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Translocation of Human # Defensin Type 3 through a Neutrally Charged Lipid Membrane: A Free Energy Study Rabeta Yeasmin, Matthias Buck, Aaron Weinberg, and Liqun Zhang J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b08285 • Publication Date (Web): 15 Nov 2018 Downloaded from http://pubs.acs.org on November 22, 2018

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The Journal of Physical Chemistry

Translocation of Human β Defensin Type 3 through a Neutrally Charged Lipid Membrane: A Free Energy Study Rabeta Yeasmin∗ Department of Chemical Engineering, Tennessee Technological University Matthias Buck† Department of Physiology and Biophysics, Case Western Reserve University, School of Medicine, Cleveland, OH, 44106 Aaron Weinberg‡ Department of Biological Sciences, Case Western Reserve University, School of Dental Medicine, Cleveland, OH, 44106 Liqun Zhang§ Department of Chemical Engineering, Tennessee Technological University, Cookeville,TN 38505 Tel: 931-372-3474

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Abstract Human β defensin type 3 (hBD-3) is a cationic (+11 charged) antimicrobial peptide. It has 3 pairs of intramolecular disulfide bonds which can break to form the linear analog of hBD-3 under reducing conditions. hBD-3 can disrupt both gram-positive and gram-negative cell membranes, and even mammalian cell membrane at high concentrations. However, the structural basis for the membrane-disrupting function of hBD-3 is still unknown. In order to understand the interaction mechanism of hBD-3 with a neutrally-charged lipid membrane, explicit solvent and lipid umbrellasampling simulations were performed using NAMD program on the hBD-3 wild-type and the linear analog, in both the monomer and dimer forms. During the insertion and translocation process, most of the protein structure changes take place near the membrane-solvent interface, while the membrane interior appears to stabilize and rigidify the native-like hBD-3 structure. An energy barrier of 20 kcal/mol(domain unit) should be overcome by hBD-3 dimer in wild-type to cross the POPC bilayer but only 13 kcal/mol(domain unit) to insert into the bilayer center, and 20 kcal/mol for hBD-3 monomers to insert into the membrane center. Significant reorientation of lipids around hBD-3 inside the membranes was observed, which suggests a toroidal model for the membrane disruption process.

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Electronic address: [email protected]

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1.

INTRODUCTION

Defensins are cationic cysteine-rich small molecules with molecular masses ranging from 3 to 5 kDa[1]. They are a critical part of the innate immune system that provides an initial antimicrobial barrier for mucosal surfaces such as the oral cavity, surface of the eyes, airways, lungs, and skin[2–8]. They have broad-spectrum anti-microbial activity against encapsulated viruses, fungi, both Gram positive and Gram negative bacteria, and even against antibiotic resistant bacteria[9, 10]. Mammalian defensins are classified into α, β, θ categories based on their size and disulfide bonding pattern. Among the class of human β defensins (hBD), hBD type 3 (hBD-3) is of special interest. hBD-3 is mainly secreted from epithelial cells[11, 12]. It has 45 residues, and is composed of one α-helix, and three anti-parallel β-strands that are held together by three intramolecular disulfide bonds formed by six cysteine residues. Its secondary structure is shown in Figure 1 (Top) and its disulfide bridge connection in Figure 1(Bottom). The disulfide bonds are very important for restraining the structure of hBD-3[13]. Under reducing conditions, those disulfide bonds break, which converts hBD-3 into the linear analog form. Interestingly, the disulfide bonding status does not influence the anti-bacterial activity of hBD-3[14–17]. Antimicrobial peptides (AMPs) can interact with the bacterial lipid membrane and disrupt the bilayer boundary of cells. Up to now, there are 4 theories to explain the disruption mechanism of AMPs on lipid membrane, which includes the carpet model, the pore formation model which can be further classified into toroidal model and barrel-stave model, and the in-plane diffusion or partial insertion model[18–20], with a sketch of different disruption mechanisms shown in Figure 2. In the carpet model, AMPs bind to the surface of bacterial membranes, cover it in a carpet-like manner, and dissolve it like a detergent[21]. In the pore formation model, the AMPs bind with the lipid membrane first, then aggregate within the membrane, and lastly form channels inside the membrane[10, 22]. If significant reorientation of lipids happens, the model is referred to as toroidal; otherwise, it is referred to as a barrel-stave model. In the in-plane diffusion or partial insertion model, AMPs only bind with the lipid membrane on the surface and partially insert into the membrane. The antibacterial activity of defensins is related to their capability to disrupt lipid membranes. Because hBD-3 is strongly positively charged, it can interact with negatively charged ACS Paragon 3Plus Environment


FIG. 1: Top).The secondary structure of hBD3, which has one α helix (shown and labeled in red), three β strands called β1 to β3 (in blue), two loops or turns called L1 to L2 (in cyan), and three coils or bends called C1 to C3 (in green). The three pairs of disulfide bonds are shown in yellow sticks. The head/N-term, tail/C-term and several connection regions between coils and loops are shown in wheat, with no assigned secondary structure; Bottom). The sequence and disulfide bridge connection of hBD-3.

lipid head groups such as POPS and POPG. Usually, bacterial lipid membranes[23] are negatively charged while the normal mammalian cells are overall neutrally charged or have the negatively charged lipid phophatidylserine on the inner side of the cell membrane. So hBD-3 can distinguish between bacterial cells and normal cells and disrupt the lipid membranes of bacterial cells only. The mechanism by which hBD-3 disrupts lipid membranes is still unknown. However, experimentally it is clear that hBD-3 disrupts freshly isolated monocyte (normal) cells which expresses negatively charged phospholipid PS on the outer membrane, but not after protecting the monocyte cells by binding the PS with other molecules[24]. Recently, Phan et al.[25] determined that hBD-3 can permeabilize different kinds of cell membranes, and even the monocyte membrane at high enough protein concentration. We are motivated to study the disruptive effect of single and oligomeric hBD-3 on non-charged membranes as those respective molecules translocate the lipid bilayer, before forming membrane disrupting structures, such as pores or carpets with the negatively charged lipids on ACS Paragon 4Plus Environment

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FIG. 2: Four kinds of disruption mechanisms of AMP with lipid membrane.

the inside of the bilayer. Molecular dynamics (MD) simulations have become a key tool in elucidating the structure, dynamics and functional mechanism of proteins [13, 26–30]. Researchers have worked to understand the interaction between cell-penetrating peptides and lipid membranes by energy calculation and by direct interaction/insertion studies of antimicrobial peptides (AMPs) with lipid membrane using MD simulations[31, 32]. General and Asciutto [33] studied the positively charged peptide Penetratin’s association and translocation to a lipid membrane using umbrella-sampling (US) simulations with restraints on the membrane. They found that the secondary structure of the peptide changed as it moved across the bilayer. By interrogating a total of 91 Bac2A-based peptides, including the wild-type and diverse mutants, through the lipid membrane using umbrella-sampling simulations, Zhao et al.[34] revealed a good correlation for the peptides between predictive transmembrane activity and antimicrobial/hemolytic activity. Cell penetrating peptides usually have multiple arginine residues. Researchers have performed umbrella-sampling simulations also on the arginine rich cell penetrating peptides across lipid bilayers[35–37]. Huang et al.[35] did long term all atom US simulation to investigate the free energy of translocation of an arginine-rich cell-penetrating peptide (a cyclic Arg9 or abbreviated as cR9) across a DOPC lipid bilayer. Their result suggested the water-pore formation translocation mechanism for cR9. Hu et al.[36] did multi-microsecond long coarse-grained umbrella-sampling simulations on translocation of positively charged linear and cyclic Arg9 into a DPPC bilayer. They systematically analyzed factors such as peptide conformation, media components including water, ion, and ACS Paragon 5Plus Environment


lipid membrane contributing to the translocation energy barrier of Arg9 peptide across the lipid bilayer. Their free energy calculation also supported the pore formation mechanism for Arg9 passing through the DPPC lipid bilayer. Based on our survey, to date, no research has been done to calculate the free energy of translocation of hBD-3 through a lipid membrane, although recently a study has been published of Scots Pine Defensin interacting with a model membrane surface using coarse grained MD[29]. In this project, umbrella sampling simulations using the NAMD program have been performed to study the free energy of hBD-3 monomer and dimer approaching and crossing a POPC lipid bilayer, using both explicit solvent and lipid. Based on potential of mean force calculations using the WHAM program from different US windows, it is found that hBD-3 insertion into the neutrally charged lipid membrane requires less energy than crossing the membrane. From the structure analysis, it is found that most of the structural changes of hBD-3 monomer and dimer occur near the solvent-lipid interface, while the membrane is thinned near the inserted protein due to the lipid head-groups coming into the membrane to form interactions with charged and polar protein sidechains. Significant reorientation of lipids relative to the protein was observed. A toroidal lipid pore formation/peptide translocation model was suggested based on the result of this project.

2.

MATERIALS AND METHODS 2.1.

Simulation Details

All-atom simulation set up. hBD-3 can form a dimer[38]. In reducing condition, the disulfide bonds on hBD-3 will break, which converts hBD-3 into the linear analog form. In order to understand the effect of hBD-3 oligomerization status and its disulfide bonding status on its translocation capability, in this project, simulations on both the monomer and dimer forms of hBD-3 were set up with the monomer in both the wild-type and linear analog states, and the dimer in wild-type. The crystal structure of hBD-3 was downloaded from the protein data bank (PDB ID of 1KJ6)[38]. The initial dimer structure of hBD-3 was predicted in the previous work[13] by aligning two hBD-3 molecules at a similar position to the NMR predicted dimer structure by Schibli et al.[38]. In the hBD-3 linear analog form, all three disulfide bonds were broken and cysteine sidechains given as -SH, compared to the ACS Paragon 6Plus Environment

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S-S bonds in the wild-type (connections shown in Figure 1). The electrostatic interaction is important to the dynamics and function of hBD-3[39, 40]. Since the N-terminus and β2 strand are positively charged and are important to the disruption of hBD-3 on membrane, in this project, the initial insertion orientation of hBD-3 is by its N-terminus with a slight tilt and with β2 strand facing the lipid membrane. The insertion structure and orientation for hBD-3 monomer and dimer are shown in Figure S1 in the Supplemental Material. The dimer was inserted with one monomer in exactly the same direction as in the monomer simulation. The hBD-3 dimer has a globular shape, thus simulations on the hBD-3 dimer translocating through the POPC lipid membrane (in total 200 lipids with 100 POPC lipids in each leaflet) were set up to cover the whole height range of hBD-3 from totally outside of the membrane to inside, then to totally outside, on the other side. However, hBD-3 monomer is faraway from a globular form as shown in Figure S1, thus, instead of setting up simulations to observe the whole translocation process, only the insertion process from outside of the membrane to the lipid bilayer center was studied. In order to set up the different windows for the umbrella-sampling simulations, the CHARMM-GUI program[41] was performed to place the hBD-3 monomer and dimer at the center of the POPC (100 in upper layer and 100 in lower layer) lipid bilayer with the orientation shown in Figure S1. The two glutamate residues present in hBD-3 sequence, were not protonated. In order to observe the whole crossing process of hBD-3 dimer and insertion process of hBD-3 monomers, enough amount of water molecules were added into each system to make sure even at the highest/lowest window there are still at least 12 ˚ A of water on the above/below COM of protein. This was accomplished by applying a water thickness of 60 ˚ A above as well as below the POPC membrane surface. The size of each system, number of atoms, and number of counter ions in system are shown in Table S1 in the Supplemental Material. After that, Steered MD (SMD) was performed to pull the hBD-3 dimer to the top and bottom of the box by applying a small enough constant force (0.5 kcal/(mol·˚ A2 ) on Ca atoms of the protein and with a restrain force of 0.3 kcal/(mol·˚ A2 ) on N, C218, and C316 atoms of the lipids to make sure no lipids come out of the membrane with the protein. Although POPC lipid is overall neutrally charged, it is zwitterionic, having both a negatively charged phosphate and a positively charged choline group, making the entire head group neutral. Importantly, the small enough constant force in pulling can also allow the protein reorient if it has strong interaction with lipids. SMD was also performed to pull ACS Paragon 7Plus Environment


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the hBD-3 monomer from the center of the membrane to the outside of the membrane. The movie generated based on the steered MD simulations on hBD-3 dimer is shown in Video S1 in the Supplemental Material. Based on the structure of hBD-3 dimer at different heights (distances to the bilayer center) in the system, in total 49 windows were generated, with the distance between the center of mass (COM) of protein to the COM of lipid bilayer to be -48 ˚ A to 48 ˚ A with a distance gap of 2 ˚ A. Based on the structure of the hBD-3 monomer at different heights in the system, in total 25 windows were generated, with the distance between the COM of protein to the COM of lipid bilayer to be 0 ˚ A to 48 ˚ A with a distance gap of 2 ˚ A. Simulations performed and details of each system are shown in Table S1. In order to calculate the potential of mean force (PMF) for the hBD-3 transferring through/insertion into the neutrally charged lipid membrane, umbrella-sampling simulations were performed in the NPAT (the abbreviation of NPz AT, here Pz is the normal pressure) ensemble with the NAMD program ver. 2.10[42] and the covar restraint. The CHARMM36 forcefield[43] was applied with dihedral cross-term corrections (CMAP)[44]. The temperature was set at 313.15 K to make sure the neutrally charged POPC lipid membrane stay in the lamellar fluid phase throughout the simulation. Since the dynamics of protein in lipid has strong temperature dependence[45], umbrella-sampling simulations at higher temperatures such as 383.15 K and 453.15 K were performed as well for hBD-3 monomer in both wild-type and analog forms. Based on our test results on protein COM position and RMSD calculation, the system in each window can reach an equilibrated state within 3 ns. Since the dynamics and capability of AMP to translocate into the membrane improve significantly at higher temperatures [45], shorter simulations were performed on hBD-3 monomers at two higher temperatures. After finishing 80 ns US simulations on hBD-3 monomers at 313 K, 36 ns US simulations at 383 K were continued in each window. After that, the system temperature was increased again to 453 K, and another 36 ns US simulations were performed in each window. The time step in MD simulations was 2 fs. Potential of Mean Force Calculation. The Potential of Mean Force (PMF) was calculated using the WHAM program[46] by combining results of hBD-3 in different windows. The biasing potential was calculated using the following form: Ebias = k ∗ (z − z0 )2

(1)

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bilayer, and z0 is the target height of the window. In the US simulations, constant k was set as 3 kcal/(mol·˚ A2 ), following tests on both hBD-3 monomer and dimer based. Besides that, the umbrella sampling histograms derived from the sampling windows were also analyzed for each system to make sure the neighboring bins have good enough amount of overlap. A representative histogram for the hBD-3 dimer is shown in Figure S2 in the Supplemental Material. The radius of gyration (Rg) in this project was calculated using the wordom program[47, 48], while the hydrophobic and hydrophilic accessible surface areas (ASA) were calculated using the CHARMM[49] program based on the Richards and Lee’s method[50] with a solvent radius of 1.4 ˚ A, following the same method as ref[26]. The CHARMM program c41b2 was also applied to calculate the membrane thickness, using the phosphate group position as reference for the lipid head position. Averaging the lipid head group positions in each layer individually, the membrane thickness is determined by subtraction of the average height of upper-layer lipid headgroups by the average of the lower-layer lipid headgroups. This method has been used before by other paper[51]. Root mean squared fluctuations (RMSF), which represents the averaged structure fluctuation of a residue Ca atom, is also calculated with CHARMM. The number of lipid heads within 6 ˚ A or 4 ˚ A to protein surface atoms was calculated using CHARMM program. The average number and the standard deviations are calculated based on snapshots output from the last 30 ns simulation trajectories at a frequency of 1 ns.

3.

RESULTS 3.1.

The Middle of the Bilayer Presents a Structure and Dynamics Stabilizing

Environment

Based on explicit solvent and lipid NAMD simulation trajectories in each US window, protein structures were analyzed for each system. The structures of hBD-3 have deviations from the original monomer/dimer structures by different amounts along the height. The average RMSD and standard deviation based on the last 70 ns for dimer and last 30 ns for monomer MD trajectories in different windows are shown in Figure 3. Analyzing the RMSD fluctuation in the last 30 ns for dimer, the result is shown in Figure S3 in the Supplemental ACS Paragon 9Plus Environment


Material. At the force constant of 3 kcal/(mol·˚ A2 ), the RMSD of hBD-3 dimer inside or around the lipid membrane are stable, with the RMSD no more than 8 ˚ A and still in a dimer form. At the faraway positions (further than 30 ˚ A), the RMSD of hBD-3 dimer can reach 10 ˚ A and above, which can agree with findings from the previous work[13]. Calculating the RMSD for the two units in the dimer separately, we have the result shown in Figure 3 (Left). As can be seen, inside the membrane (in the height range of -24 ˚ A to 24 ˚ A), the RMSD of each unit is no more than 4 ˚ A. At the membrane and water interface and also inside the solvent, the RMSD of each unit can reach 7 ˚ A. Comparing the RMSD of hBD-3 dimer with that of each unit, large deviations were observed outside the membrane. Considering the transient dissociation of the dimer as it emerges after transiting the membrane, that proves that the dimer structure deviated from the original dimer structure significantly. At the height of 48 ˚ A, 46 ˚ A, 40 ˚ A, 38 ˚ A, 32 ˚ A, -38 ˚ A, and -44 ˚ A, temporal dissociation of hBD-3 dimer was observed in the last 30 ns. That contributes to the large RMSD result for hBD-3 dimer in wild-type form at those places as shown in Figure 3 (Left). The average RMSD and the standard deviation for hBD-3 monomer in both wild-type and analog forms at different temperatures are shown in Figure 3 (Right). hBD-3 structure deviation from the original crystal structure is the smallest (around 2 ˚ A) at the center of the membrane (with height equals to zero). With the height increasing, the RMSD increases. The RMSD of analog form increases with temperature rising. However, the monomer wildtype has similar RMSD at different temperatures, except a higher RMSD at 453.15 K inside the membrane; i.e., emphasizing the importance of the disulfide bonds in restraining hBD-3’s structure. The hBD-3 dimer RMSD reaches a local minimum at around 6 ˚ A, and two local maximums with one at around -18 ˚ A and the other at around 18 ˚ A. The RMSD of the hBD-3 monomer reaches the minimum at 0 ˚ A, and has two local maximums with one at around 18 to 24 ˚ A and the other one located at around 36 to 42 ˚ A. Considering the maximum structural deviation of hBD-3 dimer and monomer are around the solvent and lipid interface, that emphasized the hydrophobic effect on the protein structure change. Those are also consistent with the radius of gyration result as shown in Figure S4 and the accessible surface area (ASA) result shown in Figure S5 and S6 in the Supplemental Material, which also show local maximums at the solvent and lipid interfaces. The larger hydrophobic ASA ACS Paragon10 Plus Environment

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FIG. 3: RMSD for hBD-3 dimer and each unit (unit1 and unit2) in wild-type at different heights in the membrane (Left) based on last 70 ns simulation trajectories, and RMSD comparison (Right) for hBD-3 monomer in wild-type (in black) and analog (in red) forms at 3 different temperatures: 313.15K shown as solid line, 383.15K shown as dashed line, and 453.15 K shown as dot-dashed line. During the RMSD calculation, the monomer structures were aligned on the crystal structure of hBD-3 monomer, while the dimer structure were aligned on the dimer structure predicted by the research lab with details explained in the Simulation Method section.

than the hydrophilic ASA of hBD-3 dimer and monomers shown in Figure S5 and S6 also justified the smaller RMSD of hBD-3 dimer and monomer inside the membrane, since the lipid membrane supplied a hydrophobic environment. Similarly, the RMSF was calculated for hBD-3 in both monomer and dimer forms, and the results are shown in Figures S7, S8 in the Supplemental Material. With height increasing, the RMSF of the residues increase. RMSF inside the membrane (0 ˚ A to ±24 ˚ A) have lower RMSF than windows in solvent. With temperature increasing, the RMSF of hBD-3 wild-type increases slightly, while it increases significantly for the hBD-3 analog. Overall, the RMSDs and RMSFs of hBD-3 molecules, especially the hBD-3 dimer, inside the lipid are much smaller than in the solvent, and the middle of the bilayer presents a structure and dynamics stabilizing environment.

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FIG. 4: Average membrane thickness of POPC lipid bilayer in the system of hBD-3 dimer in wildtype at different regions with the distance d from the protein to lipids being: smaller than 5 ˚ A; in the range of 5 ˚ A to 15 ˚ A; further than 15 ˚ A; and in all the distance range at 313 K. Result from NPT ensemble are highlighted using stars with the line patterns consistent with the corresponding original NPAT ensemble result. 3.2.

The POPC Membrane Thins around hBD-3 Insertion Site and Thickens Else-

where

Based on the US simulation trajectories in different windows, the membrane thickness at three different distance ranges from the protein was calculated, which includes a distance within 5 ˚ A, between 5 to 15 ˚ A and greater than 15 ˚ A from the protein. The average and standard deviations of membrane thickness results for hBD-3 dimer in POPC lipid bilayer are shown in Figure 4, and the result for hBD-3 monomer in the wild-type is shown in Figure 5(a) and Figure 5(b) in an analog/reduced form. Interaction between hBD-3 and nearby lipids within the distance range of 5 ˚ A from the peptide results in a significant membrane thinning. When the hBD-3 dimer is inside the membrane (in the height range of -24 ˚ A to 24 ˚ A), the membrane becomes thinner by 4 to 8 ˚ A(last structures from MD simulations in different windows are shown in Figure S9). Such ACS Paragon12 Plus Environment

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FIG. 5: Membrane thickness of POPC lipid bilayer for hBD-3 monomer in wild-type (a) and analog forms (b) at different temperatures and over different distance ranges from the protein. Results for hBD-3 monomer in analog form at 453 K in NPT ensemble are shown in blue stars. ACS Paragon13 Plus Environment


kind of membrane thinning around protein is also observed for hBD-3 monomer in both wild-type and analog forms at 313 K with an thickness decrease of up to 10 ˚ A, and also at 383 K and 453 K. Focusing on the lipids within the distance range of 5 to 15 ˚ A from the peptide, slight membrane thickening was observed by up to 3 ˚ A inside the membrane (in the range of -24 ˚ A to 24 ˚ A in height), although not so observious for hBD-3 monomers at different temperatures. But the membrane thickening was more obvious for lipids further than 15 ˚ A from the protein. The increase of membrane thickness is up to 6 ˚ A for the hBD-3 dimer inside the membrane, while up to 2 ˚ A for the hBD-3 monomers at all three temperatures within the height range of 0 ˚ A to 12 ˚ A. The stronger interaction between lipids and hBD-3 dimer than the monomer causes more lipid displacement and thus thickening away from the protein compared to the monomer. In order to see if the membrane thinning around protein and membrane thickening at distant lipids from protein was caused by NPAT ensemble effect, in total 9 windows having the heights from 0 ˚ A to 48 ˚ A with a distance gap of 6 ˚ A were selected to conduct the NPT US simulations for 15 ns continuing the last NPAT simulations at different heights. The slight decrease in z-direction then fluctuation of POPC lipid membrane area in different windows were observed, and the fluctuation reaches equilibrium within 2.5 ns. Based on last 5 ns trajectories, the membrane thickness from NPT simulations were calculated with result shown using stars in Figure 4. The membrane thinning within 5 ˚ A to the protein is still significant, so does the membrane thickening in the distance range of 5 ˚ A to 15 ˚ A. However, the membrane thickening at distance further than 15 ˚ A and in the overall membrane almost disappeared. That emphasized the reorientation of lipids to hBD-3 in lipid membrane, which is independent on ensembles. Figure 5 shows that with temperature increasing from 313 K to 383 K, the membrane thickness increased by 1 to 2 ˚ A in both hBD-3 monomer wild-type and analog systems. But it decreased from 383 K to 453 K. That could relate to the significant curvature of membrane that appears to develop at 453 K as shown in Figure S10 and Figure S11. Since the interaction between hBD-3 and lipid heads can build extra stress to disrupt the membrane and the lipid area increases with temperature increasing[52], in order to remove the NPAT effect and see if that will influence the membrane thickness result, we also did 15 ns NPT US simulations on hBD-3 analog at 453 K in nine widows, from 0 ˚ A to 48 ˚ A with a distance gap of 6 ˚ A by continuing the last NPAT system configurations in each window. Small increase ACS Paragon14 Plus Environment

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FIG. 6: Number of lipid heads within the minimum distances of 6 ˚ A or 4 ˚ A to hBD-3 dimer at 313 K (Left), and to hBD-3 monomer in both wild-type and analog forms at 313 K, 383 K, and 453 K.

in membrane surface area was observed during the first 1.5 ns MD simulations, then the membrane surface area fluctuated evenly. Based on last 5 ns simulation result, membrane thickness was calculated in each window with result shown in Figure 5(b). The membrane thinning around protein was still significant, while the membrane thickening at a distance further than 15 ˚ A disappeared since the membrane area is not fixed any more. However, the membrane thickness in different windows at 453 K in NPT ensemble are still very close to the result at 313K and 383 K in NPAT ensemble. That emphasized the lipid heads interaction with protein is independent on ensembles and test result validated the NPAT ensemble result.

3.3.

hBD-3 Molecules Inside the Membrane Are Covered by Lipid Heads

In order to see if the lipid heads are staying with hBD-3 to some extent/get pulled into the membrane, the distances from the lipid heads to the hBD-3 molecules were calculated and the number of lipid heads within the distance of 6 ˚ A or 4 ˚ A to the protein was counted. Based on the last 30 ns simulation trajectories, the average lipid head number and the standard deviation results in different windows are shown in Figure 6 (Left) for the dimer system and Figure 6 (Right) for the monomers. Inside the membrane (in the height range of -24 ˚ A to 24 ˚ A), the number of lipid heads within the distance range of 6 ˚ A or 4 ˚ A to the hBD-3 dimer and monomer is significant. Inside the membrane, the number of lipids close to hBD-3 is much higher than outside. At ACS Paragon15 Plus Environment


48 ˚ A windows, hBD-3 dimer and monomers are totally outside of the membrane, thus the number is zero. That is consistent with the last structures shown in Figure S9, Figure S10 and Figure S11. However, at -48 ˚ A window for the hBD-3 dimer, the tail of one hBD-3 still attached to the lipid membrane surface, thus the number of lipid head is around 5 at a distance range of 4 ˚ A and around 9 at a distance range of 6 ˚ A. Generally, the longer the distance range, the bigger the number of lipid heads. For hBD-3 monomers, the numbers are very similar in different forms. With temperature increasing, the fluctuation of numbers at different heights increased. The lipid head number result proves that the lipid heads have the tendency to approach hBD-3 molecules staying around. That also agrees with the membrane thickness result.

3.4.

Free Energy Calculations Show a Significant Barrier to a Dimer Translocating

through the Membrane

Translocation PMF result for the hBD-3 dimer. In this project, US simulations have been performed on hBD-3 dimer to observe the whole translocation process. The PMF vs. height result is shown in Figure 7 (Top). With the height increasing from -48 ˚ A to +48 ˚ A, an energy barrier of ∼40 kcal/mol protein (or 20 kcal/mol hBD-3 unit) needs to be overcome for hBD-3 to cross the membrane from bottom to top. Similarly, with the COM hBD-3 relative to COM membrane position decreasing from 48 ˚ A to -48 ˚ A, an energy barrier of 40 kcal/mol should also be overcome for the hBD-3 dimer to cross the POPC lipid bilayer from top to bottom. In the whole height range, an almost symmetric PMF profile was observed, with three peaks at around -12 ˚ A, -2 ˚ A, and 10 ˚ A, and two local minimums at -8 ˚ A, and 6 ˚ A. All of the PMF peak positions are inside the lipid membrane. Output structures at local maximum and local minimum positions, the result is shown in Figure 7 (Bottom). All 5 figures showed the reorientation of lipids toward the protein. Already with a dimer, it is suggested that a water pore could form at those positions based on the deformation of the lipid bilayer. Insertion PMF result for hBD-3 Calculating PMF at the height range of 0 ˚ A to 48 ˚ A, we have the result for hBD-3 monomer in the linear analog form at 3 different temperatures shown in Figure 8 (Top). Figure 8(Top) shows that at 313 K, an energy barrier of 22 kcal/mol should be overcome ACS Paragon16 Plus Environment

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FIG. 7: Top). Potential of Mean Force and standard deviation for hBD-3 dimer in wild-type form at 313.15 K at a force constant of 3 kcal/(mol·˚ A2 ). Bottom). Structures of hBD-3 dimer in wildtype form at different local maximum and local minimum positions after 140 ns explicit solvent and lipid NAMD simulations.

for hBD-3 monomer analog to insert into the POPC lipid bilayer center; while it takes only 18 kcal/mol at 383 K, and also 18 kcal/mol at 453 K for the protein to reach the same point. Figure 9 (Left) shows that the hBD-3 monomer in wild-type needs to overcome an energy barrier of 22 kcal/mol at 313.15 K to insert into the POPC lipid bilayer, while only 18 kcal/mol at 383 K and 16 kcal/mol at 453 K to reach the bilayer center. From both, it shows that once reaching 16 ˚ A above the membrane center, hBD-3 monomer can slide into the center of lipid membrane at 313 K, whereas similar barriers exist at the higher temperatures for hBD-3 monomer in wild-type and analog forms. Comparing PMF results for hBD-3 monomer in analog and wild-type forms, at the force ˚2 ), hBD-3 monomer in the linear analog form needs to overcome constant of 3 kcal/(mol·A a similar energy barrier to insert into the neutrally charged lipid bilayer comparing to the wild-type at 313 K, and also similar at higher temperatures. Thus, breaking the disulfide bonds of hBD-3 can not impair its lipid membrane insertion capability. Output the structures of hBD-3 monomer in both wild-type and analog forms at both local maximum and local minimum positions, the result is shown in Figure 8 (Bottom) and ACS Paragon17 Plus Environment


FIG. 8: Top). Potential of Mean Force and Standard Deviation for hBD-3 monomer in analog form at 313.15 K, 383 K, 453 K in US simulations; Bottom). Structures of hBD-3 monomer in analog form at different heights and different temperatures.

Figure 9 (Right). Significant curvature of lipid membrane was observed at 453 K for hBD-3 monomer analog and wild-type. More structures at the end of MD simulation at different heights are also shown in Figure S10 and Figure S11 in the Supplemental Material. Because of the interaction between hBD-3 monomer and lipids, it is not surprising to see significant curvature formed at 453 K for both hBD-3 wild-type and analog. Also, Figure 8 3(a), 3(b), and Figure 9 3(a), 3(b), and 3(c) showed the reorientation of lipids to hBD3, which agrees with the lipid membrane thickness within the distance of 5.0 ˚ A from the protein. Translocation and Insertion PMF Comparison Comparing the insertion PMF for both hBD-3 dimer and hBD-3 monomers at 313 K, the translocation PMF of dimer is shown ACS Paragon18 Plus Environment

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FIG. 9: Left). Potential of Mean Force and Standard Deviation for hBD-3 monomer in wild-type form at 313.15 K, 383 K, 453 K in US simulations; Right). Structures of hBD-3 monomer in wild-type at different heights and different temperatures.

FIG. 10: Potential of Mean Force comparison for the translocation of hBD-3 dimer through POPC lipid bilayer (dashed line), insertion of hBD-3 dimer into POPC lipid bilayer (black solid line), and insertion of hBD-3 monomer in both wild-type (red solid line) and analog forms (green solid line) at the force constant of 3 kcal/(mol·˚ A2 ) in US simulations.

in Figure 10. Comparing the insertion PMF result of hBD-3 dimer with hBD-3 monomers, similar peak heights were reached, which is only half of the translocation peak heights as shown in Figure 7. That emphasized the extra energy barrier when crossing the center of lipid membrane.



4.

DISCUSSION

General Comparison In order to transpass the zwitterionic lipid membrane, the hBD-3 dimer needs to overcome an energy barrier of 20 kcal/mol per unit, while around 20 kcal/mol are needed for hBD-3 monomers to insert into the lipid membrane center. The calculated free energies for both hBD-3 dimer and monomer are positive over the whole distance range studied, which means that hBD-3 dimer could not transpass through the neutrally charged lipid membrane to any appreciable extent, and hBD-3 monomer could not insert into the neutrally charged lipid membrane either. This observation agrees with the experimental findings from Anthony et al. [24], in which they found hBD-3 did not disrupt mammalian cell membrane. Based on hBD-3 monomer insertion free energy results from this project, the energy barriers are similar for hBD-3 in cross-linked form and after transiting into a linear analog. That proves that releasing all three disulfide bonds will not impair or facilitate the translocation capability of hBD-3, which agrees with findings from ref[14], which showed that the disulfide bonds status would not affect its antibacterial activity. Zhao et al.[34] studied the antimicrobial peptides (AMPs) transpassing across the bacterial lipid membrane (represented by POPE+POPG lipid bilayer) and normal cell (represented by POPC lipid bilayer) membrane. They found that the free energy to be overcome for AMP Bac2A translocating through the neutrally charged red blood cell lipid membrane (around 30 kcal/mol) was much higher than through the bacterial membrane. In our work, we inserted the hBD-3 in dimer and monomer forms, respectively, into the eukaryotic membrane models. It is expected that if inserting the hBD-3 molecules in negatively charged lipid bilayers, a much lower energy barrier will be observed. This in turn accounts for the selective character of hBD-3 in disrupting only bacterial membranes and thus promoting antibacterial activity. During the Steered MD process, we found that the N-terminal head of hBD-3 is very important in initial insertion, as evidenced by the final structures of hBD-3 monomer/dimer at different levels of membrane insertion from SMD plus MD simulations (Figure S9, S10 and S11), which are the last structures after long-term equilibration simulations at different temperatures. These results therefore demonstrate that the head of hBD-3 going in first/coming out last is the preferred orientation for the insertion of the defensin monomer ACS Paragon20 Plus Environment

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and dimer into the POPC lipid membrane. Thus, the initial orientation of the hBD-3 monomer and dimer in this project, chosen by our intuition that lipids may be more easily displaced first by a narrow protein edge, coincides with the preferred insertion orientation. The reverse argument can be made about coming out, where it would be thermodynamically favorable to displace many lipids at the same time to accomplish a cooperative solvation of a blunt protein surface. This result agrees with the findings with Sudheendra et al[53], who studied the conformation and mode of action of hBD-3 analogs with model bacterial and mammalian membranes using both experimental and MD simulation methods. They found that the linear analogs which only have the central and C-terminal sections of hBD3 are less active than the fully cross-linked hBD-3 interacting with bacterial membranes. Their work emphasized the importance of the N-terminal section of hBD-3 interacting with lipid membrane; i.e., consistent with our finding. That also justified our initial insertion orientation of hBD-3 monomer and dimer in this project with the structures shown in Figure S1 in the Supplemental Material. Since translocation/insertion free energy is orientation dependent[34], the PMF result would change if a different insertion orientation is taken. In this project, based on the NPT simulations on hBD-3 dimer in POPC lipid bilayer, only a slight membrane thickness change was found. That is also true as shown for hBD-3 monomer analog at 453.15 K. Based on Figure 8, the potential of mean force(pmf) for hBD-3 analog at different temperatures are similar. At 313 K, the PMF is slightly higher than those at 383 K and 453 K. Although at faraway positions such as at 48 ˚ A, the interaction between hBD-3 and lipid membrane should be zero and the PMF should be close to zero, we observed that at 313 K the PMF did not reach zero instead. The reason is that the structure of hBD3 monomer in analog form changes a lot in different windows as shown in Figure 3(Right) at three temperatures. Even at a height of 48 ˚ A, hBD-3 monomer can extend to interact with the POPC lipid membrane. Thus, even at faraway positions, hBD-3 still can interact with the lipid membrane now and then. That contributed to the large deviation of PMF data at faraway positions at three temperatures. Such kind of situations did not exist for hBD-3 monomer in the wild-type, i.e. with disulphide bonds formed. Thus, the PMF value for hBD-3 monomer in the wild-type almost reaches zero at faraway positions. Comparing the PMF profile for the hBD-3 monomer in wild-type and the analog forms, similar energy barriers were observed. That agrees with experimental findings that breaking the disulfide bonds can not impair the antimicrobial activity of hBD-3[14–17]. ACS Paragon21 Plus Environment


In this work, hBD-3 monomer in both the oxidized wild-type and reduced analog forms was used in the simulations. Comparing the hBD-3 monomer in wild-type and analog forms, they have the same sequence except that the three pairs of disulfide bonds formed between three pairs of cysteine residues in the wild-type, while they were broken in the analog form. Based on Figure 3(Right), the hBD-3 monomer wild-type has a smaller RMSD than the analog form at different temperatures, and the membrane thinning is slightly more significant in the wild-type form as shown in Figure 5. The radius of gyration of the analog form is also larger than the wild-type especially at the high temperature as shown in Figure S4(Right). The accessible surface area (ASA), hydrophobic ASA and hydrophilic ASA are similar as shown in Figure S6. The RMSF of the analog form is larger than the wild-type form especially at 383 K and 453 K as shown in Figure S8. However, we observed similar insertion PMF results for both forms at the different temperatures. Quantitative Comparisons of hBD-3 with Other AMPs hBD-3 is an arginine rich peptide. It has a total of 7 arginine (R) residues as shown in Figure 1, with three in the first α helix and the first β strand region, and the other four in the last coil region and on the third β strand. It also has six lysine (K) residues, one in the first loop region, one in the second loop region, and the other three in the third β strand region. Although it has two negatively charged residues glutamate (E) in the second β strand, they are balanced by two lysine (K) residues nearby (K26 and K32). Thus, the total charge of hBD-3 is +11. It has been found that transferring an arginine residue from water to the lipid membrane center takes around 14.5 to 19.4 kcal/mol of free energy[54–57], although the translocation energy barrier showed non-additivity on the number of arginine residues. Huang et al[35] found that the translocation of an arginine-rich peptide through a neutrally charged lipid membrane would create a solvent pore formation in a lipid membrane path based on their free energy calculations. Although hBD-3 has a total charge of +11, the free energy of membrane translocation is only 40 kcal/mol (dimer units) in our explicit solvent and lipid simulations, clearly pointing to such non-additivity. No water pore is formed in the timescale of our simulations. Interestingly the free energy of the dimer translocating through the membrane is considerably higher at 40 kcal/mol compared to the around 26 kcal/mol needed to approach to the center of the lipid bilayer. The extra amount of energy (around 7 kcal/mol per hBD-3 domain) likely originates from crossing the center of lipid bilayer, which is totally hydrophoACS Paragon22 Plus Environment

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bic. The local minima seen just before and after crossing the membrane center could lead to an accumulation of hBD-3 at these locations, but we also notice that the energy needed for the insertion of the dimer is slightly less per domain unit than for the monomer (around 22 to 24 kcal/mol), suggesting that hBD-3 association in the membrane may be favored. Possible Translocation Mechanism hBD-3 is a natural antibiotics and appears to function as a dimer[38, 58, 59]. In our simulation, steered MD was applied to pull the protein dimer and monomer out of the membrane. After long enough explicit solvent and lipid constrained simulations, with the peptide constrained to a position with a force constant of 3 kcal/(mol·˚ A2 ), different final structures of peptide in lipid membranes were generated. Significant dynamics of hBD-3 molecules were observed in each window. In total, 140 ns simulations were performed in each window in the height range of -48 ˚ A to 48 ˚ A with a distance gap of 2 ˚ A. When hBD-3 dimer is inside the lipid bilayer, reorientation of POPC lipids were observed. Significant reorientation of lipids to hBD-3 monomer was observed as well because of the strong electrostatic interaction between hBD-3 charged sidechains/termini and the POPC head groups. Because of the reorientation of lipids to the peptides, membrane thinning was observed in both hBD-3 dimer and monomer simulations as shown in Figure4, and Figure 5. Ratio of lipid heads within 6 ˚ A or 4 ˚ A to protein is significant inside the membrane. Currently, the simulation system has only hBD-3 monomer or dimer in 200 POPC lipids, thus the concentration of hBD-3 in lipids is low. No increase of protein accessible surface area, ASA, was observed inside the membrane range, as shown in Figure S5 and S6. Instead, the ASA for both hBD-3 monomer and dimer are very stable in the whole height range; RMSD and Rg of the dimer and monomer are the smallest inside the membrane. No dissociation of hBD-3 dimer inside membrane was observed either. It is intriguing to see that the RMSD and dynamics of hBD-3 are the largest at the solvent-membrane interface, even leading to transient dissociation of the dimer as it emerges after transiting the membrane. In other systems, it has been observed that solvent-membrane interfaces can partially unfold protein[60–64]. Altogether, these observations imply that membrane disruption is unlikely to follow the carpet model mechanism, and the result suggested the pore formation mechanism. Experimentally, it is found that hBD-3 can break the cell membrane[24]. Because pore formation is a cooperative behavior, based on result from this project, we can not conclude that hBD-3 takes a specific pore formation model to insert into POPC lipid membrane since ACS Paragon23 Plus Environment


we did not observe the whole translocation process in simulation directly. However, based on four translocation mechanisms available up to now, since significant lipid reorientation was observed in the translocation process in both NPAT ensemble and NPT ensemble, the result in this project suggests a toroidal model based for pore formation. Experimental work such as using solid-state NMR[65] and explicit simulations to directly watch the translocation of hBD-3 molecules through the membrane should be conducted to test this prediction in the future. We plan to carry out simulations with a larger number of hBD-3 monomers or dimers translocating through both neutrally and negatively charged membranes.

5.

CONCLUSIONS

Human β defensin type 3 is characterized by three disulfide bonds formed between six cysteine residues in its structure. In order to understand the protein’s ability to disrupt lipid bilayers which consist of neutrally charged membrane, the structure and free energy associated with hBD-3 dimer translocating through the POPC lipid bilayer and of the hBD3 monomer insertion into the POPC lipid bilayer are calculated in this project. During the translocation and insertion process, large structure deviations from the original hBD-3 monomer/dimer structures were observed based on RMSD results, with the local minimum, apparently slightly stabilizing the native-like structure and dynamics near the center of the membrane. Considerable fluctuations in the structure and dynamics occur at around the solvent and membrane interface. The hBD-3 dimer has an almost symmetric free energy vs. membrane location profile with a peak of 20 kcal/mol (domain unit); however insertion is less (and thus slightly more favorable) at 13 kcal/mol (domain unit) suggesting that dimers will accumulate in the membrane and likely further associate. By contrast, hBD-3 monomers in both the wild-type and linear analog forms need to overcome around 20 kcal/mol energy barrier to insert into the POPC lipid bilayer center. Considering the magnitude of the free energy in different hBD-3 states studied, the free energies are all well above zero. Consequently, the probability that hBD-3 can pass through or insert into the normal cell membrane in a dimer or monomer form in a spontaneous fashion is very small. It is likely that further hBD-3 association and especially anionic lipids are required to make the insertion if not passage, thermodynamically favorable.


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6.

SUPPORTING INFORMATION

The supporting Information is available free of charge on the ACS publication website. It includes one table showing the simulations performed in this project, in total 11 figures showing the insertion structures of hBD-3 monomer and dimer, histogram of hBD-3 dimer in wild-type, RMSD comparison for hBD-3 dimer, Rg of hBD-3 dimer and monomers, ASA of hBD-3 dimer and monomers, RMSF of hBD-3 dimer and monomers, structures of hBD-3 dimer and monomer at the end of the simulations. It also includes on video showing the SMD pulling process.

7.

ACKNOWLEDGMENT

This work was supported by supercomputer time mainly from Ohio Supercomputer Center, and partially from XSEDE via an award to Zhang L (MCB160041). Some of the umbrella-sampling simulations and analysis were carried out at the Computer Aided Engineering (CAE) Network system in Tennessee Technological University. The discussion with Dr. Ge Jin in the Department of Biological Sciences and Dr. Bingchen Wang from the Cancer Center in Case Western Reserve University helped the initial development of the project. The discussion with Dr. Alexander J. Sodt from NIH helped with the PMF calculation.

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