Cooperative Modes of Action of Antimicrobial Peptides Characterized

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B: Biomaterials and Membranes

Cooperative Modes of Action of Antimicrobial Peptides Characterized with Atomistic Simulations: a Study on Cecropin B Ya-Wen Hsiao, Magnus Hedström, Valeria Losasso, Sebastian Metz, Jason Crain, and Martyn D Winn J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b01957 • Publication Date (Web): 08 May 2018 Downloaded from http://pubs.acs.org on May 9, 2018

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The Journal of Physical Chemistry

Cooperative Modes of Action of Antimicrobial Peptides Characterized with Atomistic Simulations: a Study on Cecropin B ∗,†

Ya-Wen Hsiao,

‡

Magnus Hedström,

Crain,

¶

Valeria Losasso,

and Martyn Winn

†

†

Sebastian Metz,

Jason

†

†Scientic Computing Department, STFC Daresbury Laboratory, Keckwick Lane,

Daresbury, Warrington WA4 4AD, UK ‡Clay Technology AB, Ideon Science Park, SE-223 70, Lund, Sweden ¶IBM Research UK, Hartree Centre, Daresbury, WA4 4AD, UK E-mail: [email protected]

Phone: +44 (0)1925 603190

1

ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Antimicrobial peptides (AMPs) are widely-occurring host defense agents, of interest as one route for addressing the growing problem of multidrug-resistant pathogens. Understanding the mechanisms behind their anti-pathogen activity is instrumental in designing new AMPs. Herein, we present an all-atom molecular dynamics and free energy study on cecropin B (CB), and its constituent domains. We nd a cooperative mechanism in which CB inserts into an anionic model membrane with its amphipathic N-terminal segment, supported by the hydrophobic C-terminal segment of a second peptide. The two peptides interact via a Glu· · · Lys salt bridge, and together sustain a pore in the membrane. Using a modied membrane composition, we demonstrate that when the lower leaet is overall neutral, insertion of the cationic segment is retarded, and thus this mode of action is membrane specic. The observed mode of action utilizes a exible hinge, a common structural motif among AMPs, which allows CB to insert into the membrane using either or both termini. Data from both unbiased trajectories and enhanced sampling simulations indicate that a requirement for CB to be an eective AMP is the interaction of its hydrophobic C-terminal segment with the membrane. Simulations of these segments in isolation reveal their aggregation in the membrane, and a dierent mechanism of supporting pore formation. Together, our results show the complex interaction of dierent structural motifs of AMPs, and in particular a potential role for electronegative side chains in an overall cationic AMP.

INTRODUCTION Antimicrobial peptides (AMPs) form an important part of the innate host defense system. Compared to other natural or synthesized antimicrobials, such as the widely-used class of antibiotics targeting bacterial penicillin-binding proteins, AMPs act dierently 1,2 by attacking the microbial cellular membrane directly. For example, AMPs that are active against Gramnegative bacteria target the negatively charged lipopolysaccharides on the bacteria outer membrane via electrostatic and van der Waals forces. 3 Since membrane components are not 2


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as readily altered as protein targets, it is believed to be harder for microbial strains to obtain resistance against AMPs, although some examples of shifts in membrane composition have been observed. 46 Therefore, AMPs have received considerable interest over recent decades as a source of new antibiotics for treatment of multiple-drug resistant infections. Detailed accounts regarding AMPs and their medicinal potential can be seen in review articles by Zaslo, 7 Boman, 8 or Mahlapuu. 9 Generally, mature AMPs have a length in the approximate range 20-40 amino acid residues. While they are often unfolded in the aqueous environment, they assume well dened structural motifs when adsorbed on the membrane. 10 The simplest, and relatively common, motif is a single α-helix. This is typically amphipathic, with residues arranged into one hydrophobic and one hydrophilic face, allowing the peptide to insert between the hydrophobic membrane interior and the hydrophilic headgroups or aqueous environment. This simple motif may be modied in various ways, to ne tune interaction with the membrane or other membrane components, and potentially to drive oligomerization of AMPs. Cecropins from the Cecropia moth (Hyalophora cecropia) form one family of AMPs that demonstrates a broad spectrum of antibacterial activities against both Gram-negative and Gram-positive bacteria. Cecropin B (CB) possesses the highest antibacterial activity in the family, 11 with minimal inhibitory concentration (MIC) values against Gram-negative strains ranging from 0.5 to 16 µg/ml. 12 It has also been found able to kill cancer cells. 13 CB consists of 35 amino acids with the sequence shown in Table 1. Its expected secondary structure when adsorbed on the membrane consists of two α-helices, residues 1-21 and 25-35, connected by an Ala22-Gly23-Pro24 hinge. 14,15 The N-terminal helix is amphipathic, while the C-terminal helix is predominantly hydrophobic. A similar helix-hinge-helix structure has also been found in other AMPs such as melittin 16 and buforin II. 17 Experimental results on cecropins indicate that the organization of an amphipathic Nterminal helix connected to a hydrophobic C-terminal helix by a exible hinge region is required for strong, broad-spectrum antimicrobial activities. 18 Studies of synthetic cecropin 3



A-magainin II (CA-MA) hybrid peptides show that variants in which the hinge (Gly-Ile-Gly) is deleted lose their lytic ability against bacterial and tumor cells, as well as against PC/PS vesicles. 19 The pore-forming ability in model lipid bilayers is also reduced. When the hinge is substituted with a Pro residue, the peptide retains its lytic and pore-forming activities. The authors postulated that the exibility or bending potential induced by the hinge sequence or the Pro residue plays a key role in allowing the α-helix in the C-terminus to span the lipid bilayer. 19 In another study on the channel-forming properties of cecropins, Christensen et al. pointed out that a exible segment between the N-terminal amphipathic region and the C-terminal hydrophobic region of the peptide is required for the observed time-variant and voltage-dependent conductance through the channel. 20 Apart from cecropin and its derivatives, the Pro hinge has also been reported as the key to the cell-penetrating ability of buforin II. 17 In another AMP, Anal 3, derived from the amino-terminus of H. pylori ribosomal protein L1, Lee et al. substituted Glu9 in the original sequence with Pro and observed a four-fold stronger antimicrobial activity. 21 In view of these observations, one needs to consider the individual role of the N- (residues 1-21) and C- (residues 25-35) domains of CB on membrane interaction, and the interplay among these domains and the hinge region, to understand the mechanism of CB. Experiments on the interaction of AMPs with pathogens and model membranes are mostly informative on longer lengthscales and timescales, and the details of the molecular mode of action cannot be observed directly. An understanding of the dependence on sequence remains elusive, and this in turn hinders the design of new AMPs. All atom molecular dynamics (MD) simulations provide the necessary atomic level of detail. Simulations are restricted to shorter timescales, but should provide insight on the initial stages of binding or pore formation. 22 For AMPs, most MD studies have focused on general or macroscopic descriptions of the peptide-membrane interaction or on secondary structure changes, with fewer reporting detailed atomic pictures for lipid-peptide and peptide-peptide interactions. Such interactions in peptide-membrane systems are multi-faceted, and therefore there can 4


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be multiple modes of action, perhaps occurring simultaneously or under dierent conditions. Nevertheless, it is conceivable that identifying a few of them can progress the understanding of AMPs, and suggest avenues for designing more potent AMPs. With such a purpose in mind, we present results from all-atom MD simulations in a model lipid bilayer of CB, the C-domain of CB (CBC), and the N-domain of CB (CBN). In addition to following the time evolution of AMP-bilayer systems, we also performed free energy calculations to obtain the free energy barrier for a peptide to translocate the membrane (∆GT M ). Our aim is twofold: (1) to investigate whether ∆GT M diers between systems with one peptide and with multiple peptides. The results may shed light on the AMPs cooperative eect. (2) to aid interpreting our observations in MD simulations for the individual domains of CB, CBC and CBN, since the free energy information on peptides and their segments may explain how they function together. Based on the results, we propose a cooperative mechanism for CB on the bacterial membrane that requires its hinge and complementary charges on its N- and C-domains when two neighboring peptides are in a "tail-to-head" conguration. This mechanism has a comparable free energy barrier to that of a single peptide entering the membrane with its N-terminus. The addition of a C-terminus in the pore may have a supportive role to sustain the pore. This understanding can be used in designing AMPs utilizing this type of cooperative action to enhance or modulate the ecacy.

METHODS System Setup.

The structure of the peptide was built from its sequence as a single continuous α-helix using PyMOL. 23 Since the main aim of this study is to understand the peptide-membrane interaction, we started from a conguration in which one or more peptides are already embedded in the membrane. Specically, consulting the melittin results based on the X-ray diraction 5



data of Hristova et al., 16 we positioned each peptide at the glycerol region of the upper leaet of the lipid bilayer, with the helical axis parallel to the membrane surface. Furthermore, we followed the nding of Sato et al. 10 and positioned the peptides with their hydrophobic face down. The model membrane was constructed using the CHARMM-GUI 24 and consisted of 75 % 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 25 % 1,2-dilauroyl-sn-glycero-30

phospho-(1 -rac-glycerol) (DLPG), mimicking anionic bacterial membranes. The lateral size of the membrane was 120×120 Å2 which amounts to 464 lipids for both leaets together. In one control simulation, we replaced the lower leaet with 100 % DLPC, giving an asymmetric model membrane. The system has a net charge, owing to the peptides as well as the DLPG headgroups. Counterions were introduced to neutralize the system, and in addition, we added 0.15 M NaCl to mimic the salinity of the natural system. A 20 Å TIP3P 25 water layer was added on both upper- and lower-leaet surfaces. This amount of water ensures the full hydration of the membrane 26,27 and that the bulk properties including the 0.15 M salinity is reached before the simulation cell border. The membrane normal was set along the z -direction. All setups as well as gures in this article were done using VMD procedures. 28,29

MD Simulations.

The system was equilibrated at 310 K and 1 atm in three stages. (1) lipid heads and peptides were restrained to initial positions; and waters were kept xed. (2) only the backbones of peptides were restrained. (3) everything was free to move. Each equilibration phase was run for more than 10 ns with a 1 fs timestep. The CHARMM force eld was applied to both peptides 30,31 and lipids. 32,33 The particle-mesh Ewald method 34 was employed to evaluate electrostatic energy. A 12 Å cuto was applied to non-bonded interactions. The production runs were done in the NPT ensemble, using a 2 fs timestep. Furthermore, while the membrane area was allowed to vary, the ratio of the lateral dimensions was kept constant. Periodic boundary conditions were employed in all three dimensions. 6


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We investigated systems containing one or several peptides embedded in the membrane. For clarity, we refer to them as 1CB or 4CB for systems with one or four copies of the CB peptide, and similarly for other peptides or peptide domains. The systems in the MD study include 4CB, 4CBN, 10CBN and 10CBC, and individual peptides are labeled as chains A through to J. All simulations were carried out using NAMD 2.9. 35

Adaptive Biasing Forces and Metadynamics.

The free energy barrier for the peptide to translocate the membrane (∆GT M ) was estimated from the potential of mean force (PMF), which was calculated using the adaptive biasing forces (ABF) method as implemented in NAMD. 3638 In the ABF calculations, the reaction coordinate was chosen to be the z-coordinate of the center of mass (COM) of the peptide terminus inserting the membrane, relative to the instantaneous midplane of the membrane given by the z-coordinate of the COM of all phosphorus atoms. The COM of the peptide terminus was evaluated using the rst/last three residues of the N/C-terminus. We performed ABF calculations on the 1CB, 1CBC, 1CBN, and 4CB systems. The calculation was started from the endpoint of the corresponding equilibration phase, see above, so that the reaction coordinate runs from the equilibrated position of the terminal residues to the surface of the lower leaet. For an ecient free energy calculation, we employed the stratication strategy of breaking down the reaction coordinate into consecutive windows. The window size was set to 1 Å, and the simulation in each window was run for 40 ns, which gave an even distribution of event counts. A force constant of 10 kcal/mol/Å2 was used for the harmonic bias to conne the sampling within the chosen window along the reaction coordinate. The bin width for accumulating the instantaneous force was 0.1 Å, and we set a threshold of 20000 samples before applying any adaptive biasing force. The standard error in the PMF was estimated from dierent time blocks. The calculated data were split into two block sets of 20 ns per window. An upper limit of the error is estimated from the maximum dierence between 7



the two sets divided by

√ 2 since the nal PMF is calculated using both sets. The PMF

results from the two block sets are displayed in dashed lines together with the nal PMF (solid lines) in each graph. Although the chosen reaction coordinate is commonly used, 3942 there have been concerns about its suitability, since hysteresis has been observed using such collective variables. 40,43 Nevertheless, as we are to deduce mechanisms by comparing relative free energies of closely related systems, such a reaction coordinate fullls our purpose. Alternative collective variables are discussed in the article by Hub et al. 43 During the unbiased MD simulation, we observed a mode of action that seemed better described with two collective variables, i.e., moving one peptide by the N-terminus and the other by the C-terminus across the membrane (see below). Therefore, we also performed metadynamics 44 calculations to obtain two-dimensional free energy landscapes, using the NAMD implementation. The Gaussian hill width was set to 2 Å based on the choice by Ghaemi et al. 45 for a similar collective variable. The value of 0.1 kcal/mol was used for the hill height and it was varied according the rate implemented in NAMD.

RESULTS AND DISCUSSION MD Simulation on 4CB.

The peptide to lipid ratio of the 4CB system is about 1:100 in our setup using a 120×120 Å2 membrane patch. Since all four peptides are adsorbed on one leaet, which we arbitrarily label as the upper leaet, the eective ratio is about 1:50 and thus falls well within the experimentally reported range of 1:200 to 1:15. 46 At the end of the equilibration, the rst 7-8 residues of the N-terminus of chains B and D become unfolded, as do the C-terminal residues 30-35 of chains B and C (Figure 1, top panel). CB is known to be unfolded in solution, and so some partial instability is not surprising. A production run of 800 ns was started from this conguration. In the production run, the C-terminal region of chain C starts to break o from the full 8


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α-helix after around 20ns. After 80 ns, residues 1-7 of chain C become nearly parallel to the membrane normal, also breaking o from the α-helix (middle panel of Figure 1), and around 130 ns this conguration extends to residues 8-12 (ending with a Gly). There is an intra-peptide hydrogen bond between Glu9 and Arg16 (bypassing another potential partner at Arg13) which gives an arch shape to chain C. The top panel of Figure 2 shows the angles between the helical axes of peptide segments and the membrane normal. There is a clear correlation in the movements between the C-segment of chain A and the N-segment of chain C. The angles decrease from around 90◦ to ca. 45◦ before 100 ns, and the latter gives the time point when the peptides enter the membrane. The angle for the C-segment of chain C also decreases, reecting the fact that this segment tilts as it enters the membrane. While both termini of chain C are in the membrane, its hinge region is parallel to the membrane surface, albeit lower than those of other chains. A pore is formed between the N-terminus of chain C and the C-terminus of chain A, and can be seen already at the end of the equilibration phase (top panel of Figure 1). Chain C fully embeds into the membrane, with the COM of residues 1-3 1.6 Å above the membrane center at this moment. Water comes under the upper leaet headgroups and above the hydrophilic side of chain C, and the whole peptide appears to immerse in the membrane. Initially, the pore is lined mainly by phospholipid headgroups, with associated solvating water molecules forming a water defect in the membrane which develops into a trans-membrane pore. At the same time, peptides also start to participate in the pore formation process. The N-terminal region of chain C continues to dive down over 70 ns, carrying lipid headgroups and water on its hydrophilic side (middle panel of Figure 1). Local membrane thinning arises because of the lowering of the upper leaet lipid headgroups. The lipid headgroups interact with the peptide mainly through the cationic residues Lys/Arg, either directly via salt bridges or through a water-mediated hydrogen bond network, similar to the suggestion by Oh. et al. 19 on the structure-function relationships of the hybrid CA-MA (cecropin-magainin). The C-terminal segment of chain C also has water surrounding it inside the membrane, albeit to 9



a lesser extent. This is conceivable since the Glu31 and Lys33 on the C-segment are solvated by the water and Lys33 can interact with the headgroups as well. The neighboring chain A enters the membrane with its C-terminus in close proximity to the N-terminal segment of chain C. A steady interaction between Lys10 of chain C and Glu31 of chain A is established, mediated by water and the lipid headgroups. The middle panel of Figure 2 shows the z-coordinate (relative to the bilayer midplane) of NNH3+ of Lys10 of chain C and CCOO− of Glu31 of chain A. It shows that the motions of these two side chains become fully correlated after 130 ns, with their z-coordinates almost identical as the peptides move deeper into the membrane. The bottom panel of Figure 2 shows that the actual distance between the charged side chain groups of these residues (NNH3+ · · · OCOO− ) is longer than 35 Å initially, but shortens over the course of the simulation. Between 130 ns and 400 ns, the distance is 10-15 Å with the side chains interacting via water or lipid headgroups. After 400 ns, the separation decreases rapidly to around 3.0 Å as a salt bridge is formed. The nal conguration is shown in the bottom panel of Figure 1. It is possible that a neutral residue with an electronegative side chain may form an inter-peptide hydrogen bond with a lysine partner and exhibit similar correlation. We tested this by scoping simulations on Gln31 and Ser31 mutated systems, starting from the 4CB conguration with a well-established salt bridge. Details can be seen in the Supporting Information, Table S1 and Figures S1S2. Briey, the Gln31 variant shows a weaker interaction by having twice as long distance compared to CB, between the side chains of Gln31(A) and Lys10(C): they are indirectly engaged through a hydrogen bond network consisting of water and headgroups. Whereas for the Ser31 mutant, the two side chains are so far apart that there is in principle no interaction between the two peptides. The hydroxyl group of Ser31 interacts mainly with the backbone oxygen of Ala27 of the same chain forming an intra-peptide hydrogen bond and maintaining its α-helical structure. After the salt bridge is forged, there are still water molecules around the side chains but not along the bridge. Importantly, the hydrophilic residues of the AMP are not at all 10


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bare inside the membrane, which would have resulted in unfavorable energetics as have been speculated. 47 Rather, they are surrounded by lipid headgroups which are solvated by water. The radial distribution function shows that there are on average four oxygens within 3.55 Å of the NNH3 + of each lysine. These are oxygens from the lipid phosphate groups or from water, by which the Coulomb interactions are largely screened. At the end of the simulation after ≈ 800 ns, chain C is embedded deep in the membrane, with both termini directed towards the lower surface and the backbone of residues 14-21 lying in the hydrophobic region and parallel to the membrane surface. Some lipid headgroups from the upper leaet associate with the N-terminal lysines of chain C, and ip to the lower leaet (see yellow spheres in the bottom panel of Figure 1). The two domains from chains A and C appear to collaborate in keeping the headgroups between them and maintaining the pore during the time course. Figure 3 shows the amount of water in the pore as a function of time. To quantify the dynamics of the pore, we obtain the water count within 6 Å from the residues 1-10 of chain C, and ±12 Å from the membrane midplane. It corroborates that the pore comes into existence in the nal stage of equilibration and remains open throughout the whole 800 ns time span of the simulation. The radius of the pore uctuates between 5.9 and 2.9 Å, as determined from the water count and assuming a cylindrical pore. The lower curve shows the water count in the lower half of the membrane, which takes a slightly greater proportion of the total as the simulation progresses. To demonstrate how electrostatic and hydrophobic interactions inuence the membrane translocation of the peptides, we did a 100 ns scoping simulation of the 4CB system but now on a membrane whose lower leaet consists of DLPC only, while the upper leaet maintained the original mixed composition. Dierent from what we have seen, chain A enters the membrane with its C-terminus immediately, followed by chain C also with its C-terminus at close to 100 ns, whereas the N-terminus of chain C stays bound to the upper leaet (Figure 4). Recall that in the original 4CB system, chain C has entered the membrane 11



before 80 ns with its N-terminus, ahead of chain A. A pore of about 8 Å in radius is formed near the C-terminus of chain C, lined with headgroups from the upper leaet. The neutral lower leaet interacts well with the hydrophobic C-domain, but not with the amphipathic N-domain, since the lower leaet lacks the electrostatic interactions to attract it down, or to overcome its high binding anity to the upper leaet. As a result, there is less driving force for the N-terminus to enter the membrane. A similar idea was tested by Sengupta et al. on melittin. 48 Instead of manipulating the membrane composition, they removed side-chain charges to eliminate the electrostatic interaction and found no pore formation.

∆GT M

of 1CB, 1CBC, and 1CBN.

To help understand the behavior observed in the MD simulations, we investigated the free energy barrier to translocating the membrane (∆GT M ) for a single CB peptide, as well as the constituent domains CBN and CBC. The PMF curves were estimated using the adaptive biasing force method, and are shown in Figures 5, 6, and 7. Positive (negative) z-distances indicate that the COM of the group dening the reaction coordinate, i.e., the rst or last 3 residues of the chosen peptide, lies above (below) the bilayer center along the membrane normal. Except for the C-terminal insertion of 1CB, there are no clear maxima seen in the PMF curves, although most show multiple shoulders or plateaus. Almost all of the systems show pore formation during the ABF calculation, although the details may depend on the choice of the reaction coordinate. 40,43 We use the water count to help determine at which ABF window pore formation occurs, and dene the corresponding PMF value as ∆GT M . Specically, we use the water count between the membrane center and the lower leaet, in a range 2-12 Å below the membrane midplane, and an increase in this value is often correlated with a kink on the PMF curve. An example can be seen in the case of the C-terminal insertion of 1CBC, see bottom panel of Figure 6. A jump in the water count occurs at z ≈ -8 Å, where there is a small kink in the PMF curve, and the associated simulation snapshot shows that water 12


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has gone through the membrane at this point. Note that the water count may also uctuate due to local membrane thinning and undulation. The estimated ∆GT M s are compiled in Table 2, and the comparisons are summarized as follows: (1) For 1CB, the free energy of N-terminal insertion (∆GT MN ) is clearly lower than that of C-terminal insertion (∆GT MC ), 0 and 7.5 - 11.0 kcal/mol, respectively. (2) 1CBC follows a similar trend: ∆GT MN < ∆GT MC , 4.8, and ≥ 11.0 kcal/mol, respectively. Note that less water enters the pore created by the N-terminal insertion of 1CBC, due to the hydrophobic nature of this end. (3) For 1CBN, membrane insertion by the N-terminus results in higher energy barrier: 12 kcal/mol and ≥21 kcal/mol for ∆GT MC and ∆GT MN , respectively. (4) ∆GT M s of CB and CBC are lower than those of CBN, regardless which end inserts the membrane. A pore is formed near the N-terminus at the end of the equilibration of 1CB (see upper snapshot of top panel of Figure 5), incorporating lipid headgroups from both upper and lower leaets, and only partially lined by the peptide. This pore develops further, up to ca. -5 Å, during the ABF calculation for the N-terminal insertion of 1CB. However, the pore appears to decrease in size, judging from the decrease of water count shown in Figure 5. In contrast, the pore in the 4CB system maintains its size throughout both the unbiased MD simulation (Figure 3) and the ABF calculation (discussed in the next section, see Figure 8, upper panel). It may be of interest to further explore the involvement of the C-terminus in supporting pore stability. At any rate, the process of peptide N-terminus membrane insertion is seemingly a completely spontaneous one, as was observed for chain C in the unbiased MD simulation. We tentatively assign ∆GT MN = 0 kcal/mol in this case. Although on deeper insertion there is a shallow barrier of 0.5 kcal/mol around -4 Å, and a plateau of 2 kcal/mol 13



around -8 Å, these are low compared with other PMF curves. In particular, C-terminal insertion (∆GT MC ) of 1CB has a substantially higher barrier of 11 kcal/mol around -11 Å. While this is the only clear maximum among all PMF results, it occurs in the nal stages of insertion when the C-terminus of 1CB immerses into the layer of lower leaet headgroups. The high water count (red bars in Figure 5) is articial in this case, as it includes bulk waters solvating the lower lipid headgroups. Omitting such waters by restricting the z-range to 2-8 Å below the membrane midplane (blue bars) makes the pore closure more apparent. This is understandable, since the hydrophilic residues are no longer in the membrane core to support waters or internal lipid headgroups. The lowering of the PMF after -11 Å is likely associated to the stabilization when the peptide starts to adsorb to the lower membrane surface. For CBC, the result of the higher ∆GT MC than ∆GT MN is in line with spin-labeled (SL) ESR experiments on the synthetic peptide CB3 consisting of two CBC segments. It was found that C5SL-CB3 has higher binding anity, mainly of hydrophobic origin, than C30SL-CB3 at a lipid composition similar to the present work. 49 The situation for the CBN domain is remarkably dierent, with ∆GT M considerably higher and with C-terminal insertion the energetically preferred mode. In the case of Nterminal insertion, the upper leaet is drawn down with the peptide, but the lower leaet retreats away from the peptide terminus and makes a sharp local dent. No water goes in between the lower leaet and the peptide until the peptide has nearly reached the lower leaet: z ≈ -12 Å (Figure 7). It has been shown that the peptide CB1 (consisting of two CBNs) is in fact less eective in lysing lipid bilayers compared to the wild type, although the binding anity of CB1 to the lipid heads is high. 50 Perhaps it is the high anity of electrostatic origin that limits the ability of the peptides to enter the membrane. Furthermore, as CB1 has a hinge region by design, it is structurally exible as is CB. Thus, one may infer that lacking a hydrophobic domain in CB1 causes the lower lysing activity, in analogy to the relatively high value of ∆GT M found for CBN (Figure 7). It is conceivable that the hydrophobic segment of the full-length CB can facilitate the peptide interacting with the 14


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lipid tails and hence the membrane permeation. A hydrophobic domain can both interact with the membrane core and reduce the chance for the amphipathic peptides to re-partition into water. Nevertheless, the role of a hydrophobic domain might be even more involved: Shin et al. suggested an intracellular mode of action where hydrophobic domains enter the membrane and accumulate inside the cell; 51 whereas Wang et al. 50 suggested that these largely nonpolar parts can accumulate on the membrane and disrupt it in a cooperative manner. We do see the C-terminus of CB entering the membrane with a moderate ∆GT MC , and a pore is formed by it as well. To understand the nature of the C-terminal insertion of CB, we further investigated the behaviors of CBC (see section: MD on 10CBC and 4CBN).

∆GT M

of 4CB.

Cooperative eects have been reported for some AMPs, e.g., melittin. 52 Conversely, Gazit et al. speculated for cecropins that non-cooperative binding of the peptides may facilitate their diusion eciently into the membrane center. 53 Similarly, Hung et al. reported that peptide aggregation was not observed. 49 In our MD simulation of the 4CB system, we observed specic interactions between two peptides which may have functional signicance. To clarify this behavior, and to examine whether it is related to certain cooperative eects of CB, we investigated the free energy of insertion of one peptide molecule in the presence of the other three, and compared the resulting prole to that of the 1CB system. We are also able to follow the response of the three unrestrained peptides as the chosen peptide progresses along the reaction coordinate. Since it is chain C that inserts its N-terminus into the membrane in the MD simulation for the 4CB system, we calculated the PMF prole for the N-terminal insertion of chain C, dening the reaction coordinate in the same manner as for 1CB (upper panel of Figure 5). At the same time, we allow the other peptides to react to the inuence brought by the movement of chain C. The PMF of 4CB can be seen in the upper panel of Figure 8, showing a small 15



barrier for 4CB of 0.4 kcal/mol near z = 0, and a shoulder just below 1 kcal/mol, whereas the 1CB system has a shoulder at 2.0 kcal/mol. Nevertheless, a pore is already present at the beginning of the ABF calculation, similar to the situation of 1CB. We therefore assign

∆GT MN = 0. Interestingly, while the N-terminus of chain C is moved down stepwise as the ABF calculation progresses, we see that the C-terminus of chain A also inserts in step with chain C, showing a two-peptide tail-to-head cooperative action similar to that seen in the MD simulation. At around -4 Å chain A passes chain C, and remains on average 4 Å ahead until the end of the calculation, seen in the lower panel of Figure 8. While the early stage PMFs of 4CB and 1CB (N-terminal insertion) share common features, the PMF of 4CB rises steeply after the rst shoulder, reecting the inuence of inserting a second copy of the peptide. The conguration in the nal window of the ABF calculations again shows a salt bridge between the C terminal region of chain A and the N terminal region of chain C, although in this case Glu31 of chain A interacts with Lys3 of chain C, rather than Lys10 as seen in the MD simulation. Since the N-terminal region of CB possesses several lysines, with long positively charged side chains, it is likely that there are multiple congurations which reduce the energy cost for C-terminal insertion of a partner peptide. For comparison with the MD trajectory sampling, a histogram of the water content obtained in the ABF calculations is shown in Figure 8, upper panel. As trajectories in ABF have dierent meaning from those of unbiased MD, we instead take the average amount of water within each ABF window which in this gure is related to the z-coordinate of the N-terminus. We only count the water between 2 and 12 Å below the bilayer center, since we would like to use this quantity to decide when the pore formation takes place in the same way as for the other systems in this study. Observing the trajectories, we know that residues 1-12 break o the α-helix from the rest and stands nearly vertical inside the membrane, so the selection of water close to the peptide segment is related to the pore and shows that it remains open at the end of the ABF calculations. The radius of an equivalent cylindrical 16


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pore varies between 2.4 Å and 4.6 Å which is within the range seen in the MD simulation. The system might not be best represented using one reaction coordinate when two peptides are involved. To further explore the free energy landscape, we used the position of the C-terminus of chain A as the second collective variable and performed metadynamics calculations to get the two-dimensional PMF, shown in Figure 9. CV1 is the reaction coordinate from chain C and the CV2 refers to chain A. A lower energy band containing the minimum runs along the diagonal between the two collective variables. The minimum is located at (CV1, CV2)=(-2.05, -2.25) Å. The free energy landscape corroborates a close correlation between the two peptides, namely that the two chains going through the membrane together is energetically favorable. Figure 9 also shows that the free energy is in comparison higher when either A or C chain is ahead of the other. This is in line with that seen in the unbiased MD simulation. When comparing to the one-dimensional PMF result of 4CB N-terminal insertion, the latter appears not to follow the minimum path (lower panel of Figures 8 and S3). The observation points out a viable mode of action. We would like to stress that owing to the complexity of peptide-membrane interaction, our nding does not exclude other modes of action. Based on the MD and ABF results, we hypothesize that, in addition to the single peptide insertion by one of the two termini (N-terminus in the case of CB), CB can insert in pairs nearly simultaneously, using complementary segments. The resulting ∆GT M is comparable or slightly lower, compared to 1CB, so the presence of two other peptides does not seem to inuence the energetics. In fact, there is no clear conguration change for the other two chains. The PMFs of 1CBC imply that the C-terminus of CB can enter the membrane with moderate free energy cost as it is lower than that of 1CBN (Figures 6 and 7). Furthermore, the hinge segment grants the exibility, so both termini can simultaneously insert the membrane. If there is a Lys-containing amphipathic domain of another peptide nearby, then the Glu-containing hydrophobic domain will be able to bring the other peptide down the membrane via electrostatic interaction and the energy cost of C-terminal insertion 17



may be lowered by the aid of the amphipathic domain. The higher water count at z < -10 Å for 4CB compared to 1CB (either N- or C- terminal insertion) suggests that two peptides are able to support a larger pore than a single peptide.

MD on 10CBC and 4CBN.

To further dissect the mode of action of CB, we studied the behavior of individual segments of CB, namely CBC and CBN, using unbiased MD. Due to the shorter length of CBC, we included ten copies of the peptide in this case in a 440 ns simulation. CBC is largely hydrophobic apart from two charged residues (a Glu and a Lys). During the rst 200 ns, eight out of the ten peptides form dimers with a neighbor using the Glu· · · Lys and hydrophobic interactions, see Figure 10. The exceptions are chain C, which becomes unfolded during equilibration and subsequently desorbs into the bulk water, and chain H which becomes transmembrane shortly before 140 ns. Although partnered with chain E, chain D also adopts a transmembrane orientation. Both chains D and H enter the membrane with their N-termini, consistent with the PMF curves shown in Figure 6, whereas their C-termini stay close to the upper leaet, presumably due to direct or water-mediated Lys-headgroup interactions. Chains D and H behave similarly to cell-penetrating peptides, 54 namely, they enter the membrane with hydrophobic interaction and introduce no clear pore. The time evolution of the angle between the membrane normal and the helical axes of peptides D and H is shown in Figure 11. Chain D adopts a transmembrane orientation early on, which it retains for the whole simulation. The orientation of the isolated chain H uctuates more, until around 280 ns when it begins to participate in an aggregate with chains D, E, I, and J (and later chain C). The CBC aggregate is shown in Figure 12, and the side view shows that some lipid headgroups and water are within this aggregate. Although largely hydrophobic, there are two charged residues on each chain and the collective electrostatic interaction gives a higher chance to bring down the nearby lipid headgroups and the water solvating them. These peptides aggregate rather than distributing evenly in the membrane, 18


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and this is driven by a combination of hydrophobic interactions and Glu· · · Lys interactions, partially mediated by inserted waters. The trajectory of chain C provides a concrete example of an expected feature of AMPs. This chain desorbs from the membrane surface into water with a random coil structure during the rst 200 ns, but binds to the membrane by its N-terminus after ca. 375 ns. When it rebinds to the membrane, chain C joins chains D, G, H, I and J in the aggregate described above. As expected for many AMPs in a membrane environment, it partially refolds into an

α-helix again. The desorption of a peptide was only seen in the 10CBC simulation but not in systems containing CB or CBN. Although this may simply be the stochastic nature of the simulations, it is in line with the higher binding anity to the lipid headgroups of CB1 (two CBNs) compared to CB3 (two CBCs). 50 In contrast to CB and CBC systems, which show examples of translocation within 100 ns, CBN shows almost no action during the 500 ns simulation of the 4CBN system. The binding to the anionic membrane surface is very stable, and all four CBN peptides stay embedded in this region. The distribution of the angle between CBNs' helical axes and the membrane normal is limited between 70◦ and 140◦ , indicating that all peptides remain in their surface state and move according to the membrane undulation. It is known that the mode of action of AMPs can be concentration-dependent, 55 and so we also investigated a higher peptide concentration with a simulation of nearly 300 ns on a 10CBN system. We again failed to observe signicant membrane penetration, and thus this increased concentration did not alter the result. The inertness of CBN agrees with the high ∆GT M seen in the ABF calculations (Figure 7).

CONCLUSIONS We performed MD simulations and free energy calculations for the membrane translocation of cecropin B and its individual domains, aiming to elucidate the mechanism of interaction

19



with an anionic membrane that leads to pore formation. We investigated systems with four or ten peptides to examine whether there is a cooperative eect when multiple peptides are present. In the unbiased MD simulation of the 4CB system, we identied a pore-forming mode of action, namely that the C-terminus of one peptide engages the N-terminus of a neighboring peptide by forming a salt bridge between them in a tail-to-head conguration. The pore formation is spontaneous, as was conrmed by an ABF calculation where the energetics of insertion of one of the four peptides was followed. The ABF result for N-terminal insertion of 1CB also shows a favorable free energy change for initial penetration of the membrane, although in this case the rest of the peptide stays surface bound and the pore is not so well developed. This result illustrates how cooperation between peptides can help to support membrane pores. We note that this is not the formation of a symmetric multimer. This interaction relies on the presence of an anionic side chain for CB. A Gln31 mutant may also exhibit cooperative eect, however, to a much weaker extent. While AMPs are usually considered to be cationic, and indeed CB has an overall positive charge, the structural role of anionic residues is not usually considered. The cooperative behavior we have seen in CB relies on complementary charges, and would not be possible for purely cationic AMPs. In addition to its role in inter-peptide interactions, Glu was also observed to form intra-peptide links. We carried out a metadynamics simulation with two reaction coordinates for the two participating peptides, and this gives a clear picture of how the two participating peptides couple with each other and form a potential energy minimum when they insert the membrane together. Nevertheless, the full set of ABF calculations supplemented with water counts show clear trends which are in agreement with what is observed in unbiased MD simulations. For the full-length CB, the PMF curves suggest a preference for N-terminal insertion, as seen for chain C in the 4CB simulation. We have also used unbiased MD and ABF simulations to study the role of the N- and C20


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terminal domains. The sequence implies dierent physico-chemical characteristics for these domains, and indeed we see quite distinct behavior. Although, full-length CB has a preference for N-terminal insertion, the N-terminal domain on its own shows a signicantly higher PMF for insertion, which is in line with unbiased simulations of CBN where no insertion was observed. This implies a crucial role for the C-terminal domain. Unbiased simulations of CBC showed the formation of a cluster supporting a small pore, and this may imply an alternative mode of action for cecropins. The full-length CB may incorporate both N-terminal and C-terminal led modes of action, and the connecting hinge is crucial in this regard. It is known from experiments that AMPs may lose their activity when hinge regions are removed by mutation, or conversely, insertion of a new hinge region may boost the activity. 18,19,21 Indeed, it is expected that AMPs may have multiple modes of action, dependent on various factors including local concentration and lipid composition. This study provides one illustration of this, with C-terminal insertion preferred when the lower leaet is composed of DLPC-only. The results presented here oer an atomic resolution view of AMPs in action, and a step forward in understanding their activity. Molecular simulations illustrate the ne balance of forces governing the behavior of AMPs in lipid membranes. Designing sequence variants for therapeutic use requires an understanding of these subtleties. 56 Our results highlight the distinct roles of the two domains in cecropins, the role of both positively and negatively charged side chains, and cooperative behavior between peptides. There are hints at multiple modes of action, and whether there are additional modes is an interesting question that deserves further work. These results may also be relevant in other elds, for example aggregation of helices in a membrane context is implicated in some dementia diseases. 57

21



Table 1: Amino acid sequences of CB, CBC, and CBN

peptide CB CBC CBN

amino acid sequence KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL−COCH3 AIAVLGEAKAL−COCH3 KWKVFKKIEKMGRNIRNGIVK−COCH3

22


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Table 2: Estimated ∆GT M (kcal/mol)

peptide 1CB 1CBC 1CBN 4CB

N-term inserts 0.0 4.8 ≥ 21.0 0.0

C-term inserts 7.5 - 11.0 ≥ 11.0 12.0 n/a

Acknowledgement This work was supported by the STFC Hartree Centre's Innovation Return on Research programme, funded by the Department for Business, Energy and Industrial Strategy. The STFC Hartree Centre is a research collaboratory in association with IBM providing High Performance Computing platforms funded by the UK's investment in e-Infrastructure. The Centre aims to develop and demonstrate next generation software, optimized to take advantage of the move towards exa-scale computing. We also acknowledge computing resources provided by STFC Scientic Computing Department's SCARF cluster. We thank Alin Elena and Max Ryadnov for useful discussions.

Supporting Information Available BLAST Search Concerning Glu31, MD Simulations on Gln31 and Ser31 Mutants, and MD and ABF Trajectories Mapped onto the 2-CV PMF are supplied as Supporting Information.

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Figure 1: Snapshots from the MD simulation of the 4CB system, taken at 0 ns (top panel, top view is shown in the inset to the left), 80 ns (middle panel), and 800 ns (bottom panel, top view is shown in the inset to the left). Yellow spheres: upper leaet phospholipid heads; silver: lower leaet phospholipid heads; cyan: water. Color codes for the peptide residues: red: negatively charged; blue: positively charged; green: polar; white: hydrophobic.

31



Figure 2: Selected geometric measures from molecular dynamics simulations of the 4CB system. The top panel shows angles between the helical axes of peptide segments and the membrane normal. N-segment refers to residues 1 to 21, C-segment refers to residues 25 to 35, and letters in parentheses identify a specic peptide chain. The middle panel shows atomic depths along the membrane normal of Glu31 on chains A and Lys10 on chain C. The bottom panel shows the time course of the distance between these two residues. 32


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Figure 3: The number of water molecules close to the N-terminus of chain C of 4CB over the course of the MD simulation. The count only includes water molecules that are within 6 Å of the residues 1-10 of chain C. The count is further limited to those ±12 Å from the membrane midplane (black line) or 2-12 Å below the membrane midplane (red line). No smoothing has been done on the raw counts, and the steps reect groups of waters crossing the thresholds.

33



Figure 4: 4CB on the membrane with DLPC-only lower leaet. The C-termini of chains A and C enter the membrane before the N-terminus of chain C. A pore near the C-terminus of chain C starts to appear around 50 ns, consisting of the headgroups. At nearly 100 ns the C-terminus(C) also enters the membrane.

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Figure 5: PMF for 1CB: a pore has already formed at the beginning of the N-terminal insertion so it is spontaneous (upper panel); and for the C-terminal insertion (bottom panel). The corresponding estimated error: 1.0 and 1.6 kcal/mol, respectively. Red bars are the average water count within 6 Å from the N-(C-)segment and 2-12 Å below the membrane midplane, within each ABF stratication window. Blue bars are the water count within 2-8 Å below the membrane midplane showing pore closure since the C-terminus adsorbs onto the lower leaet after it reaches z = -11 Å. Snapshots show the congurations at the moment of pore formation and at the end of the calculation.

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Figure 6: PMFs for 1CBC N-terminal insertion (top) and C-terminal insertion (bottom). The corresponding estimated error: 0.4 and 2.5 kcal/mol, respectively. Red bars are the average water count within 6 Å from the protein and 2-12 Å below the membrane midplane, within each ABF stratication window. Snapshots show the congurations when water counts reach maximum.

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Figure 7: PMFs for 1CBN N-terminal insertion (top) and C-terminal insertion (bottom). The corresponding estimated errors: 1.3 and 1.1 kcal/mol, respectively. Red bars are the average water count within 6 Å from the protein and between 4-12 Å above the COM of phosphorus atoms of the lower leaet near the peptide, within each ABF stratication window. The lower start is due to the sharp local dent feature (see text). Snapshots show the congurations when water counts reach maximum.

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Figure 8: Upper panel: PMF for the 4CB system, and the average water count within each window of ABF. The corresponding estimated error: 0.6 kcal/mol. Red bars are the average water count within 6 Å from the N-terminus of chain C and 2-12 Å below the membrane midplane, within each ABF stratication window. The lower snapshot shows the starting conguration for the simulation, which is also the moment of pore formation. The upper snapshot shows the end conguration of the peptides around the pore. Lower panel: Membrane depths of the termini of chains A and C as the ABF calculation progresses.The N-terminus of chain C is actively pulled, hence the steady increase in depth, while the Cterminus of chain A is responding spontaneously. 38


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10

12

10

5

8 CV2 (Å)

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0 6 −5

4

−10

2

0 −10

−5

0 CV1 (Å)

5

10

Figure 9: Two-dimensional PMF (kcal/mol) of 4CB. CV1: z-distance of N-terminus of chain C; CV2: z-distance of C-terminus of chain A to the membrane center. Positive values indicate that the terminus is above the membrane center.

Figure 10: The 10CBC system after 200 ns of unbiased simulation. Top (left panel) and side views (right panel) are shown. The color coding is as in Figure 1.

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Figure 11: Fluctuation of the angles between the helical axes and the membrane normal for chains D and H of the 10CBC system over the course of an unbiased MD simulation. An angle of 90◦ implies the peptide is parallel to the membrane surface, while smaller angles indicate vertical penetration of the membrane.

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Figure 12: 10CBC aggregation after 400 ns, top (left panel) and side view (right panel). The aggregate of six peptides is visible on the right-hand side of the latter. The color coding is as in Figure 1.

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