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. Author manuscript; available in PMC: 2021 Jul 1.
Published in final edited form as: Alcohol Clin Exp Res. 2020 Jun 18;44(7):1344–1355. doi: 10.1111/acer.14363

Effect of ethanol on Munc13-1 C1 in membrane: A Molecular Dynamics Simulation Study

Youngki You 1, Joydip Das 1,*
PMCID: PMC7572713  NIHMSID: NIHMS1605822  PMID: 32424866

Abstract

Background:

Ethanol has a significant effect on synaptic plasticity. Munc13-1 is an essential presynaptic active zone protein involved in priming the synaptic vesicle and releasing neurotransmitter in the brain. It is a peripheral membrane protein and binds to the activator, diacylglycerol (DAG)/phorbol ester at its membrane-targeting C1 domain. Our previous studies identified Glu-582 of C1 domain as the alcohol-binding residue.

Methods:

Here we describe a 250 ns molecular dynamics (MD) simulation study on the interaction of ethanol and the activator-bound Munc13-1 C1 in the presence of varying concentrations of phosphatidylserine (PS).

Results:

In this study, Munc13-1 C1 shows higher conformational stability in ethanol than in water. It forms fewer hydrogen bonds with phorbol 13-acetate in the presence of ethanol than in water. Ethanol also affected the interaction between the protein and the membrane and between the activator and the membrane. Similar studies in a E582A mutant suggest that these effects of ethanol are mostly mediated through Glu-582.

Conclusions:

Ethanol forms hydrogen bonds with Glu-582. While occupancy of the ethanol molecules at the vicinity (4Å) of Glu-582 is 34.4 %, the occupancy in the E582A mutant is 26.5 % of the simulation time. In addition, the amount of PS in the membrane influences the conformational stability of the C1 domain and interactions in the ternary complex. This study is important in providing the structural basis of ethanol’s effects on synaptic plasticity.

Keywords: alcoholism, molecular dynamics, presynaptic, Munc13-1, alcohol-binding site, lipid membrane, phorbol ester, diacylglycerol

Introduction

Defining the molecular mechanism of ethanol action is key to developing an intervention to alcohol addiction. At relatively low concentrations, ethanol affects the function of many central and peripheral synapses (Liu and Hunt, 1999, McCool, 2011, Lovinger and Roberto, 2013, Roberto and Varodayan, 2017). Although clear effects of ethanol have been identified in postsynaptic compartments, its effects on the presynaptic compartments is less-studied. Ethanol acts on multiple targets (Harris et al., 2008, Howard et al., 2011) and affects many neuronal circuitries in the brain (Abrahao et al., 2017). While ethanol’s effects on postsynaptic receptors, such as GABAA (Olsen and Liang, 2017), glycine (Soderpalm et al., 2017), and glutamate (Rao et al., 2015) are well-established, there has been evidence of a significant effect of ethanol on presynaptic function (Siggins et al., 1987, Roberto et al., 2003, Nie et al., 2004, Diamond and Gordon, 1997). It has been suggested that ethanol may be directly affecting synaptic transmission by altering vesicle fusion and neurotransmitter release (Barclay et al., 2010), possibly by interacting with the proteins associated with the neurotransmitter release machinery.

Munc13-1 is a presynaptic active zone protein essential for synaptic vesicle priming (Betz et al., 1997, Sassa et al., 1999) and neurotransmitter release (Betz et al., 1998, Brose et al., 2000). A central function of Munc13-1 is to bridge the synaptic vesicle and the plasma membrane and the C1 and C2 domains play major roles in this anchoring process (Quade et al., 2019). Munc13-2, Munc13-3, and Munc13-4 (Chen et al., 2013) are the other members of the Munc13 family known to date. Munc13-1 is expressed primarily in the hippocampus, cerebellum, cortex, and striatum regions of the brain (Augustin et al., 1999a) and modulates short-term presynaptic plasticity (Rosenmund et al., 2002, Lipstein et al., 2013) and long-term potentiation through its interactions with an active zone protein RIM (Yang and Calakos, 2011). Munc13-1 and 13–2 double- knockout mice result in complete abolishment of neurotransmitter release (Augustin et al., 1999b, Varoqueaux et al., 2002, Aravamudan et al., 1999, Richmond et al., 1999).

Structurally, Munc13-1 is a large peripheral membrane protein having several modulatory domains (Fig. 1A). There are three C2 domains, one C1 domain and one MUN domain (Shin et al., 2010). The N-terminal C2A domain is followed by a high-affinity diacylglycerol (DAG)/phorbol ester binding C1 domain, a Ca2+ binding C2 domain (C2B), its characteristic MUN domain, and a C-terminal C2 domain (C2C) (Aravamudan et al., 1999, Ma et al., 2011). Binding of DAG/phorbol ester to the Munc13-1 C1 domain stimulates synaptic vesicle priming as well as the translocation of Munc13-1 from the cytoplasm to the plasma membrane (Andrews-Zwilling et al., 2006). There are strong sequence similarities among the C1 domains of Munc13, Dunc-13, Unc13 and the C1 domain of protein kinase C’s (PKCs) (Das and Rahman, 2014). While homologous, the C1 domains of Unc13 and Munc13 show a substantial difference in net charge, which may contribute to the difference in observed binding affinities. Further, binding of the ligand to the C1 domain is dependent on the presence of phosphatidylserine (PS) in the membrane. PKC C1 showed highest ligand binding in membrane containing 100% PS, but Munc13-1 C1 showed highest ligand binding in membrane containing 20% PS (Das et al., 2018). The binding of DAG to the Munc13 C1 domain lowers the energy barrier for vesicle fusion and promotes neurotransmitter release (Basu et al., 2007). The H567K mutation in the C1 domain prevents the binding of phorbol ester, thereby inhibiting the activation of vesicle fusion (Betz et al., 1998, Basu et al., 2007).

Figure 1. Structure of Munc13-1 and membrane - phorbol 13-acetate - Munc13-1 C1 ternary complex system for MD simulation.

Figure 1.

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(A) Domain structure of Munc13-1. The C1 domain binds DAG/phorbol ester and lipids. C2 domains bind lipids and Ca2+. MUN is a self-folding domain consisting of two Munc13 homology domains. (B) Phorbol 13-acetate-bound C1 domain was embedded into the lipid membrane in the 1% ethanol solvent. Four different systems, Munc13-1 C1 + phorbol 13-acetate + water; Munc13-1 C1 + phorbol 13-acetate + ethanol; Munc13-1 C1 (E582A) + phorbol 13-acetate + water; Munc13-1 C1 (E582A) + phorbol 13-acetate + ethanol, were used. C1 domain is represented by the solid ribbon. The phorbol 13-acetate and Glu-582 are represented by the yellow CPK and green CPK, respectively. The lipid membrane, PS, is represented by the gray line. Magenta colored sticks indicate the ethanol molecules. Water molecules in the system are not shown.

The C1 domain of Munc13-1 has close structural homology with the C1 domain of PKC. A comparison of the PKCδC1B and Munc13-1 C1 structures shows that both structures have two β-sheets, a short C-terminal α-helix and two Zn+2 binding sites. DAG binds inside a groove formed by two loops (Shen et al., 2005, Zhang et al., 1995, Das and Rahman, 2014). The NMR structure of Munc13-1 also revealed that the side chain of Trp-588 in the C1 domain occludes the ligand binding site (Shen et al., 2005). The orientation of the tryptophan residue at the homologous position in the C1 domain of PKCδ (Zhang et al., 1995) and PKCθ (Rahman et al., 2013) is different than that of Munc13-1.

Using diazirine-based photoaffinity labeling followed by mass spectrometric analysis, we showed alcohol binds at the Glu-582 of the Munc13-1 C1 domain. We also showed that deficits in dunc13 lead to defects in alcohol sensitivity, tolerance and self-administration in Drosophila (Das et al., 2013, Xu et al., 2018).

Here we report a 250 ns molecular dynamics (MD) simulation study of ethanol’s interaction with the activator-bound Munc13-1 C1 and its E582A mutant in the presence of varying amounts of PS (Fig. 1B). Our results show that ethanol stabilizes the C1-activator-membrane ternary complex and Glu-582 plays a role in this stabilization. Ethanol affects protein-activator, protein-membrane and activator-membrane interactions. Occupancy of ethanol molecules around Glu-582 was dependent on the PS concentration and point mutation at this site.

Material and Methods

Molecular Docking

Phorbol 13-acetate was docked into the Munc13-1 C1 domain WT and mutant using AUTODOCK 4.2 (Morris et al., 2009). The docking simulations were prepared, run, and analyzed using AutoDockTools (ADT). The NMR structure of Munc13-1 C1 domain (PDB: 1Y8F) was selected for the molecular docking (Shen et al., 2005). The E582A mutant of C1 domain was created by using Discovery Studio Visualizer 4.5 (DS, Biovia Inc.). The structure of phorbol 13-acetate was created using ChemBioDraw version 12.0 and then energy minimization was carried out for the ligand using 0.1 RMS kcal/mol/Å2 gradient level in MOE 2018 (MOE; Chemical Computing Group). Kollman charges and Gasteiger charges were assigned to the proteins and the ligand, respectively. For the docking space of ligand, a grid was generated and centered on the geometry of the phorbol ester in phorbol ester-bound PKCδ C1B (PDB: 1PTR) which is a homologue of Munc13-1 C1 (Zhang et al., 1995). The grid included the active site between the two loops of the C1 domain within a box size set at x = 40 Å, y = 55 Å, and z = 40 Å. The Lamarckian Genetic Algorithm (LGA) was used to search for the best twenty conformers during molecular docking. The phorbol 13-acetate docked structures were visualized using DS.

Building the phorbol 13-acetate-Munc13-1 C1 complex in the phospholipid membrane

The orientation of the phorbol 13-acetate-bound Munc13-1 C1 domain in the lipid membrane was calculated using the PPM server (Lomize et al., 2011). The conformation of bound ligand that marked the lowest binding energy in the largest cluster was used. The model of PS/PC - phorbol 13-acetate - C1 ternary complex in a rectangle box filled by TIP3P water molecules was constructed using the CHARMM-GUI Bilayer Builder (Jo et al., 2008). N-termini and C-termini were terminated with acetylation and methyl amination, respectively. The initial simulation box was generated with side lengths 61 Å × 61 Å for XY-axis and Z-length was determined by water thickness. Minimum water height on the top and bottom of the system was set to 20 Å. The net charge on the system was neutralized by adding potassium ions. For 100% PS, the number of lipid molecules in the lower and upper leaflets were 58 and 64, respectively. For the 20% PS, the number of lipid molecules in the lower and upper leaflets were 12 and 13, respectively. The number of PC in the lower and upper leaflets were 46 and 51, respectively. The charmm36 force field was used to describe the system (Lee et al., 2015). For the ethanol solvated system, 1% ethanol molecules were added to the system using GROMACS 5.1.2. This ethanol concentration was used earlier for the MD simulation studies on glycine receptors and GLICs (Murail et al., 2011, Murail et al., 2012). By adding 1% ethanol, we approximated a 1 mol-% or 555 mM ethanol concentration. This value is about 3 times the concentration to induce anesthesia in tadpoles (Alifimoff et al., 1989) and 30 times above the legal threshold for intoxication in humans (Harris et al., 2008). When one molecule of ethanol was docked near Glu-582, we observed random movement of the ethanol molecule right after the start of the simulation. This is not surprising for a low-affinity ligand like ethanol. Therefore, instead of docking one molecule of ethanol, we added 1% ethanol molecules for our MD studies. MD simulation studies were done in two different PS concentrations, 100% PS and 20% PS in PC. In mammalian cell membranes the phosphatidylserine (PS) comprises 2–15% of total phospholipid (Leventis and Grinstein, 2010, Newton, 1993b) and C1 domain-containing proteins show specificity for PS. Earlier all our biochemical studies on C1 domains were conducted in 100% and 20% PS (Das et al., 2018, Blanco et al., 2019, Das and Rahman, 2014). 100% PS was used earlier in the molecular dynamics study of C1 domains (Ashida et al., 2016).

Molecular Dynamics Simulation

Molecular dynamics (MD) simulations were performed on the ternary complex of the lipid membrane - phorbol 13-acetate - C1 domain using GROMACS 5.1.2 package of programs (Hess et al., 2008). Energy minimization was completed using the steepest descent method to remove steric clashes generated while solvating the ternary system. The minimized system was equilibrated for maintaining the temperature and pressure of systems and relaxing the solvent. The equilibration was carried out in two serial phases. NVT optimization with 300 K was performed for 100 ps in the first phase, and then NPT optimization was performed at 1 bar for 10 ns in the second phase. After equilibration, the MD production run was conducted using the Nose-Hoover thermostat algorithm and Parrinello-Rahman barostat algorithm allowing a time step of 2 fs. The time constant was kept at 0.5 ps for the temperature coupling and 5 ps for the pressure coupling. Electrostatic interactions was computed by the particle mesh Ewald (PME) method with fourth-order cubic interpolation and 1.6 Å grid spacing (Essmann et al., 1995). The Coulombic interactions, electrostatic, and van der Waals interactions were calculated with a 12 Å cutoff. All bonds were constrained using the parallel LINCS method. For analysis, the atomic coordinates were saved every 2 ps during the MD simulation.

Analysis of MD simulation and statistics

The MD trajectories were analyzed by GROMACS analysis tools, including gmx rms, gmx hbond, gmx distance, and gmx select. The horizontal movement of the C1 domain along the plasma membrane was quantified from the images of movement on the x,y-plane using ImageJ (http://rsb.info.nih.gov/ij/). Occupancy was defined as the time the ethanol molecules reside within 4 Å of Glu-582 (Ala-582). We examined 125,001 frames including the frame at time 0 (with 2 ps intervals between consecutive frames) for the occupancy of the ethanol. The occupancy was calculated as the percentage (%) of the studied frames in which the ethanol molecule was seen in the vicinity (4Å) of Glu-582 (Ala-582) during the 250 ns simulation. For clustering, ethanol molecules within 6 Å of Glu-582 (Ala-582) were identified at every 1.0 ns over 250 ns. The center of geometry (cog) of identified ethanol molecule was calculated, and then the ethanol molecules were clustered based on the cog by k-means clustering using RStudio version 1.2 (Team, 2015, Mustata and Briggs, 2004). Twenty initial configurations were used to find optimal clusters. Data analysis was performed using Prism 7.0 software (GraphPad Software, Inc., San Diego, CA). All statistical analyses were performed on the basis of three independent simulations. Each simulation was reproduced for the MD production starting from the same equilibrium data. Standard error of the mean (SEM) of three independent simulations was used for the statistics. The graphs were plotted with QtGrace. The structures and trajectories were visualized using PyMol version 1.7 (Schrodinger, LLC) and DS 4.5.

Results

Effect of ethanol on conformational dynamics of Munc13-1 C1 in membrane containing 100% PS

In our previous studies, we found that alcohol binds to Glu-582 of Munc13-1C1 and mutating Glu-582 to alanine reduced alcohol binding (Das et al., 2013, Xu et al., 2018). In order to gain insight into the effect of ethanol on structural stability of the ternary complex of protein (Munc13-1 C1/ E582A), ligand (phorbol 13-acetate), and plasma membrane, MD simulations were performed at membrane containing 100% PS. The four different systems for this study are: WT C1 + phorbol 13-acetate + water; WT C1 + phorbol 13-acetate + ethanol; E582A C1 + phorbol 13-acetate + water, and E582A C1 + phorbol 13-acetate + ethanol, where WT represents wild-type Munc13-1 C1. The systems were stabilized quickly and maintained the stable status during 250 ns. Ethanol also affected the conformational dynamics of the Munc13-1 C1, which is represented by the changes in the root mean square deviations (RMSD). The WT C1 domain in water showed higher fluctuation of RMSD than WT C1 in ethanol after 115 ns (Fig. 2) in two of the three simulations (Fig. S1). Comparison of the three simulations suggest that there could be two distinct conformational states with RMSD value of ~0.25 nm and ~ 0.5 nm. However, WT C1 in ethanol changed its conformation slowly till 50 ns, and then remained steady through the remainder of the 250 ns. The orientation of both the C-terminus and N-terminus of WT C1 in water changed significantly as compared to the systems containing ethanol. To understand the role of Glu-582 and its neighboring residues in maintaining the conformational dynamics, the RMSD plots of Glu-582 and four residues in the vicinity of Glu-582, from Cys-580 to Glu-584, were also monitored (Fig. S2). For the WT in water we see large differences in RMSD for all the simulations until ~75 ns and then the RMDS values were more or less stabilized in all the three simulations. In the WT in ethanol, however, till 75 ns all the three simulations look similar, and after that the RMSD values go up and stabilized for all the three simulations, except a fluctuation from 200 to 230 ns in one replicate. The two loops of the C1 domain, between which the ligand binds, maintained their initial poses in both water and ethanol because the loops were capped by ligand and embedded in the plasma membrane.

Figure 2. Comparison of structural deviation of Munc13-1 C1 during 250 ns MD simulation in 100% PS.

Figure 2.

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Plot of RMSD of Munc13-1 C1 versus time for the four different systems: Munc13-1 C1 + phorbol 13-acetate + water; Munc13-1 C1 + phorbol 13-acetate + ethanol; Munc13-1 C1 (E582A) + phorbol 13-acetate + water and Munc13-1 C1 (E582A) + phorbol 13-acetate + ethanol. In the systems containing ethanol, 1% ethanol of water molecules is added. RMSD was calculated after linear square fit to the C1 domain. The plot is 30 ps running averages of RMSD. The WT Munc13-1 C1 in ethanol showed less fluctuation than the WT C1 in water. WT, wild-type; EtOH, ethanol.

In the RMSD plot of the mutant, E582A C1 in water showed high fluctuation from the beginning of the simulation which was not observed in WT C1 (Fig. 2 and Fig. S1), suggesting a possible role of Glu-582 in maintaining the protein structure. E582A C1 in ethanol also changed its conformation slowly and remains steady throughout the simulation period. However, it showed less fluctuation than E582A C1 in water. In water, the RMSD values of the five residues E582A C1 indicate the presence of several states and ethanol does not appear to stabilize as it did for the five residues in WT (Fig. S2).

In summary, ethanol affects the conformational dynamics of the C1 domain in membrane.

Effect of ethanol on the mobility of the C1 domain in membrane containing 100% PS

Munc13-1 is peripheral membrane protein and is activated by binding with DAG/phorbol ester at its C1 domain in the plasma membrane. The DAG/phorbol ester binding site of the C1 domain is composed of two loops embedded into the plasma membrane. To understand the effect of ethanol on conformational changes and its movements in the membrane, we monitored the changes in distance between the center of geometry (cog) of the C1 domain and the cog of the membrane along X, Y and Z axes. During the 250 ns MD simulation, ethanol influenced the vertical movement of the C1 slightly, but it affected the horizontal movement in the plasma membrane (Fig. 3 and Fig. S3). The horizontal movement is represented by the movement along the X and Y-axis and the C1 domain in water moved more inside the membrane than in ethanol during the 250 ns simulation time. The total area that the WT C1 traversed in the XY plane in the presence of water and ethanol were 2.211 ± 0.389 nm2 and 1.710 ± 0.282 nm2, respectively. For the E582A C1, the values were 1.657 ± 0.191 nm2 and 1.959 ± 0.104 nm2 (Fig. S4). The lower value for ethanol in WT suggests that ethanol prevents horizontal movement in WT and the higher value in E582A suggests that ethanol does not prevent the horizontal movement of E582A C1 in the membrane.

Figure 3. Movement of the C1 domain along the lipid membrane during 250 ns MD simulation in 100% PS.

Figure 3.

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The change in the distance between the center of geometry (cog) of the lipid membrane and the cog of the C1 domain is measured in four systems: Munc13-1 C1 + phorbol 13-acetate + water; Munc13-1 C1 + phorbol 13-acetate + ethanol; Munc13-1 C1 (E582A) + phorbol 13-acetate + water and Munc13-1 C1 (E582A) + phorbol 13-acetate + ethanol. The distance was monitored along X (upper panel), Y (middle panel) and Z (lower panel) - axes, respectively. The WT Munc13-1 C1 in water moved significantly in the membrane along the X and Y-axis, but in EtOH it moved less in the membrane. The mutant in water also moved less along the X and Y-axis than WT in water. Right, schematic diagram showing the Munc13-1 C1 (purple), membrane and the directions of three axes. cog, center of geometry; WT, Wild-type; EtOH, ethanol.

In summary, Glu-582 appears to play a role in dictating the movement of the C1 domain by interacting with ethanol.

Effect of ethanol on the interactions between Munc13-1 C1 and phorbol 13-acetate in membrane containing 100% PS

The binding of DAG/phorbol ester and the Munc13-1 C1 in the plasma membrane is an important step in the Munc13-1 activation process. To understand if ethanol could affect this interaction, we monitored the number of hydrogen bond formations between the ligand (phorbol 13-acetate) and the C1 domain. The WT C1 formed hydrogen bonds to phorbol 13-acetate in water continuously during 250 ns, but the same continuity was not observed in the presence of ethanol (Fig. 4A). This is observed for all the three simulations (Fig. S5). In water, WT C1 formed hydrogen bonds with Trp-588, but in ethanol it formed a hydrogen bond with Leu-586, as well as with Trp-588 (Fig. 4B). In ethanol, phorbol 13-acetate moved slightly from the top of the two loops of the C1 domain toward Leu-587 during the pressure coupling. Phorbol 13-acetate formed a hydrogen bond with the indole NH group of Trp-588 until about 45 ns of the MD production time. It moved slightly toward Leu-586 again, and then it formed a hydrogen bond with the amino group of Leu-586 at around 60 ns.

Figure 4. Hydrogen bond formations between phorbol 13-acetate and the C1 domain during 250 ns MD simulation in 100% PS.

Figure 4.

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(A) Plot of the number of hydrogen bonds between phorbol 13-acetate and the C1 domain during 250 ns MD simulation for four different systems: Munc13-1 C1 + phorbol 13-acetate + water; Munc13-1 C1 + phorbol 13-acetate + ethanol; Munc13-1 C1 (E582A) + phorbol 13-acetate + water and Munc13-1 C1 (E582A) + phorbol 13-acetate + ethanol. The initial docking poses of the phorbol 13-acetate and C1 domain are the same in water and in EtOH before the systems reach equilibrium. Ethanol affected hydrogen bond formation between the C1 domain and phorbol 13-acetate. The plot is the 30 ps running averages of the number of hydrogen bonds. (B) Snapshots of the phorbol 13-acetate-bound C1 domain at the point of energy minimized system (EM), 0, 100, 200, and 250 ns. C1 domain at 0 ns is represented by the solid ribbon. The colored stick indicates phorbol 13-acetate; EM (gray), 0 ns (yellow), 100 ns (cyan), 200 ns (green), and 250 ns (orange). The residues which formed a hydrogen bond are shown in magenta. The dotted line represents the hydrogen bond formed at 0 ns. WT, Wild-type; EtOH, ethanol.

E582A C1 in water and ethanol formed less hydrogen bonds with phorbol 13-acetate than WT C1 (Fig. 4A and Fig. S5). Fig. 4B shows differential interaction of phorbol 13-acetate with WT and E582A C1 in ethanol.

In summary, ethanol affects the number of hydrogen bonds between phorbol 13-acetate and C1 domain residues. Mutation of Glu-582 to alanine also influenced the number of hydrogen bonds between phorbol 13-acetate and the C1 domain.

Effect of ethanol on the interactions between the ligand and the membrane containing 100% PS

After determining the effect of ethanol on the interaction between the ligand and the protein, we also investigated if ethanol could affect the interaction between the ligand and the membrane. WT C1 in water and in ethanol showed the similar number of hydrogen bonds initially for all the three simulations. But in ethanol, the number of hydrogen bond went up to 4, at least for one simulation (Fig. 5 and Fig. S6). The possibility is that the phorbol 13-acetate cannot form many hydrogen bonds with the membrane since the membrane is composed of the hydrophobic tails of the lipid molecule. In water, phorbol 13-acetate stayed within the membrane throughout 250 ns. In ethanol, phorbol 13-acetate slowly comes to the surface of the membrane till 100 ns and remains there till 250 ns, allowing it to interact with the phosphatidylserine head groups. However, the other replica in ethanol did not show hydrogen bonds after 65 ns. Three replicas of WT C1 in ethanol showed three different results unlike WT C1 in water (Fig. S6). This indicates that ethanol influenced the number of hydrogen bonds between phorbol 13-acetate and the membrane in WT C1. E582A in water and ethanol showed less number of hydrogen bonds between phorbol 13-acetate and the membrane than WT. The number of hydrogen bonds with E582A in water were changed only from 0 to 1. However, the number of hydrogen bonds with E582A in ethanol kept changing continuously from 0 to 4 through 250 ns.

Figure 5. Hydrogen bond formations between phorbol 13-acetate and the membrane during 250 ns MD simulation in 100% PS.

Figure 5.

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A plot of the number of hydrogen bond between phorbol 13-acetate and the membrane during 250 ns. The initial docking poses of the C1 domain - phorbol 13-acetate complex to the membrane are the same in water and in EtOH before the systems reach equilibrium. The presence of EtOH increased the number of hydrogen bonds between phorbol 13-acetate and membrane compared to WT in water. The plot is the 30 ps running averages of the number of hydrogen bonds. WT, Wild-type; EtOH, ethanol.

In summary, ethanol affected the number of hydrogen bonds between of phorbol 13-acetate and membrane for both WT C1 and E582A. However, such effect was not prominent in E582A, indicating a possible role of Glu-582 in hydrogen bond formation.

Occupancy of ethanol in Munc13-1 C1 in the membrane containing 100% PS

Ethanol molecules were found to move around the system during the 250 ns simulation. To determine if ethanol molecules have a preference for any particular site(s) in Munc13-1 C1, and if a Glu-582 to alanine mutation could affect this preference, we measured the occupancy of the ethanol molecule in the vicinity (4Å) of Glu-582/Ala-582 in Munc13-1 C1. The occupancy was measured by calculating the percentage of the time ethanol molecules spend in the vicinity of the Glu-582/Ala-582 during the timescale (250 ns) of the MD simulation. The occupancy of ethanol was found to be 34.4 ± 3.0 % for WT C1 domain and 26.5 ± 5.4 % E582A C1 at 100% PS.

In summary, the occupancy of ethanol in the vicinity of Glu-582 is influenced by mutation at this site.

Binding site of ethanol on Munc13-1 C1

To understand how ethanol molecules bind to the Munc13-1 C1 residues, we did cluster analysis of the ethanol molecules by creating 20 clusters within 6 Å of Glu-582 (Fig. 6). The bigger the size of the cluster, the higher is the number of ethanol molecule in the cluster. We selected a distance of 6 Å to cover most of the residues surrounding the alcohol-binding residue, Glu-582. 689 ethanol molecules were observed in 753 frames of ethanol and WT C1. On the other hand, 619 ethanol molecules were observed in E582A C1.

Figure 6. The cluster of ethanol molecules abutted to Glu-582/Ala-582 in 100% PS.

Figure 6.

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20 Clusters (represented as balls) chosen to represent the site of the ethanol retained within 6 Å of Glu-582 (Ala-582). The bigger size of a ball indicates a higher number of ethanol molecules in a cluster. The yellow and green stick indicate the Glu-582 and Ala-582. The arrow indicates the fluctuation of the Glu-582 and Ala-582 during the simulation. C1 domain at 0 ns is represented by the solid ribbon WT C1 showed larger size clusters as compared to E582A C1. WT, Wild-type.

The cluster analysis revealed that ethanol molecules interacted with the C1 domain at multiple sites during 250 ns MD simulation, although some sites are preferred over others. While the biggest cluster in WT C1 had 107 (15.5%) ethanol molecules, E582A C1 had 68 (10.9%) ethanol molecules in its biggest cluster. Smaller clusters were more scattered around E582A C1 as compared to WT C1, indicating that ethanol had preferred regions for binding to the WT C1 domain but not the mutant. The range of the fluctuation of Ala-582 during simulation time was close to the small clusters. The minimum distance between the Ala-582 and the biggest cluster is 4.5 Å. However, Glu-582 is in the vicinity of the biggest cluster and the minimum distance between the Glu-582 and the biggest cluster is 3.1 Å. It was shown in the previous section that ethanol molecules had higher occupancy in WT C1 than in E582A C1. Taken together, then, this results suggest that ethanol molecules reside around Ala-582 in E582A C1 at many regions for a shorter duration, but reside around Glu-582 in WT C1 at fewer regions for a longer duration. A snap shot of the 127.71 ns MD simulation shows that there are two ethanol molecules nearby Glu-582, each forming a hydrogen bond with its carboxyl group or the backbone amino group. Leu-611, Tyr-581 and Asn-613 provided hydrophobic interaction to the lower ethanol molecule and the –CH-CH2 group of serine moiety of PS provided hydrophobic interaction to the upper ethanol molecule (Fig. S7).

In summary, ethanol molecules showed preferred binding regions near Glu-582 in WT C1 as compared to Ala-582 in E582A.

Effect of ethanol on WT/E582A C1 in membrane containing 20% PS

We also measured the protein-ligand, protein-membrane and ligand-membrane interactions, and the occupancy of ethanol in membrane containing 20% PS in PC. The fluctuation of RMSD of WT C1 and E582A C1 in water was slightly reduced in the 20% PS membrane, as compared to 100% PS (Fig. S8). WT C1 in water moved along the X and Y-axis in the membrane, but E582A did not move much (Fig. S9). However, both WT and E582A C1 moved along the X and Y-axis in the membrane in the presence of ethanol (Fig. S9). The total area that the WT C1 traversed in the XY plane in the presence of water and ethanol were 2.080 ± 0.287 nm2 and 2.016 ± 0.469 nm2, respectively. For the E582A C1, the values were 1.471 ± 0.498 nm2 and 2.256 ± 0.122 nm2 (Fig. S9).

As for the hydrogen bond formation is concerned, WT C1 formed slightly more hydrogen bonds (maximum 4) with phorbol 13-acetate than in 100% PS (maximum 3) and the binding site of the ligand was slightly changed (Fig. S10). Ethanol affected the hydrogen bond formation between WT C1 residues and phorbol 13-acetate. E582A C1 in ethanol showed higher number of hydrogen bonds (maximum 3) than 100% PS (maximum 2). There were slightly more hydrogen bonds between phorbol 13-acetate and the membrane for WT C1 in ethanol than water (Fig. S11). However, for the mutant in ethanol shows lower number of hydrogen bonds.

In the cluster analysis, 733 ethanol molecules were observed within 6 Å of Glu-582 for WT C1 and 463 ethanol molecules for E582A C1 in 753 frames. The size of the clusters of WT C1 was smaller than the cluster size in 100 % PS (Fig. S12). At 20% PS, the occupancy of ethanol for of E582A (37.5 ± 7.4 %) was higher than the WT (22.3 ± 3.2 %).

Discussion

The objective of the present investigation is to gain structural insights into how ethanol interacts with proteins in a lipid membrane environment. To this end, we have performed MD simulation studies of the membrane-targeting C1 domain of the presynaptic protein Munc13-1 in its activator-bound state in the presence of ethanol in membrane. The C1 domain-containing proteins specifically interact with PS, whose concentration in the plasma membrane varies depending on the cell type (Newton, 1993a, Ingolfsson et al., 2014). In our previous binding studies using photolabeling, mass spectrometry and FRET analysis, we identified Glu-582 of Munc13-1 C1 as the alcohol-binding residue (Das et al., 2013, Xu et al., 2018). However, the binding studies were done in the membrane-free condition. Here we studied the ethanol-protein interactions in the presence of lipid membrane containing PS. For the MD studies, we first docked the ligand phorbol 13-acetate into Munc13-1 C1 and built the membrane around it and ran simulation in the presence and the absence of ethanol. The docking site of the phorbol ester in Munc13-1 C1 was slightly different than that of PKC C1 because of the occlusion of the binding site by Trp-588 in Munc13-1 C1, but not by the homologous Trp in PKC C1 (Shen et al., 2005, Zhang et al., 1995). Our earlier studies suggest that there are no significant movement of Trp-588 during the MD simulations (Das et al., 2018, Blanco et al., 2019).

The current study showed that ethanol increased the stability of Munc13-1 C1 as measured by its lower fluctuation of RMSD values in ethanol as compared to water (Fig. 2B and Fig. S1). Similar observations were made in a study with the Glycine receptor (GlyR), in which ethanol stabilized the open state of the receptor (Murail et al., 2011). Ethanol also reduced the horizontal movement of the WT C1 domain in the membrane with 100% PS as compared to water, again suggesting ethanol’s stabilizing effect (Fig. 3, Fig. S3, and Fig. S4).

To understand the role of Glu-582 in mediating ethanol’s effects, a comparison was made between the WT C1 and the E582A C1 for their stability, protein-ligand, protein-membrane and ligand-membrane interactions. Ethanol’s effect on the increased stability was not observed in E582A C1, suggesting that Glu-582 mediates the interaction between ethanol and the C1 domain (Figs. 2, 3, S1, S3, and S4). As for the activator-C1 interactions, in the presence of ethanol, there were fewer hydrogen bonds between phorbol-13-acetate and the C1 domain than in water, suggesting reduced affinity due to movement of different residues at the activator binding site in the presence of ethanol (Fig. 4A and B). Our previous FRET data using the fluorescent ligand, dansyl-DAG, also supported this result (Xu et al., 2018). However, ethanol influenced (increase or decrease) in hydrogen bonds between the phorbol 13-acetate and membrane (Fig. 5 and Fig S6). Further, ethanol did not reduce the movement of E582A C1 in the membrane, unlike the WT C1, again suggesting the role of an ethanol-Glu-582 interaction in the mobility of the protein in the membrane (Fig. 3 and Fig. S4). As for the occupancy, ethanol molecules reside in the vicinity of Glu-582 longer in WT C1 than in the vicinity of Ala-582 in E582A C1 in 100% PS. The carboxyl group of Glu-582 forms up to three hydrogen bonds with ethanol molecules during 250 ns. Because of relatively shallow pocket around Glu-582, many different ethanol molecules, not a specific one, interact with Glu-582 continually during 250 ns.

The interaction between the C1 domain and membrane is affected by the net charge of the C1 domain. PS has a net negative charge and we previously reported that Munc13-1 C1 with its net negative charge shows higher binding affinity to the ligand at 20% PS than at 100% PS (Das et al., 2018), while the PKCδ C1B and PKCθ C1B with net positive charge show higher binding affinity at 100% PS (Rahman et al., 2013). In 20% PS, the repulsion between C1 domain and the membrane was reduced and Glu-582 could interact with the positively charged choline moiety of the lipid membrane. Further, because of this varying charge of the membrane, the orientation of the C1 domain and the Glu-582 was also somewhat altered. The orientation of the C1 domain in 20% PS, where seemingly a ~90° rotation of the entire domain, placing the phorbol ester site in the plane of the lipid head groups and rigidifying the mutation site throughout the simulation (Fig. S13A). It is difficult to predict from our studies if this pose could be of any physiological relevance, since it would presumably place the remainder of the Munc13-1 protein in proximity to or conflict with the membrane leaflet. (Fig. S13). As a result, in 20% PS, the Glu-582 could come closer to membrane, making it less accessible to ethanol molecules than in 100% PS. Indeed, distance measurements show that Glu-582 is closer to the membrane in 20% PS than in 100% PS (Fig. S13B). For E582A C1, there was no difference in the distance between Ala-582 and the membrane at either PS concentration because of the absence of Glu-582. This could be the reason why higher occupancy was observed for E582A in 20% PS than WT C1.

In Munc13-1 C1, the alcohol-binding residue Glu-582 is located close to a short α-helix away from both membrane and the activator binding site (Das et al., 2013). This α-helix however, changed its conformation during the MD simulation. The surface diagram showed that there are shallow and solvent-exposed pockets near Glu-582 where ethanol can bind. The hydroxyl group of ethanol forms hydrogen bond with the carboxylic acid group of Glu-582 and hydrophobic interactions are exerted by Leu-611, Tyr-581 and Asn-613 or by the CH-CH2 group of the PS’s serine moiety (Fig. S7). Although this is based on one frame shown for the representation purposes, analysis of multiple frames suggests that ethanol, protein and membrane are involved in atomic interactions. As per the recent crystal structure of Munc13-1, the C2B domain lies adjacent to the C1 domain and forms charge pairs with several of the negative charges on the C1 domain. This helps the C1 domain to reorient and contribute to membrane binding. Of these, positively charged Arg-750 and Arg-754 of the C2B domain are in close proximity to the alcohol-binding residue Glu-582 of C1 (Xu et al., 2017). Therefore, it is highly likely that domain-domain interactions will affect ethanol binding at this site. Is there a commonality of the ethanol binding site of Munc13-1 C1 and other alcohol-binding proteins? Glutamate has the propensity of forming hydrogen bonds and has been implicated in alcohol binding for several proteins, such as Glu-262 in acetylcholine receptor (Pratt et al., 2000); Glu-33 in L1 cell adhesion molecule (Arevalo et al., 2008); Glu-163 and Glu-193 in Rho GDP dissociation inhibitor (Ho et al., 2008); Glu-146 in lignin peroxidase (Ambert-Balay et al., 1998); and Glu-13 in pepsin (Andreeva et al., 1984). However, Glu-582 is not a conserved residue in homologous C1 domains. The alcohol-binding residues of PKCε C1B are His-248 and Tyr-250 (Das et al., 2009, Pany and Das, 2015) and in PKCδ C1B, the alcohol-binding residue is Tyr-236, which forms a hydrogen bond with the hydroxyl group of cyclopropyl methanol (CPM) and Met-239 exerts a hydrophobic interaction with the methylene groups of CPM (Shanmugasundararaj et al., 2012). Like Munc13-1C1, alcohol binding site here is also a shallow pocket and somewhat solvent-exposed. In the Drosophila odorant-binding protein LUSH, the ethanol binding site is between two α-helices, α3 and α6. Ser-52 and Thr-57 of α3 form a hydrogen bond with the hydroxyl group of the ethanol molecule and Trp-123, Phe-113 and Phe-64 of α6 exert a hydrophobic interaction with the ethyl group of ethanol (Kruse et al., 2003). In the proton-activated G. violaceus ligand-gated ion channel (GLIC), the ethanol binding site is at the inter-subunit interface. Glu-19’, Asn-15’ and Asn-200 can form hydrogen bonds with the hydroxyl group, and Leu-17’ and Ilu-16’ provide hydrophobic interactions (Sauguet et al., 2013). In IRK1, a variant of K+ channel GIRK2, 2-methyl-2,4 pentanediol (MPD) binds at a hydrophobic pocket located on the surface of the cytoplasmic domain. Tyrosine residues and backbone carbonyls form hydrogen bonds with the hydroxyl group of MPD (Aryal et al., 2009). Taken together, the structural motif of the alcohol binding site may differ from protein to protein, but the common feature of all the sites is that the hydrogen bond and hydrophobic interactions are the key atomic forces for alcohol’s binding to protein.

Munc13-1 is a peripheral membrane protein that facilitates assembly formation by bridging the vesicle and the plasma membrane and acts as a master regulator of vesicle exocytosis (Rizo, 2018). Our results showing that ethanol influences the interaction between the activator and the protein, between the protein and the membrane, and between the activator and the membrane, indicate that ethanol may affect vesicle exocytosis by modulating protein activity. Ethanol can directly bind to the vesicle membrane and alter lipid bilayer properties; it can affect the binding of active zone proteins to the membrane, thereby altering its activity; and it can directly bind with the proteins and regulate their activities. Several studies indicated such ethanol-lipid-protein interactions (Crowley et al., 2003, Yuan et al., 2007, Ghare et al., 2011, Bettinger et al., 2012). Further experiments are needed to determine whether these effects are sufficient to alter protein function at the concentration at which ethanol exerts its intoxicating effects.

In conclusion, ethanol stabilizes the ternary complex, C1 domain-phorbol 13-acetate-lipid membrane through Glu-582. Multiple ethanol molecules can bind to the Glu-582 continually. This study is important in providing structural basis of interaction between ethanol and Munc13-1 in a membrane environment and will contribute to elucidating role of ethanol in synaptic plasticity.

Supplementary Material

Supp FigS1-13

Acknowledgements

MD simulations were performed using the server at the Center for Advanced Computing and Data Systems (CACDS) of the University of Houston. This research has been supported by funding from National Institutes of Health Grant 1R01 AA022414−01A1 to J.D. We thank Courtney Hunt, Ph.D. for editing the manuscript.

Footnotes

Notes

The authors declare no competing financial interest.

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