposing two switching packing modes which were consistent with the previous NMR revolution.7 Through a series of site-directed mutations, we further emphasized the key role of the G540xxxG544 motif at the N-terminal in inducing the righthanded configuration. In addition, the interacting motifs of the alternative parallel mode were proved to cover multiple residues distributing along the whole TMD scale. Other than the heptad repeat motif discussed previously, the FF557xH559 domain locating on the C-terminal was found to positively consolidate the parallel configuration by synergistic anchoring mechanisms. The represented configurations in the CG simulations were further investigated under the atomistic restriction to estimate the switching dimer structure of the EphA2.

■

RESULTS AND DISCUSSION Coarse-Grained Simulation for the EphA2 TMD Dimerization. The TM region (LAVIGGVAVGVVLLLVLAGVGFFIH) of the EphA2 receptor was obtained based on the sequence alignment from the structural and dynamic NMR data (PDB ID 2K9Y).7 The initial structure of the EphA2 receptor TM domain (all involved TM sequences are seen in Table 1) was constructed on PYMOL software and subsequently inserted into a pre-equilibrated and hydrated DPPC bilayer with an approximately 55 Å distance between the separated monomers, which could rule out the effect of the

■

MATERIALS AND METHODS Coarse-Grained Model. The coarse-grained model was established using the Martini force field in which four heavy atoms and associated hydrogens are represented by one CG bead on average. All simulations were performed on GROMACS 4.5.5 software.31 The TM regions of the wild 7817

DOI: 10.1021/acs.jpcb.5b01116 J. Phys. Chem. B 2015, 119, 7816−7824

Article

The Journal of Physical Chemistry B Table 1. Sequences of the EphA2 TM Helix and Its Mutants TM helix

TM sequence

EphA2-WT G539IA542IV546I G540IG544I F556IF557I H559I F556IF557IH559I

LAVIGGVAVGVVLLLVLAGVGFFIH LAVII GVI VGV ILLLVLAGVGFFIH LAVIG IVAV IVVLLLVLAGVGFFIH LAVIGGVAVGVVLLLVLAGVGII IH LAVIGGVAVGVVLLLVLAGVGFFII LAVIGGVAVGVVLLLVLAGVGI III

electrostatic interaction and the van der Waals force at the beginning of the simulation. The separated helices spontaneously approached and interacted mutually to form a dimeric assembly, which was dynamically influenced and consolidated by the bilayer environment. The two TMD monomers were confirmed to form a stable packing assembly when the distance between their backbones was less than 10 Å. To investigate the structural features of the wild-type TMDs of the EphA2 receptor during the assembling process, the crossing angle of the two monomers was monitored and calculated (Figure 1A). Unlike the GpA38 TMD dimer that solely presents the right-handed configuration, the crossing angle of the EphA2 exhibited a bimodal distribution in which the peaks were located at about −18° and 0°, implying that the dimer transformed from the right-handed to the parallel mode alternatively, which was consistent with the switching packing case under the experimental conditions. Further, the residue contacting distributions of the two alternative conformations were explored. The interacting residues involved in the righthanded conformation were detected mainly locating on the Nterminal region, especially of the G540 and G544 positions (Figure 1B), while for the parallel conformation, the contacting residues appeared balance located and scattered along the entire transmembrane domain (Figure 1C). Specifically, the residues of L535, G539, A542, V546, L549, G553, and FF557 notably occupied the interface of the dimer and were highly in line with the proposed heptad repeat motif L535xxxG539xxA542xxxV546xxL549xxxG553, which controlled the parallel configuration identified from the NMR study. Right-Handed Conformation within the EphA2 TMD Dimer. The N-terminal G540xxxG544 motif was proposed as the determinant regulatory domain for the right-handed conformation within EphA2 TMD dimer. To further estimate the structural function of the G540xxxG544 motif, a typical simulation trajectory was selected to reveal the distance between the two G540 residues and the crossing angle tendency along with time. The results demonstrated that both of the curves presented two distinct stages during 0.5−2.3 μs consistently (Figure 1D). When the dimeric structure predominantly exhibited the righthanded conformation during 0.5−1.6 μs, the G540 residue pair was close to interact mutually; following definite steering within the packing helices, the dimeric helices stretched and alternatively formed the parallel conformation, showing a further gap between the opposite G540 residues. In this regard, there is an intimate correlation between the local domain around the G540 and the configuration transformation of the dimer. To clearly demonstrate the structural dynamics of the switching restrained dimeric situations, two typical packing helices together with the G540 and G544 are presented in two views (Figure 1D). According to the residue contact distributions (Figure 1B) within the right-handed mode of the wild type, except for the marked association of G540 and G544, some of the C-terminal

Figure 1. (A) All dimer trajectories of CG-WT simulations were converged, and the crossing angles were calculated. The zero of the crossing angle corresponds to the parallel helix packing and the negative crossing angle to the right-handed mode. The residue contact distributions were analyzed for the right-handed (B) and parallel (C) modes. Only the residue backbones were analyzed to obtain the estimation. Similarly, hereinafter, (D) for a typical dimer trajectory, the crossing angle and the G540−G540 distance in terms of time were analyzed, and the tendency lines are colored blue and red, respectively; the two dimerization modes were drawn by VMD software, with the G540 and G544 labeled in yellow and the FF559 labeled in green.

residues were also presented in the right-handed packing interface, which implied that the normal expression of the righthanded mode might be intermittently affected by the interaction of the heptad repeat motif. To test this hypothesis we substituted the highly hydrophobic residue isoleucine for the G539, A542, and V546 residues which constituted the pivotal domain of the heptad repeat motif. Consequently, the parallel packing mode mainly vanished in the mutant, retaining the right-handed formation of −20° (Figure 2A). In addition, the overall contact region was found to shift toward the N-terminal, 7818

DOI: 10.1021/acs.jpcb.5b01116 J. Phys. Chem. B 2015, 119, 7816−7824

Article

The Journal of Physical Chemistry B

Figure 3. Homodimerization simulation of the G540IG544I mutant. (A) Crossing angle distributions of the variant and the WT are, respectively, presented by green and gray columns. (B) Residue contact distribution of the mutant. (C and D) Spatial distributions of the N-terminal and C-terminal regions of one monomer referenced to the center of the other one, respectively, which was generated from the wild type. (E and F) Generated from the G540IG544I mutant. Figure 2. Dimer structure of the G539IA542IV546I mutant. (A) Crossing angle distributions of the mutant and wild type, which are shown by green and gray columns, respectively. (B) Residue contact distribution of the mutant. (C) Right-handed dimer structure assembled by the two mutational monomers, which, respectively, colored blue and red; the packing residues G540, G544 and the I539, I542, I546 residues are marked with yellow and gray beads, respectively; the FF557 residues are colored green.

wild ype (Figure 3B). Notably, the relatively prominent interaction of FF557 residues attracted attention, which was proposed previously for its potential function in the parallel assembling mode.7 On the basis of the results, it was suspected that the abolishment of the G540xxxG544 induced a loosened contact within the N-terminals of the dimer, while the binding of the C-terminals was strengthened, which bundled the two helices. To demonstrate the potentially discrepant contact of the two half parts of the TMDs, the spatial distributions of the two regions on the x−y plane were calculated by measuring one helix relative to the reference one which was moved to the center. Compared with the wild type that revealed a nonsignificant difference between the two parts (Figure 3C and 3D), the spatial distribution of the N-terminal region of the G540IG544I spread and exhibited a large-scale projective area, whereas the distribution of the C-terminal appeared compact (Figure 3E and 3F). Accordingly, an anchoring interaction within the C-terminal regions was confirmed to exist in the packing of the G540IG544I mutant. Combined with the above discussion, the anchoring performance probably derived from the FF557 residues, which contain the aromatic rings, by which the peptide chains could hardly freely rotate to dissociate from each other. To this end, we then calculated the distances of F556−F556 and F557−F557 of the G540IG544I and the wild type (Figure 4A and 4D). A remarkable discordance was observed between the two distance trends of the wild type that F557−F557 stayed at a larger value of about 15 Å while F556−F556 maintained a minor distance of about 5 Å (Figure 4B). The

especially the G540xxxG544 motif (Figure 2B). As shown in Figure 2C, the mutation enabled the two monomers to constantly assemble as a right-handed configuration via the G540xxxG544 motif, while the isoleucine residues arranged outside. It was concluded that the G540xxxG544 motif played a governing role in the formation of the right-handed dimeric structure of the EphA2. Parallel Packing Mode within the EphA2 TMD Dimer. In order to obtain a better understanding of the intermolecular mechanisms of the parallel mode which was regulated by complicated regions or residues (Figure 1C), the mutation of G540IG544I was subsequently performed to exclude the other interference factors. As expected, the proportion of the righthanded configuration decreased evidently and the peak of the crossing angle distribution was found to shift to 0° under the mutation treatment (Figure 3A). Moreover, the wide crossing angle distribution of the mutation that varied from −40° to 40° symmetrically suggested that a multi-interfaced and switching assembling event existed in this case. Moreover, the compact packing domain was found mainly concentrated on the Cterminal instead of the whole transmembrane presented in the 7819

DOI: 10.1021/acs.jpcb.5b01116 J. Phys. Chem. B 2015, 119, 7816−7824

Article

The Journal of Physical Chemistry B

occurred: the mutational helices solely formed the right-handed dimer and some of the conformations exhibited a larger crossing angle of about −35° (Figure 5A), which implied that

Figure 4. (A and B) Distance plots of the F556−F556 (black line) and F557−F557 (red line) of one random trajectory, derived from the WT and the G540IG544I, respectively. The initial 0.3 μs period during which the packing was unstable is not shown in the figures. (C and D) Packing snapshots of the WT and the G540I544I are, respectively, exhibited with the two helix backbones colored blue and orange; the Gly and the mutational Ile located at 540th and 544th are highlighted with yellow and gray beads, respectively; the FF557 residues are colored green, and the residue distance is presented with red dotted lines, and value was mechanically calculated by the VMD software, adopting the magnitude of Angstroms. The olive and purple arrows are used to link the distance evolutions derived from the WT and the G540I544I with their bottom views of the C-terminals, respectively.

Figure 5. (A) Crossing angle distributions of the F556IF557I mutant and the wild-type control, respectively, shown with green and gray columns; the right figure represents the residue contact distribution of the corresponding mutant. Similar cases are presented in B and C, which demonstrate the mutants of H559I and F556IF557IH559I.

incomplete binding between the FF557 blocks mainly resulted from the underlying interaction of the G540xxxG544 motif. In contrast, for the G540IG544I that formed the parallel mode, both of the residue pairs were found to be tightly interacting (apart about 4.4 Å) during most of the assembling (Figure 4C). Meanwhile, the binding within the N-terminals became weaker (9.39 Å of I540−I540 larger than 7.11 Å of G540−G540) since the polar attraction derived from the G540xxxG544 was removed, which corresponds to the loosened contact as described above. Further, in the parallel mode of the G540I544I, the packing efficiency within the G539xxA542xxxV546 regions was seriously weakened (Figure 3B) for the N-terminal regions and kept incompact and switching freely (Figure 3A). Therefore, comparing the residue contact of the parallel mode with the wild type (Figure 1C), it was surmised that during the regular signal transduction of the EphA2 receptor under the physiological condition the existence of the G540xxxG544 motif was partly positive rather than harmful for the full performance of the parallel packing mode within the TMDs. C-Terminal Anchoring Domain within the EphA2 TMD Dimer. Given the possibly pivotal role of the FF557 residues in maintaining the EphA2 parallel configuration, a mutant of F556IF557I was implemented to obtain the structural assessment of this domain. A significant structure transformation

the spatial restriction derived from the C-terminals was largely released. Notably, similar to the G540IG544I, the heptad repeat motif which acts as the important packing module of the parallel mode almost buried its efficacy in this case. In this regard, it was inferred that the FF557 residues act as a comparable packing domain to the heptad repeat motif in inducing the parallel configuration. Upon closer inspection, a square area of residues contact distribution around the G540xxxG544 motif attracted attention (Figure 5A). Apart from the direct interaction of G540−G540 and G544−G544 in the F556IF557I dimerization, asymmetrical packing modes of G540−G540 and G540−G544 simultaneously occurred on a certain amount (Figure S1, Supporting Information). In other words, the N-terminals of the two peptides could swing with a definite lateral angle, which brought about the contacting motif switching alternatively. For the EphA2 TM domain, the conserved polar residue H559 was discovered at the C-terminal and probably involved in the F556IF557I swinging packing process for its specific biophysical properties.39,40 To detect its potential role, the mutation of H559I was performed and the conformation was 7820

DOI: 10.1021/acs.jpcb.5b01116 J. Phys. Chem. B 2015, 119, 7816−7824

Article

The Journal of Physical Chemistry B

Figure 6. Spatial distributions of the tail domains of one targeting chain relative to the other one which was moved to the center. The tail domain being calculated consisted of six consecutive residue backbones located at the C-terminal. (A, B, C, and D) Tail spatial distributions of the WT, F556IF557I, H558I, and F556IF557IH559I mutants, respectively.

Figure 7. (A, B, C, and D) Dimer configurations of the wild-type, F556IF557I, H559I, and F556IF557IH559I mutants, respectively. Figures inserted the middle demonstrate the bottom views of the wild type, F556IF557I, and H559I, respectively. Brown beads exhibited in B represent the lipid phosphates, and the distance units are Angstroms. The residue backbones are displayed with quicksurf style and tan in color. G540, G544 residues and the substituted Ile residues are marked with yellow and gray beads; the FF557 residues are green, and the H559 residue is purple.

charged lipid phosphates (defined with particle mode of “Qa”, described in “martini_v2.0_lipids.itp”) of the lower membrane leaflet, by which the H559 assisted the FF557 to stabilize the parallel mode. The inference was further supported by the distance evolutions of the F556−F556 and F557−F557 that more often a detached state between the FF557 blocks was sustained for the H559I mutant (Figure S2, Supporting Information). The affinity between the membrane-attractive residues and the lipid phosphates was also concerned in the EGFR dimerization that the assembly tilt and location in membrane could be altered under the mutation of G380R.16 Furthermore, note that other homogeneous human Eph receptors (like EphA1−4, -7, -8) also contain such residues (R or H) at the C-terminal positions that are adjacent to the membrane surface,7 which implied the underlying common roles of the R or H in regulating the assembly configurations of Eph receptors. To clearly illustrate the packing functions of FF557 and H559 residues in the EphA2, the cluster method was applied to provide typical representations of the three mutants as well as the wild type to demonstrate their dimeric conformations (Figure 7). For each helix of the wild type, the FF557 aromatic rings symmetrically arranged outside, forming a riveting structure. The two riveting structures packed together solidly with their openings almost facing the same direction, followed by the backbones of the monomers arranged in a parallel mode (Figure 7A). When the riveting structures were unlocked as the

transformational similar to the wild type (Figure 5B). However, the right-handed configuration of the H559I became predominant, while the proportion of the parallel mode decreased apparently. Additionally, the complementary threepoint F556IF557IH559I mutant did not exhibit any parallel mode and the crossing angle profile drastically shifted leftward (Figure 5C), showing a larger angle alteration than the F556IF557I (Figure 5A). Furthermore, taking the measure of the spatial freedom of the C-terminal domains around the FF557IH559 residues (the last six successive backbones), compared with the wild type (Figure 6A), the tails of the three mutants wandered more actively as the spatial distributions covered most of the circle area and the distances between the tails of the mutants became further (Figure 6B− D). Accordingly, it was confirmed that the H559 residue played a significant role in regulating the configuration of the dimer in lipid bilayer, which was attributed to the positively charged state of its imidazole ring near the anionic membrane surface at low ionic strength.41 Despite the histidine residue being nonprotonated in the CG model, the high polarity of the imidazole side chain (defined with a particle mode of “SP1” in “martini_v2.1_amino acids.itp”, which is available from the Martini homepage) still performed a strong interaction (“attraction” degree, the second strongest nonbonded interaction of all 10 classes, described in “martini_v2.1.itp”) with the 7821

DOI: 10.1021/acs.jpcb.5b01116 J. Phys. Chem. B 2015, 119, 7816−7824

Article

The Journal of Physical Chemistry B FF557 residues were substituted, the monomers, especially their tails, achieved much more spatial freedom (Figure 6B); then a right-handed dimer packed by the G540xxxG544 motif was favorable to form (Figure 7B). In addition, even if the FF557 residues existed, when the strong affinity (associating distance of 5−7 Å in the bottom view of Figure 6B) between the lipid phosphates and the H559 polar side chain was abolished, the monomers could also gain more avidity and release the FF557 riveting blocks (Figure 7C). Nevertheless, the FF557 riveting state could not be completely eliminated under this condition, but maintained intermittent interaction causing the dimer dynamic switch (Figure 5B). Moreover, the spatial estimation (Figure 6D) further demonstrated the increase of the Cterminal freedom under the combining mutations of the FF557 and H559 residues (Figure 7D). Therefore, we concluded that the H559 residue has an assistant role to the FF557 residues, synergistically preserving the parallel dimeric structure of the EphA2. Model Refinement with Atomistic Simulations. To better refine and evaluate the dynamics of the dimeric conformations of the EphA2 TMDs, two independent atomistic simulations derived from the two CG dimeric modes were performed to estimate their packing stabilities. Representations with increased resolution could be captured from the all-atom model to demonstrate the definite packing motifs. The Cα root-mean-squared deviation (RMSD) from the initial structure in terms of the simulation time was calculated to evaluate the conformation flexibility and stability of the dimeric structure. Under the atomistic force field condition, the original chiral features of the two modes basically sustained within 100 ns, though some dynamic fluctuations during the simulation appeared (Figure 8A). However, different stability states of the two configurations were observed from the Cα RMSD graphs. The Cα RMSD tendency of the parallel mode was quite flat at about 1.5 Å, while the value of the right-handed mode increased to a relative plateau after 40 ns, showing a higher deviation about 4.0 Å (Figure 8B). One more comparison of the different packing modes is shown in Figure S3, Supporting Information, which supported the verdict further. Accordingly, the packing state of the parallel mode was proved to be more stable than the right-handed configuration, which was in agreement with the NMR structure that the dimer structure was mainly a parallel configuration with multiple interacting residues residing on the interface. The dimer conformations were further illustrated by the cluster method, which analyzed all simulation data and showed the statistical substantial interacting residues that participate in forming the definite configurations. In the right-handed conformation, the G540xxxG544 motif was verified to be located on the dimer interface, facilitating the formation and stability of the crossing interacting mode (Figure 8C). The overall spatial arrangement of the FF557 residues was basically in agreement with that of the CG model, tending to wander to some degree to adapt the formation of the right-handed mode (bottom view of Figure 8C). As for the parallel mode, because of the riveting structure formed by the FF557 residues, the associating monomers were sterically restrained and hardly steered (Figure 8D). Moreover, the binding efficiency of the G540xxxG544 motif was buried on account of the improper spatial condition. Despite the represented packing structure exhibiting some differences from the available NMR structure the heptad repeat motif and the FF557 residues were located on the packing interface simultaneously. It was speculated that such parallel

Figure 8. (A) Time dependence of the crossing angles of the parallel and right-handed modes. (B) Backbone RMSD of the two packing modes in terms of time. (C and D) Right-handed and parallel modes of the EphA2 TMDs in atomistic model. Monomers are red, the residues of G540, G544 are colored according to the type of CPK, the FF557 residues are colored with blur, and the H559 is shown according to its type of atom. C-Terminals of the two modes are bottom viewed and exhibited on the upper right.

mode was rational because of the appropriate interspace condition generated by the packing peptides, since it was increased in energy for the FF557 residues with large side chains moving to directly interface to adapt the interacting within the heptad repeat motifs. In addition, the assistant role of the H559 residue could not be neglected for its ability in maintaining the parallel mode as discussed above. Note that the histidine model defined herein was highly polar but not positively charged, which left enhancing scope for its specific potency in adhering membrane. In summary, the theoretical estimation inferred that the parallel conformation was more stable depending on the spatial anchoring force derived from the C-terminal domains, which expressed a mighty binding potency despite the obscure packing state of the heptad repeat motif.

■

CONCLUSIONS It has been proposed that the EphA2 TMD dimer could adopt two alternative packing conformations by which the arrangement of the extracellular region was regulated to respond to the signal transduction. In this study, we utilized multiple simulation strategies including CG and atomistic models to reveal the interacting motifs or residues involved in the switching packing modes, which was in good agreement with the NMR structure and recent mutational experiments. The dynamic simulations allowed for a deep insight as well as an improved understanding in the dimeric transformations with switching interfaces that were difficult to obtain in experiments. The CG results confirmed the governing role of the G540xxxG544 motif in inducing the right-handed conformation. The alternative parallel mode was also detected with the contacting residues including the proposed heptad repeat motif 7822

DOI: 10.1021/acs.jpcb.5b01116 J. Phys. Chem. B 2015, 119, 7816−7824

Article

The Journal of Physical Chemistry B and the FF557 region. Notably, further exploration certified the remarkable contribution of the FF557 riveting structures in maintaining the parallel configuration of the EphA2 TMD dimer, which was believed to be equally important to the heptad repeat motif. In addition, the definite affinity of the H559 residue with the lipid phosphates of the lower leaflet proved favorable for maintenance of the parallel conformation. By means of restricting the spatial freedom of the dimer Cterminals, the combined FF557 xH 559 region is verified synergistic in anchoring the parallel configuration. Although there is a seemingly isolated function of the G539xxA542xxxV546 and FF557xH559 domains in inducing the formation of the parallel packing mode, the dimeric configuration consolidated by the FF557xH559 region was certified to be more stable than the right-handed type. In summary, the packing motifs involved in the switching dimerization of the EphA2 TMDs are spread distributed and sophisticated, expressing competitive but also interdependent correlations in regulating its two alternative dimeric configurations. The flexible and sensitive packing mechanisms are believed to be significant to guarantee the normal biological responses of the EphA2 receptor.

■

helix packing diversity among receptor tyrosine kinases. Biophys. J. 2010, 98, 881−889. (8) Himanen, J. P.; Yermekbayeva, L.; Janes, P. W.; Walker, J. R.; Xu, K.; Atapattu, L.; Rajashankar, K. R.; Mensinga, A.; Lackmann, M.; Nikolov, D. B. Architecture of Eph receptor clusters. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 10860−10865. (9) Nikolov, D. B.; Xu, K.; Himanen, J. P. Eph/ephrin recognition and the role of Eph/ephrin clusters in signaling initiation. Biochim. Biophys. Acta, Proteins Proteomics 2013, 1834, 2160−2165. (10) Seiradake, E.; Harlos, K.; Sutton, G.; Aricescu, A. R.; Jones, E. Y. An extracellular steric seeding mechanism for Eph-ephrin signaling platform assembly. Nat. Struct. Mol. Biol. 2010, 17, 398−402. (11) Gauthier, L. R.; Robbins, S. M. Ephrin signaling: One raft to rule them all? One raft to sort them? One raft to spread their call and in signaling bind them? Life. Sci. 2003, 74, 207−216. (12) Wimmer-Kleikamp, S. H.; Janes, P. W.; Squire, A.; Bastiaens, P. I.; Lackmann, M. Recruitment of Eph receptors into signaling clusters does not require ephrin contact. J. Cell. Biol. 2004, 164, 661−666. (13) Bocharov, E. V.; Mayzel, M. L.; Volynsky, P. E.; Goncharuk, M. V.; Ermolyuk, Y. S.; Schulga, A. A.; Artemenko, E. O.; Efremov, R. G.; Arseniev, A. S. Spatial structure and pH-dependent conformational diversity of dimeric transmembrane domain of the receptor tyrosine kinase EphA1. J. Biol. Chem. 2008, 283, 29385−29395. (14) Artemenko, E. O.; Egorova, N. S.; Arseniev, A. S.; Feofanov, A. V. Transmembrane domain of EphA1 receptor forms dimers in membrane-like environment. Biochim. Biophys. Acta, Biomembranes. 2008, 1778, 2361−2367. (15) Bocharov, E. V.; Lesovoy, D. M.; Goncharuk, S. A.; Goncharuk, M. V.; Hristova, K.; Arseniev, A. S. Structure of FGFR3 transmembrane domain dimer: implications for signaling and human pathologies. Structure 2013, 21, 2087−2093. (16) Reddy, T.; Manrique, S.; Buyan, A.; Hall, B. A.; Chetwynd, A.; Sansom, M. S. Primary and secondary dimer interfaces of the fibroblast growth factor receptor 3 transmembrane domain: characterization via multiscale molecular dynamics simulations. Biochemistry 2014, 53, 323−332. (17) Sharonov, G. V.; Bocharov, E. V.; Kolosov, P. M.; Astapova, M. V.; Arseniev, A. S.; Feofanov, A. V. Point mutations in dimerization motifs of the transmembrane domain stabilize active or inactive state of the EphA2 receptor tyrosine kinase. J. Biol. Chem. 2014, 289, 14955−14964. (18) Chavent, M.; Chetwynd, A. P.; Stansfeld, P. J.; Sansom, M. S. Dimerization of the EphA1 receptor tyrosine kinase transmembrane domain: insights into the mechanism of receptor activation. Biochemistry 2014, 53, 6641−6652. (19) Finger, C.; Escher, C.; Schneider, D. The single transmembrane domains of human receptor tyrosine kinases encode self-interactions. Sci. Signal. 2009, 2, ra56. (20) Wei, P.; Zheng, B.-K.; Guo, P.-R.; Kawakami, T.; Luo, S.-Z. The association of polar residues in the DAP12 homodimer: TOXCAT and molecular dynamics simulation studies. Biophys. J. 2013, 104, 1435− 1444. (21) Wei, P.; Liu, X.; Hu, M. H.; Zuo, L. M.; Kai, M.; Wang, R.; Luo, S. Z. The dimerization interface of the glycoprotein Ibβ transmembrane domain corresponds to polar residues within a leucine zipper motif. Protein Sci. 2011, 20, 1814−1823. (22) You, M.; Li, E.; Wimley, W. C.; Hristova, K. Förster resonance energy transfer in liposomes: measurements of transmembrane helix dimerization in the native bilayer environment. Anal. Biochem. 2005, 340, 154−164. (23) Hsin, J.; LaPointe, L. M.; Kazy, A.; Chipot, C.; Senes, A.; Schulten, K. Oligomerization state of photosynthetic core complexes is correlated with the dimerization affinity of a transmembrane helix. J. Am. Chem. Soc. 2011, 133, 14071−14081. (24) Zhao, J.; Zhao, C.; Liang, G.; Zhang, M.; Zheng, J. Engineering Antimicrobial Peptides with Improved Antimicrobial and Hemolytic Activities. J. Chem. Inf. Model. 2013, 53, 3280−3296. (25) Wei, P.; Xu, L.; Li, C.-D.; Sun, F.-D.; Chen, L.; Tan, T.; Luo, S.Z. Molecular dynamic simulation of the self-assembly of DAP12-

ASSOCIATED CONTENT

* Supporting Information S

Figures showing the swaying packing presentations of the F556IF557I N-terminals; dynamics of the dimeric structures. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b01116.

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +8610 64438015. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (973 program) (2013CB910700), the National Natural Science Foundation of China (No. 21372026; 61304147), Beijing NOVA Programme (Z131102000413010), and the Fundamental Research Funds for the Central Universities (YS1407). This work was partly supported by CHEMCLOUDCOMPUTING.

■

REFERENCES

(1) Pasquale, E. B. Eph receptor signalling casts a wide net on cell behaviour. Nat. Rev. Mol. Cell Biol. 2005, 6, 462−475. (2) Lin, S.; Wang, B.; Getsios, S. Eph/ephrin signaling in epidermal differentiation and disease. Semin. Cell Dev. Biol. 2012, 23, 92−101. (3) Lackmann, M.; Boyd, A. W. Eph, a protein family coming of age: more confusion, insight, or complexity? Sci. Signal. 2008, 1, re2. (4) Alfaro, D.; García-Ceca, J. J.; Cejalvo, T.; Jiménez, E.; Jenkinson, E. J.; Anderson, G.; Muñoz, J. J.; Zapata, A. EphrinB1-EphB signaling regulates thymocyte-epithelium interactions involved in functional T cell development. Eur. J. Immunol. 2007, 37, 2596−2605. (5) Pasquale, E. B. Eph-ephrin bidirectional signaling in physiology and disease. Cell. 2008, 133, 38−52. (6) Carles-Kinch, K.; Kilpatrick, K. E.; Stewart, J. C.; Kinch, M. S. Antibody targeting of the EphA2 tyrosine kinase inhibits malignant cell behavior. Cancer Res. 2002, 62, 2840−2847. (7) Bocharov, E. V.; Mayzel, M. L.; Volynsky, P. E.; Mineev, K. S.; Tkach, E. N.; Ermolyuk, Y. S.; Schulga, A. A.; Efremov, R. G.; Arseniev, A. S. Left-handed dimer of EphA2 transmembrane domain: 7823

DOI: 10.1021/acs.jpcb.5b01116 J. Phys. Chem. B 2015, 119, 7816−7824

Article

The Journal of Physical Chemistry B NKG2C activating immunoreceptor complex. PloS One 2014, 9, e105560. (26) Zhang, H.; Tan, T.; Hetényi, C.; van der Spoel, D. Quantification of solvent contribution to the stability of noncovalent complexes. J. Chem. Theory Comput. 2013, 9, 4542−4551. (27) Ingólfsson, H. I.; Lopez, C. A.; Uusitalo, J. J.; de Jong, D. H.; Gopal, S. M.; Periole, X.; Marrink, S. J. The power of coarse graining in biomolecular simulations. Wires Comput. Mol. Sci. 2014, 4, 225− 248. (28) Monticelli, L.; Kandasamy, S. K.; Periole, X.; Larson, R. G.; Tieleman, D. P.; Marrink, S.-J. The MARTINI coarse-grained force field: extension to proteins. J. Chem. Theory Comput. 2008, 4, 819− 834. (29) Bucci, M. Lipids: Flexible fill-ins. Nat. Chem. Biol. 2014, 10, 794−794. (30) Ingólfsson, H. I.; Melo, M. N.; van Eerden, F. J.; Arnarez, C.; Lopez, C. A.; Wassenaar, T. A.; Periole, X.; De Vries, A. H.; Tieleman, D. P.; Marrink, S. J. Lipid organization of the plasma membrane. J. Am. Chem. Soc. 2014, 136, 14554−14559. (31) Van Der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A. E.; Berendsen, H. J. GROMACS: fast, flexible, and free. J. Comput. Chem. 2005, 26, 1701−1718. (32) DeLano, W. L. The PyMOL Molecular Graphics System; DeLano Scientific: San Carlos, CA, 2002. (33) Berendsen, H. J.; Postma, J. P. M.; van Gunsteren, W. F.; DiNola, A.; Haak, J. Molecular dynamics with coupling to an external bath. J. Chem. Phys. 1984, 81, 3684−3690. (34) Rotkiewicz, P.; Skolnick, J. Fast procedure for reconstruction of full - atom protein models from reduced representations. J. Comput. Chem. 2008, 29, 1460−1465. (35) Kandt, C.; Ash, W. L.; Peter Tieleman, D. Setting up and running molecular dynamics simulations of membrane proteins. Methods 2007, 41, 475−488. (36) Oostenbrink, C.; Villa, A.; Mark, A. E.; Van Gunsteren, W. F. A biomolecular force field based on the free enthalpy of hydration and solvation: The GROMOS force-field parameter sets 53A5 and 53A6. J. Comput. Chem. 2004, 25, 1656−1676. (37) Humphrey, W.; Dalke, A.; Schulten, K. VMD: visual molecular dynamics. J. Mol. Graphics Modell. 1996, 14, 33−38. (38) Psachoulia, E.; Fowler, P. W.; Bond, P. J.; Sansom, M. S. Helix− Helix interactions in membrane proteins: coarse-grained simulations of glycophorin a helix dimerization. Biochemistry 2008, 47, 10503−10512. (39) Wang, Y.; Park, S. H.; Tian, Y.; Opella, S. J. Impact of histidine residues on the transmembrane helices of viroporins. Mol. Membr. Biol. 2013, 30, 360−369. (40) Georgescu, J.; Munhoz, V. H.; Bechinger, B. NMR structures of the histidine-rich peptide LAH4 in micellar environments: membrane insertion, pH-dependent mode of antimicrobial action, and DNA transfection. Biophys. J. 2010, 99, 2507−2515. (41) Ikeda, K.; Matsuzaki, K. Driving force of binding of amyloid βprotein to lipid bilayers. Biochem. Biophys. Res. Commun. 2008, 370, 525−529.

7824

DOI: 10.1021/acs.jpcb.5b01116 J. Phys. Chem. B 2015, 119, 7816−7824