Deconstruction of the human connexin 26 hemichannel due to an applied electric field; A molecular dynamics simulation study

Accepted Manuscript Title: Deconstruction of the human connexin 26 hemichannel due to an applied electric field; A molecular dynamics simulation study Authors: Hadi Alizadeh, Jamal Davoodi, Hashem Rafii Tabar PII: DOI: Reference:

S1093-3263(16)30457-0 http://dx.doi.org/doi:10.1016/j.jmgm.2017.02.006 JMG 6848

To appear in:

Journal of Molecular Graphics and Modelling

Received date: Revised date: Accepted date:

9-12-2016 12-2-2017 13-2-2017

Please cite this article as: Hadi Alizadeh, Jamal Davoodi, Hashem Rafii Tabar, Deconstruction of the human connexin 26 hemichannel due to an applied electric field; A molecular dynamics simulation study, Journal of Molecular Graphics and Modelling http://dx.doi.org/10.1016/j.jmgm.2017.02.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Deconstruction of the human connexin 26 hemichannel due to an applied electric field; A molecular dynamics simulation study Hadi Alizadeh1, Jamal Davoodi1, and Hashem Rafii Tabar2 * 1

Department of Physics, Faculty of Sciences, University of Zanjan, Zanajn, Iran

2

Department of Medical Physics and Biomedical Engineering, School of Medicine, Shahid Beheshti University of Medical Science, Tehran, Iran

Graphical abstract

Highlights: 

The deconstructive effects of external electric fields on connexin26 hemichannel have been studied.



The external electric fields used in this study disturbed the tertiary and quaternary structures before the secondary structure disruption.



The deconstructive effects were found to be dependent on field strength and frequency.



For higher frequencies the deconstructive effects were averaged out.

Abstract Connexins are a 21-member membrane protein family constituting channels evolved in direct communication between adjacent cells by passaging cytoplasmic molecules and ions. Hexametrical assembly of connexin proteins in plasma membrane forms a wide aqueous pore known as connexin hemichannel. These hemichannels mediate cytoplasm and extracellular milieu communication both in many external tissues and in the central nervous system. In this study, a series of molecular dynamics simulations have been performed to investigate the effect of applied static and alternating electric fields on the stability and conformation of human connexin26 hemichannel. The root mean square deviations of C-alpha atoms, the dipole moment distribution, the number of inter-protein hydrogen bonds and the number of water-protein hydrogen bonds were used to assess connexin26 hemichannel stability. In the static field case, our results show that although the lowest field used in this study (0.1 V/nm) does not lead to the hemichannel deconstruction, stronger fields (>0.1 V/nm), however, disrupt the protein structure during the simulations time period. Furthermore, in the alternating case, compared to static field case, field effects on the connexin26 hemichannel conformation are reduced and consequently the protein maintains its native structure for longer times. Specifically, for the highest frequency used in this study (50 GHz), the hemichannel keeps its structure even under the effect of the strongest field (0.4 V/nm). According to our results, the protein secondary structure is preserved in the characteristic times determined for the protein deconstruction. Consequently, we suggest that the protein deconstruction is due to the tertiary and quaternary structure loss. Keywords: Connexin 26, Electric fields effects, Molecular Dynamics Simulation, Protein deconstruction

1-Introduction Due to the widespread and ever increasing application of electric/electronic devices in our everyday lives,

the

investigation

of

the

effects

of

radio

frequency(RF)

and

microwave(MW)

electric/electromagnetic radiation fields on biological tissues has become a very interesting research area during the last few decades [1-10]. Both theoretical and experimental studies, have confirmed that in addition to the thermal effects, originating from absorption of energy from the electric/electromagnetic fields by polar media surrounding biomolecules and leading to apparent temperature changes, macromolecules undergo electric/electromagnetic field induced non-thermal conformational changes [1,2,8,11]. Although computational techniques such as molecular dynamics (MD) simulations widely have been used to investigate the effects of external electric fields on biomolecules, most of these studies have been devoted to solvated proteins and peptides or pure lipid membranes [12]. Budi et al. performed a series of MD simulations to explore the effect of both static and oscillating electric fields of different strengths on insulin behavior [ 13-16]. Their findings showed

that an electric field with a strength of 0.5 V/nm is capable of disrupting the secondary structure of the insulin chain-B. Moreover, in contrast to static and oscillating fields with 1.2 and 2.4 GHz frequencies, they did not observe an appreciable change in the protein secondary structure for a field frequency of 4.5 GHz with 0.5 V/nm amplitude. English and co-workers used MD simulations to study the effect of static and low frequency (2.4 GHz) MW electromagnetic fields of varying intensities on hen egg white lysozyme (HEWL) [17-19]. Their results showed that non-thermal field effects such as marked changes in the secondary structure are insignificant for the field strengths below 0.5 V/nm. It was also shown that applying electric fields with an intensity of 0.1 V/nm did not lead to statistically distinguishable RMSDs (root mean square deviations) in both static and alternating cases compared to no-field condition. In another study on a short alanine-based peptide, they found that the exposure of an electric field with a field strength of greater than 0.1 V/nm was capable to perturb peptide conformational states [20]. Based on extended MD simulations, Marracino and co-workers investigated the effects of external pulsed and static electric fields on structural-conformational properties of solvated myoglobin [21]. They observed that myoglobin undergoes a fast unfolding (denaturation) process under highest applied fields used in their study (1 V/nm). On the other hand, no appreciable denaturation was detected under the application of a 0.1 V/nm external electric field. Accordingly, the critical external electric field to induce a significant geometrical alternation must lie between 0.1 V/nm and 1 V/nm. Very recently, Wang et al. carried out long-time (up to 1 micro-second) MD simulations to study conformational integrity of insulin under the effect of external static electric fields [22]. They reported that the insulin secondary structure is disrupted by applying electrical field with strength of 0.25 V/nm. Astrakas et al. performed MD simulations to investigate the response of chignolin, a typical beta-hairpain peptide, to the external static electric fields and showed that the peptide aligned its total dipole moment with the external field within the simulation time period used in their study [23]. Electric fields with strengths below 0.5 V/nm, had mixed effects on the creation, destruction or strength of hydrogen bonds. However, the application of sufficiently strong fields (0.5 V/nm or higher) led to destabilizing the chignolin secondary structure. In their later study focusing on the chignolin response to 1 V/nm oscillating field they found some frequency dependent conformational changes [24]. According to the authors, the protein destabilizes under fields with frequencies comparable with or smaller than the orientation selfdiffusion rate. For higher frequencies, however, the field effects are diminished and chignolin preserves its native conformation. Although there are several studies concerning the effects of both external static and alternating electric fields on proteins and peptides conformation, fewer investigations have focused on exploring the membrane proteins responses to external electric fields. Recently, an attempt was made by Garate and co-workers to study water self-diffusion within human aquaporin4 under external electric fields employing MD simulations [25]. By applying an electric field strength of 0.065 V/nm, they found that both static and alternating electric fields change the water molecules dynamics within the lumen of the pore. In particular, it was shown that the applied electric field can disrupt the well-defined bipolar orientation of water. Also, it was found that switches in the dihedral angle of the human aquaporin 4

selectivity filter, led to the boosting of water permeation events within the channel under the effect of identical external static and oscillating electric fields. In their other study, they performed MD simulations under application of pulsed electric fields and showed that the dipolar orientation of the histadine-201 residue in the selectivity filter of human aquaporin4 influences water self-diffusion by governing the dihedral angle [26]. Connexins(Cxs) are a 21-member family of tetraspan transmembrane proteins with their hexametric assemblies forming hemichannels or Connexons. The head- to- head docking of two Connexon proteins forms inter-cellular channels which mediate the communication between adjacent cells by directly passaging the cytoplasmic molecules between the two neighboring cells. All members of connexin family has a common topology, composing of four transmembrane helices (TM1-TM4) connected by two extracellular loops (EL) and one cytoplasmic loop (CL) [27]. The wide aqueous pore formed by connexon hemichannels in the plasma membrane allows the transfer of cytoplasmic molecules such as ions, second messengers, amino acids and glucose. This property leads to the widespread biological function of connexin hemichannels [28-31]. In addition to their presence in the central nervous system comprising neuronal gap junctions, connexin hemichannels are expressed in almost all hard and many external tissues and play an essential role in a variety of biological processes [32-33]. It has been shown that Cx26 gene mutation and dysfunction is linked to many human diseases such as skin diseases and hearing loss [34-35]. Recently, the crystal structure of Cx26 hemichannel at 3.5 Å resolution by X-ray diffraction was proposed by Maeda et. al [36]. This opened the window for many researchers to employ computational techniques such as molecular dynamics (MD) and Brownian dynamics simulations to study structuralfunctional properties of Cx26 hemichannel. [37-41]. In this study, we have used MD simulations to explore the effects of static and alternating electric fields, varying in amplitude and frequency, on Cx26 hemichannel conformation and its deconstruction. This work is organized as follows: following the introduction in section 1, the methodology including the construction of the simulation box and molecular dynamics simulation details are described in section 2. The MD simulation results regarding the effects of static and oscillating electric fields on Cx26 hemichannel are presented in section 3. The MD simulation results are discussed in section 4. Section 5 contains the concluding remarks of this study. 2-Methods. 2.1-Construction of the simulation box The structure used in our simulations was based on the crystal structure given by Maeda et al [36] (PDB code 2ZW3). This structure missed the atomic coordinates of Met1, cytoplasmic loop residues (110124), C-terminus residues and K15, S17 and S19 residues side chains. The structure was completed as described by Kwon et. al. [37]. The completed structure was inserted into a 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC) lipid membrane. The dimensions of our tetra angle simulation box was approximately 14 nm×14 nm×11 nm in x,y and z directions, respectively. TIP3 waters were added to the simulation box. 150 mM KCl ions were added to the simulation box to mimic physiological

conditions and the whole system was neutralized by adding appropriate counter ions. Inserting protein into POPC lipid membrane, adding water molecules and ions were achieved by using CHARMM-GUI on-line software [42-43]. The final simulation system consisted of 490 POPC molecules, 39735 TIP3 water molecules and 86 K and 140 Cl ions. 2.2-Molecular dynamics simulation details All MD simulations were performed in GROMACS 5.0.4.software [44-45] using all-atom CHARMM 36 force field [46-47]. After energy minimization of the system using the steepest descent method, NVT ensemble simulations with force constraints on heavy atoms were performed with Brendsen thermostat [48] where the time-step was set at 1fs. Then the force constraints were reduced gradually and afterwards, 80 ns isothermal-isobaric (NPT) simulations were performed using the Nose-Hoover [4950] thermostat and the Parrinello-Rahman barostat [51] for the equilibration stage. In NPT simulations the pressure was fixed at 1atm. All MD simulations were performed at T=310 K. In the NPT simulations we used a 2fs time-step. In all simulations periodic boundary conditions were applied in all three directions. The centre of mass motion removal was used for the lipid membrane and solution media separately. Long range electrostatic interactions were calculated using the particle mesh Ewald (PME) method. Following the equilibration stage, electric fields with different strengths and frequencies were applied. All electric fields were applied along the channel axis direction and from intercellular part to the cytoplasmic part.

3- Results The conformational stability of the Cx26 hemichannel during simulation time was examined by calculating the root mean square deviation (RMSD) trajectory of Cx26 C-alpha atoms relative to the starting structure. As it can be seen in Fig.1, the RMSD of C-alpha atoms reached its plateau in 80ns in the absence of electric field, indicating that our model protein had been equilibrated within this period of time (equilibration stage). In addition, the Cx26 hemichannel keeps its tsuzumi shape [36] during this simulation time (Fig. 1. b). Following the equilibration phase, a series of MD simulations (up to 50ns) of Cx26 hemichannel both in the absence of external electric field (no-field condition) and under the exposure of external electric fields with different strengths (0.1, 0.175, 0.25, 0.325 and 0.4 V/nm) and frequencies (0, 2.4 and 50GHz), were performed to probe the characteristic time wherein different electric fields could cause the destruction of the protein structure. To insure the reproducibility of the results, each simulation was repeated three times under the same conditions, except for the initial condition in which different random seeds were used.

3.1- Application of Static Electric Fields Fig 2.a. shows the time evolution of RMSDs of C-alpha atoms of the Cx26 under an applied static electric field with different strengths, relative to the end of the equilibration stage. From this result, one can notice that there are negligible deviations in the RMSD plot of the C-alpha atoms under the influence of 0.1 V/nm field compared to the no-filed simulation. Also, visualizing the Cx26 structure shows that the protein keeps its tsuzumi shape at the end of 50ns simulation under applied electric field with the strength of 0.1 V/nm. However, a jump in the RMSD values is observed for higher fields which

can be considered as an indication of the disruption of the Cx26 native structure. The time at which the jump in RMSDs value occurs decreases with increasing the applied electric field strength. This time is called the characteristic time. The effect of electrical fields on the z component of the Cx26 dipole moment is shown in Fig 2. b. The z component of the Cx26 dipole moment in the absence of an electric field is also presented for comparison. According to this figure, the general effect of applied electric fields on z component of Cx26 dipole moment is to increase its magnitude whose extent becomes more pronounced for stronger fields. The number of inter-protein and water-protein hydrogen bonds (HBs) calculated from the MD simulations under 0.1 V/nm electric field during 50ns and those calculated for 0.175, 0.25, 0.325 and 0.4 V/nm fields before the deconstruction time are shown in Fig. 3. The number of hydrogen bonds were calculated based on geometrical criteria implemented in GROMACS 5.0.4 software [44-45]. It can be seen that other than statistical differences, the number of inter-protein HBs and water-protein HBs under the 0.1 V/nm applied electric field resemble those quantities in the zero field simulations. However, higher fields led to a significant decrease in the number of inter-protein HBs and an increase in the number of water-protein HBs before the destruction time. 3.2- Application of Oscillating Electric Fields To investigate the effect of the frequency of oscillation of the electric fields on the Cx26 stability, a series of MD simulations were carried out by applying 2.4GHz and 50GHz electric fields with different strengths. For applied electric fields with frequency of 2.4GHz, similar to the static case, the RMSDs of the Calpha atoms were used as the first criterion to probe the stability of the protein. Fig.4 shows the Cx26 C-alpha atoms RMSDs time evolution under the exposure of 2.4GHz electric field with different strengths. According to this figure, the field with 0.1 V/nm amplitude does not cause a substantial change in the Cx26 structure. This result was found to be similar to that of the static field. Furthermore, the Cx26 retains its structure under exposure of 2.4GHz field with 0.17 and 0.25 V/nm amplitudes during the simulation time used in this study (50ns). This is in contrast to the static case where the Cx26 hemichannel structure was disrupted under the same strengths. For higher amplitude fields (>0.25 V/nm), although a shift in the RMSD plot corresponding to the protein deconstruction occurs, the characteristic times for the 2.4GHz oscillating fields are higher compared to those in the static case. This characteristic time is approximately 18ns and 2ns for 0.325 and 0.4 V/nm fields respectively in comparison to 1.7ns and 1ns in the static field simulations. The effects of an external 2.4GHz oscillating field on the z component of the Cx26 dipole moment are depicted in Fig. 4. The main effect of the oscillating field is the oscillation of the z component of Cx26 dipole moment with the same oscillation period. The number of inter-protein and water-protein HBs during the 50ns MD simulation for 0.1, 0.175, 0.25V/nm fields and those before the destruction times for 0.325 and 0.4 V/nm fields are shown in Fig. 5. These results show that for the lower amplitude fields (<0.325), the number of inter-protein and

water-protein HBs are the same as those calculated from the no-field simulation. However, for higher fields (0.325V/nm and 0.4V/nm), similar to static cases, the number of inter-protein hydrogen bonds was reduced while the number of water-protein HBs was increased before the deconstruction time. To further investigate the frequency dependency of the effect of an external electric field on the Cx26 hemichannel structure, the electrical fields with a frequency of 50GHz and different amplitudes were also applied. Similar to the static and 2.4GHz cases, the RMSDs of the Cx26 C-alpha atoms were calculated relative to the end of equilibration stage (Fig. 6). As it can be noticed, there is no appreciable difference between the RMSD plot obtained for 50 GHz electric fields simulations compared to that of zero field simulation. Also, the number of inter-protein and water-protein HBs were not affected by 50 GHz electric fields. However, compared to no-field simulations, stronger fluctuations in z component of dipole moment magnitude could be observed for 50 GHz electric fields (Fig. 7).

4- Discussion The completed structure of Cx26 hemichannel has a positive net charge [36]. However, the calculated distance between the center of mass of Cx26 hemichannel relative to the surrounding lipid membrane center of mass did not show an appreciable deviation from that of calculated from no-field simulation (see Table 1). From this, one can conclude that the external electric fields used in this study are not strong enough to dominate the resisting forces originated from interacting lipid membranes to move the protein along the applied fields. The time evolution of RMSDs shown in Fig. 2, indicates that 0.1 V/nm applied static electric field is not capable of the deconstruction of the Cx26 hemichannel up to the end of our simulation time (50ns). On the other hand, the jump observed in RMSD plot for the higher applied static electric fields is an indication of deconstruction of Cx26 hemichannel. Apparently, the higher the electrical field strength, the shorter the deconstruction characteristic time (Table 1). STRIDE analysis using VMD software (not shown here) showed that in a similar fashion to other proteins and peptides, the secondary structure of the Cx26 is not noticeably disrupted at these characteristic times. Accordingly, we suggest that the jump in the RMSD plot observed for higher fields (>0.1 V/nm) is due to tertiary and quaternary structure disruptions. Whereas the Cx26 hemichannel is radially symmetric, it has a strong electric dipole moment along the z direction (its axis). While the negatively charged residues are accumulated in the extracellular side of the Cx26 hemichannel, the cytoplasmic side contains many positively charged residues [36,52]. Therefore, an external electric field applied along its axis can affect the Cx26 conformation by changing its axial component (z component) of the dipole moment. Generally, static electric fields applied in our simulations increase the magnitude of the dipole moment along the z direction. This is because due to the diploe moment orientation, the exposure of a static electric field through the extracellular side of Cx26 hemichanne stretches the protein along its axis. Although the lowest field used in this study (0.1 V/nm) could increase the magnitude of the z component of dipole moment, its effectiveness was not strong enough to destruct the protein. However, higher fields have a more pronounced effect on the z component of the protein dipole moment so that by increasing the field strength, the z component of the diploe moment magnitude rises more rapidly and more intensively.

This can lead to more stretching of the residues and probably the deconstruction of the protein structure. Furthermore, the characteristic times observed in the dipole moment plot (the times above which an appreciable increase occurs in the dipole moment magnitude) were found to correlate with the conjugate destruction times observed in the RMSD plot. Moreover, the decrease in the number of inter-protein HBs before the characteristic times observed in the RMSD and the dipole moment plots could also be considered as an evidence for protein deconstruction. The deconstruction of the protein and the loss of its inter-protein HBs leave more positions for water molecules to make HBs with the protein residues which was seen in Fig. 3 A comparison made between the RMSD, dipole moment, number of inter-protein HBs and the waterprotein HBs plots as well as the determined protein deconstruction times obtained for applied 2.4 GHz oscillating fields with those calculated for the static fields showed that static fields have stronger deconstructive effects on Cx26 hemichannel (Table 1). Whereas the characteristic time is increased for 0.325 and 0.4 V/nm fields, the protein keeps its structure till the end of simulation time for lower fields. Furthermore, protein dipole moment oscillates with oscillating field period and its oscillation amplitude increases with increasing electric field strength. It is worth noting that, in contrast to solvated proteins and peptides [12,24], the Cx26 hemichannel, due to the presence of lipid membranes surrounding the protein, cannot rotate to align its dipole moment with that of the field. Consequently, the observed oscillations in dipole moment plot can be attributed to the protein residues stretching/contracting along the z axis. The results showed that, in contrast to two other cases, under a 50GHz applied field the protein preserved its structure until the end of the simulation time for all the amplitudes considered in this study. This indicates that the effectiveness of electric fields is reduced by increasing the field frequency. This is partly because by increasing the frequency of the electric field, there would not be enough time for protein residues to stretch and contract along hemichannel axis up to an extent that causes protein deconstruction.

5- Conclusions According to our MD simulation results, exogenous electric fields stronger than 0.1 eV/nm can deconstruct the Cx26 hemichannel in the simulation time available in this study. The RMSD time evolution of protein C-alpha atoms, the axial component of protein dipole moment magnitude, the number of inter-protein and the number of water-protein HBs all compared to no-filed simulation have been used as the criteria for protein deconstruction. The STRIDE analysis showed that the secondary structure of the Cx26 hemichannel is preserved before the deconstruction times determined in this study. Thus we suggest that protein deconstruction is mainly due to the tertiary and quaternary structure loss. The characteristic time long enough to deconstruct the protein structure, was found to be dependent on the field strength and frequency. As it was expected, the characteristic time of protein deconstruction was decreased with increasing the external filed strength. In addition, compared to static fields, the protein retains its structure for longer times when we used oscillating fields in our simulations. Specifically, for highest field frequency (50GHz), we could hardly observe any conformational change

in the field strength range and the simulation time used in this study, which indicated that by increasing field frequency, the field effects were averaged out.

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(a)

(b)

Figure 1. a) Cx26 hemichannel C-alpha atoms RMSD during equilibration stage, b) Cx26 shape after 80ns equilibration time.

(a)

(b)

Figure 2. a) Cx26 hemichannel C-alpha atoms RMSDs under different static electric fields, b) Cx26 hemichannel dipole moment along the z axis under static electric fields.

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(b)

Figure 3. a) Number of inter-protein H-bonds of Cx26 under static electric fields, b) Number of water-protein Hbonds under static electric fields.

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(b)

Figure 4. a) Cx26 C-alpha atoms RMSDs under 2.4GHz electric fields, b) Cx26 dipole moment along the z axis under 2.4GHz electric fields (for clarification purposes, just first 4 ns is depicted in dipole moment plot).

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(b)

Figure 5. a) Number of inter-protein H-bonds of Cx26 under 2.4GHz electric fields, b) Number of water-protein H-bonds under 2.4GHz electric fields. Weaker fields are emitted for clarification.

Figure 6. ) Cx26 C-alpha atoms RMSDs under 50GHz electric fields.

Table 1. The characteristic times determined for Cx26 deconstruction under the effect of various electric fields and protein-lipid membrane center of masses distance deviation from that of calculated from no-field simulation (COM distance deviation).

Field strength Field frequency Characteristic time COM distance deviation Number of replicates (V/nm)

(GHz)

(ns)

(nm)

0.1

0 2.4 50

-------------------

0.2±0.4 0.3±0.3 0.4±0.3

3 3 3

0.175

0 2.4 50

34±6.4 -------------

0.3±0.4 0.2±0.5 0.4±0.4

3 3 3

0.25

0 2.4 50

3±1.3 -------------

0.1±0.4 0.2±0.4 0.3±0.5

3 3 3

0.325

0 2.4 50

1.7±0.4 18±3.5 -------

0.3±0.4 0.3±0.3 0.2±0.5

3 3 3

0.4

0 2.4 50

1±0.2 2±0.7 -------

0.4±0.4 0.3±0.3 0.2±0.5

3 3 3