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A Molecular Dynamics Study of the Short-Helical-Cytolytic Peptide Assembling and Bioactive on Membrane Interface Fude Sun, Xiufang Ding, Lida Xu, Jun-Feng Liang, Long Chen, and Shi-Zhong Luo J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b04347 • Publication Date (Web): 22 Jul 2017 Downloaded from http://pubs.acs.org on July 29, 2017

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The Journal of Physical Chemistry

A Molecular Dynamics Study of the Short-Helical-Cytolytic Peptide Assembling and Bioactive on Membrane Interface Fude Suna, Xiufang Dinga, Lida Xua, Jun F. Liangb, Long Chena*, Shi-Zhong Luoa*

a Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China. b Department of Biomedical Engineering, Chemistry, and Biological Sciences, Stevens Institute of Technology, Castle Point on Hudson, Hoboken, NJ 07030, USA.

Abstract: Cytolytic peptides (CPs) have long been employed as broad-spectrum antibiotic agents to overcome multidrug resistance. However, the development of novel peptide drugs is still limited by elusive molecular understanding of the membrane-lysis mechanism and modeling of CPs, especially of the short helical species. In this study, a known anticancer CP named PTP-7b (FLGALFKALSHLL) in disrupting membranes via self-assembling approach was studied combining experiments and time-extended coarse-grained dynamic simulations. Effective membrane disintegration was induced by aggregation of the membrane-bound peptide individuals, rather than the preassembled peptide clusters. The disturbance level of lipid bilayers depended on the peptide concentrations, which was responsible for the known long time-costing of PTP-7b in killing cells. Based on lines of simulations and energy landscape calculations, the dynamics of membrane deformation evolving toward preliminary leakage resulted from the aggregated PTP-7b was demonstrated, which was subjected to the spatiotemporal cooperation of the membrane-inserted and the periplasmic peptides. The molecular mechanism incorporated the 11th histidine interaction coupled with the peptide amphiphilicity in accelerating phospholipid migration outwards. This study revealed elaborate modeling and dynamics information about the short helical CPs in membrane lysis, which would be helpful for understanding about the underlying mechanisms and rational design of CPs for drug application.

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INTRODUCTION The increasing incidence of drug-resistance, both in microbes and cancer cells, has become a serious public health problem worldwide1-2. Cytolytic peptides (CPs), which belong to a major class of antimicrobial peptides (AMPs), have long been discussed as potential drugs to overcome multidrug resistance3-4. Known as the broad-spectrum resistance against various guest pathogens, some peptide analogues show medical selectivity in anticancer treatment5-6, providing an alternative approach with mild and sustainable advantages. Among the AMPs/CPs in APD2 database, the α-helical structure acts as the most general class occupying ~14% of the total7. In most cases, the helicity property strengthens when they approach to and attach on cell membranes, which plays as the crucial phase in measuring their antibiotic efficiency8. On account of the diversity of CPs in length, sequence and structure, a number of membrane disrupting models have been proposed to illustrate their action principles 9-10. However, there are still many limitations to obtain consensus in deciphering the modeling and mechanism of the short helical CPs in membrane lysis. To our knowledge, the models of membrane disruption affected by AMPs/CPs can be classified into the transmembrane-pore and non-pore models. The former includes the “barrel-stave” model11 in which peptides interact laterally to form ion-channel-like transmembrane pores, and the “toroidal” model12 that peptides lead to local curvature of cell membranes and the lipid bilayer bends continuously through the pore neighboring with peptides. Due to the length requirement in formation of transmembrane pore, the involved peptide candidates are mostly long α-helical peptides, such as alamethicin13, magainins14 and mellitin15. In addition, a“double-belt” pore structure was proposed as a novel model for the long amphiphilic peptides16. However, for the majority of short CPs, it is difficult to parallel bundle together to form fixed channel-like peptide polymers. Depending on residue composition, the short CPs show alternative penetrating through or lying on the phospholipid bilayer to prepare for the further activities. A typical penetrating peptide is substrate of trans-activating transcriptor (TAT) which consists of 9-16 basic residues17. A recent study of penetrating peptide R9 (nine arginines) shows it adopts alternative pore-forming or pore-absent model affected by lipid compositions18. For the rest short CPs with helical structures, the “carpet” model is the most accepted style, in which the peptides parallel distribute on membranes to transport the cytoplasmic components via specific membrane perturbation19. The “detergents” model is a “carpet” dependent phase when a peptide concentration threshold reaches, then the peptides form patches to damage bilayers 20-21. Self-assembling in solution and membrane has been observed for many CPs22-23. Peptide aggregation help to stabilize and sustainable release of peptide drugs6, 24. In our previous work, a CP named PTP-7b with 13 residues is found possessing bioactive significance in killing lung cancer cells relying on aggregation 25. However, the membrane lysis by PTP-7b shows an evident time-costing feature compared with the rapid potency of its parent peptide PTP-7 (K11H forms PTP-7b)26. It is speculated that PTP-7b exerts its anticancer bioactivity based on peptide aggregation upon membrane association. Considering the typical prototype of PTP-7b being amphipathic, α-helical, cationic and short in sequence, to reveal the molecular details underlying the membrane deformation triggered by peptide aggregation appears quite meaningful for the following improvement. However, to date it remains a challenge to describe the subtle membrane disruption by conventional experimental means. Recent years, relying on the advantages of time-scale expansion and accuracy elevation, the coarse-grained (CG) dynamic molecular simulations, especially the CG model based on the Martini 2


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force filed27-30, have been extensively employed in exploring the molecular basis in various biological processes. Martini force filed in principle integrates four heavy atoms into one CG bead that is classified into 18 subtypes to optimize their interaction. Extensive calibration of the non-bonded interactions between building blocks is established to match with the experimental data31, especially the thermodynamic property such as oil/water partitioning energy. Various processes like peptide–membrane binding32, lipid organization33, and protein–protein interaction34-35 are substantially dependent on the preference of the constituent partition between polar and non-polar environments. Based on good practicability of phospholipid bilayers and updating advances for proteins, it has been successfully used to model different aspects of helical bioactive peptides when interacting with membranes 36-37, including investigation of the membrane-pore modeling38-40. In addition, CG models of β-amyloid peptide 41 and cyclic peptide 32, 42 have been employed to present their effects on membranes by forming assembly on the surface or acyl core. Energy landscapes within the peptide-bilayer interplay generated from Martini CG model is used to establish the correlation between structural characteristics and membrane disruption mode43-44. Herein, we investigated the membrane-lytic process of aggregated peptide PTP-7b by Martini CG model combining with experiment results to interpret the peptide-membrane interaction and the underlying driving forces. A stepwise “outstretch” cytolytic process was presented and analyzed, which incorporated the participations of the membrane-bound and the periplasmic peptides. The structural reorientation details and the crucial residues were explored to figure out the inherent principles. The detailed information revealed in the study will deepen our comprehension into the membrane-lysis approach of short linear aggregated peptides and the future design. METHODS Experiment Section Atomic Force Microscopy. The non-assembled peptide PTP-7b (40 µM) was prepared freshly from the stock solution (10 mM). The self-assembled peptide was prepared by incubation at 37 °C for 24 h. In brief, 0.1 mL of PTP-7b solution was dropped on the top of a freshly cleaved mica sheet glued to a steel AFM sample plate. Samples were incubated under ambient conditions for 10 min, washed to remove unbound peptides, and then air-dried. The non-assembled and self-assembled PTP-7b samples were examined using an atomic force microscope (AFM) (Veeco Instruments Inc., USA). Measurements were performed using a silicon nitride probe mounted on a NanoScope V Controller (Veeco Instruments Inc., USA) and in ScanAsyst mode (scan size of 2×2 µm, scan rate of 1.0 Hz). MTT Assay. The cytotoxicity of the peptides was determined using the MTT assay as described previously. Briefly, cells in a complete medium were added into 96-well plates (5 × 103 cells/well) and cultured at 37 °C for 14−16 h. After being washed, cells were fed with serum-free medium containing various concentrations of non-assembled and self-assembled PTP-7b and incubated at 37 °C for 2 h. After that, 10 µL of MTT (5 mg/mL) were added into each well. Cell viability was determined after 4 h of incubation by dissolving crystallized MTT with 10% SDS solution containing 5% isopropanol and 0.1% HCl and measuring the absorbance at 570 nm. Peptide Stability Determination The peptide stock (10 mM) was diluted by deionized water to the final peptide concentration of 100 µM. The peptide solution was incubated at 37 °C and was tested at time points of 0 h, 24 h respectively. The peptide stability was then analyzed by reverse-phase high-pressure liquid chromatography, and the intact peptides were identified by ESI-MS (Thermo LTQ Orbitrap XL equipped with an electrospray ionization 3



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source). Confocal Microscopy. Freshly trypsinized human cervical cancer Hela cell line were seeded in a collagen-coated eight-well glass chamber (2 × 104 cells/well) and cultured in DMEM medium containing 10% FBS at 37 °C (5% CO2) overnight. Before the assay, cell membranes were stained with Di-8-ANEPPS (2.5 µM) for 10 min. After being washed three times with PBS to remove the excess Di-8-ANEPPS, cells were exposed to fluorescein isothiocyanate (FITC)-labeled PTP-7b. Peptide accumulation and self-assembly on cell surfaces were investigated using a Leica TCS SP8 confocal microscope (Leica Microsystems, Germany) by setting the excitation wavelength at 488 nm and emission wavelength at 505−530 nm (for peptides) and 560-600 nm (for cell membranes), respectively.

CG Simulations Model set-up. All the simulations were performed on the Gromacs-4.6.6 package45. The Martini CG force field, in which four heavy atoms and associated hydrogens are considered as one bead 29-31, was employed to investigate the molecular basis of the membrane-peptide system. The input parameters were obtained from the Martini homepage (www.cgmartini.nl). A simulation box size 10 × 10 × 15 nm3 containing phosphatidylcholine (POPC)/phosphatidylserine (POPS) with a 3:1 ratio was constructed using the insane.py46-47 to imitate the cancer lipid bilayers48-49. The small simulation box was used to avoid the unnecessary spontaneous curvature of the mixed bilayers happening in large scale. The atomistic structure of PTP-7b and its mutant were firstly constructed by Pymol software50, then the peptide CG model together with the topology restrains were generated by Martinize script from the atomistic structures. The secondary structure of the peptides studied here was restrained as α-helical that was confirmed in our previous study6. Peptide individuals were randomly filled into an oblate box with a size of 10×10×2 nm3, then the peptide groups were integrated into the upside of the membrane model with a distance of 2 nm between peptides down-edge and membrane surface. Alternatively, the distance was set with -1 nm to insert the peptide in membrane. The lipid bilayers were solvated with standard CG water6. Sodium ions were added to keep the system neutral. Simulation details. The systems were firstly energy minimized for 5000 steps by the steepest descent method. Then NPT equilibration for 50 ns was conducted with the bilayer lipids, peptides, solvent and ions coupled separately in semi-isotropic style. The temperature adopted the weak-coupling scheme of Berendsen thermostat51 at 310 K with constant τT of 1 ps. The pressure was controlled with the Berendsen barostat, with a pressure reference of 1.0 bar, a compressibility of 4.5×10−5 bar−1 and a constant τP of 5 ps. Standard cut-off schemes were used for the non-bonded potentials: LJ interaction was shifted to zero in the range 0.9-1.2 nm, and the electrostatic interactions was in the range 0.0-1.2 nm. Periodic boundary condition was employed. The step time of the simulation was set as 20 fs. The neighbor list for the pairwise non-bonded interactions was updated every 10 steps. The pair-list cut-off was set as 1.4 nm, which ensured that the non-bonded forces between nearly all atoms within the cut-off distance were calculated as the pair-list updated. Each simulation ran for an effective time of 6.0 µs. All the simulation representations were performed by VMD software52. Potential of mean force (PMF). To compute the PMF between two packed peptides on the membrane surface, the same system set-up in the simulation that contained the peptide couple, bilayer and solvent was used. The selected structures were first energy minimized by the steepest descent method. The PMF was calculated using the umbrella sampling mehods53. For each system, more than 20 windows were selected to simulate according to an average distance interval of 0.2 nm between the two peptide helixes. The initial configurations for each window were generated by pulling the two peptides from their associated state to their window location using the umbrella potentials with a force constant of 1500 kJ·mol-1·nm-2 and pulling rate of 20 nm/ns for 250 ps. The fixed chain was constrained by force constant of 1000 kJ·mol-1·nm-2. Finally, 4


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we obtained the successive peptide-bilayer initial configurations with peptides remaining on the membrane surface showing successive separation distances. Each window was then equilibrated for 100 ns with the force constant of 1000 kJ·mol-1·nm-2, followed by a 1.0 µs production simulation. The WHAM method54 was used to unbias the umbrella potentials.

RESULTS Membrane disintegration caused by peptide self-assembling PTP-7b (FLGALFKALSHLL) formed evident peptide aggregates after overnight incubation compared with the fresh soluble peptide counterpart (Figure 1A). The results were consistent with the SEM views of our previous study, in which PTP-7b was proved showing comparable anticancer activity to its parent peptide PTP-7 after 60 minutes25. Relying on confocal microscope, no peptide attachment on cell surface occurred (within the first half an hour) until 60 minutes when obvious peptide aggregates emerged on membrane localization, followed by a rapid cell lysis (Figure 1B). The biphasic cell lysis caused by PTP-7b was therefore interrelated with its self-assembling upon interacting with membranes. However, it was questioned that whether it was the preassembled PTP-7b or peptide aggregates formed by the membrane-inserted individuals that actually executed the subsequent cell lysis, since PTP-7b self-assembling property was tested here in absence of the membrane environment.

Figure 1. (A) The AFM images of fresh (non-assembled) and overnight-incubated (assembled) PTP-7b dissolved in pure water. (B) PTP-7b aggregation on cell membranes induced lysis of Hela cells. The cell membranes were labeled by di-8-ANEPPS (red) and the peptide was labeled by FITC (green). Peptide concentration was fixed at 40 µM. (C) The 5



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cytotoxicity of non-assembled and assembled PTP-7b measured on Hela cells. Three independent measurements were conducted. (D) Peptide-induced surface pressure changes of lipid monolayer with a composition of DPPC-cholesterol-DPPS (50: 10: 2.5 in molar ratio) mimicking mammalian cell membranes. The initial surface pressure of the lipid monolayer was set at about 30 mN/m. The final concentration of loaded peptides was 10 µΜ.

In order to evaluate the bioactivity of the membrane-inserted and preassembled PTP-7b, the cytotoxicity of the non-assembled (fresh) and preassembled (overnight incubation) peptide was respectively tested using MTT assays. The IC50 value of the preassembled PTP-7b was nearly double folds of the non-assembled peptide (Figure 1C), suggesting the cytotoxicity of PTP-7b decreased severely when it preassembled to associate with cell surface. As a further inspection, the different cases of PTP-7b were estimated by the pressure shifts of a model lipid monolayer subjected by peptides (Figure 1D). The incubation of fresh PTP-7b induced a biphasic change of surface pressure: a rapid increase of 27.4 mN/m and a subsequent decrease of 2.7 mN/m, indicating the evident membrane binding of peptides followed by events of membrane-insertion. In contrast, the preassembled PTP-7b could not produce an equivalent membrane attachment (δ3=16.9 mN/m) either the membrane perturbation. PTP-7b could maintain intact in 94.1% after the overnight incubation, which excluded the interference of peptide degradation for estimations (Figure S1 and Table S1). Based on the results, the time-costing cell lysis caused by PTP-7b mostly or initially attributed to the membrane-bound peptide individuals which packed to aggregates subsequently. Table1. An overview of peptide variants at different concentrations in associating with membrane.

P/L α (peptide/lipids) 15/168 20/168 20/168 PTP-7b (FLGALFKALSHLL) 40/168

Peptide Assembling × √ × √

Membrane ϒ Distortion Level – + – ++

√

+++

15/168 20/168

Initial Position β (with upper leaflet) Inserted Inserted Upside Inserted 20-inserted 20-upside Inserted Upside

× ×

– –

20/168

Inserted

×

–

20/168

Inserted

√

–

20/168

Inserted

×

–

Peptide analogues

40/168 PTP-7 (FLGALFKALSKLL) PTP-7b-H11A (FLGALFKALSALL) PTP-7b-H11Y (FLGALFKALSYLL) ν PTP-7b-H11 (+) (FLGALFKALSHLL) α

The lipid number in the “P/L” indicated the lipids in the upper leaflet.

β

The initial position of “Inserted” and “Upside” mean the distance between the lower edge of peptide group and the

upper membranes surface of 2.0 nm and -1.0 nm, respectively. ϒ

The “–” symbol mean the membrane was normal; the “+” mean membrane deformation caused by peptides, and

multiply of it represented the membrane deformation degree. ν

Protonation of the 11th histidine residue.

Molecular characterization by CG simulations 6


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The membrane-lytic process occurs at a molecular level in long time-scale, which remains a challenge to be captured by the atomistic simulation and current experiment methods. In order to obtain the molecular perspective of the dynamic membrane deformation triggered by peptide aggregation, the Martini CG force field suitable for long-term biological process was employed in the study. The membrane model was composed of 436 POPC/POPS with a ratio of 3/1 to mimic the cancer cell membrane with anionic surface (all simulations have been listed in Table 1). Based on the above discussion, we first investigated the temporal-spatial relevance of PTP-7b in membrane disruption by peptide assembling inserted and outside the membrane respectively. To this end, 20 separated peptides were placed upon the membrane with respective distances to the upper leaflet of 2 nm and -1 nm to build two simulation systems (the left images of Figure 2A and 2D). The peptide concentration in simulations did not exceed the saturated concentration calculated in our previous experiments6 and at a comparable magnitude order. After 6.0 µs simulation, the peptides in both systems were able to form aggregates. Peptides outside the membranes self-packed rapidly before binding to the lipid bilayer (Figure S2). However, it showed an insignificant influence on the membrane structure (Figure 2A). In contrast, evident bilayer curvature was induced by aggregation of the membrane-inserted peptides (Figure 2B). Meanwhile, the lateral displacement of the lipid acyl chains of the upper leaflet was much closer to zero, showing a severer membrane distortion (Figure 2C) impacted by the membrane-inserted peptide block. Therefore, the simulation results matched well to the above experiments, indicating a reduced membrane-break ability of the preassembled PTP-7b aggregate.

Figure 2. Presentations of membrane deformations affected by association of PTP-7b in different situations. Peptides

were respectively placed outside (A) and inserting (B, D, E) the membrane. Only backbones of peptides were shown with blue chains. Tails of POPC and POPS were distinguished in green and purple, and the head groups were presented in red. For clarification, the water solvents were omitted. The upper images in A, B, D and E represented the final states of the four lipid-bilayer systems at different concentrations. The lower images showed the mass density profiles 55 of the 7



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upper lipid leaflet incorporating peptides within the last 1.0 µs, which were generated in the same view to the upper snapshots. (C) Disorder parameters (Sz) of the acyl carbon bonds of the lipids in the upper leaflet of the four systems.

Plenty of evidence supports that the membrane disturbance by antimicrobial peptides requires a high peptide concentration56-57. For this purpose, three systems with peptide concentration gradient of (P/L) 15/168, 20/168 and 40/168 were constructed. At the lower P/L of 15/168, the bilayer structure kept normal (Figure 2D) and self-assembling of PTP-7b was no observed (Figure S3). In comparison, notable aggregates of peptides formed when the P/L ratio elevated to 20/168. The upper leaflet showed an apparent curvature with the peptide aggregate protruding out to solvent phase (Figure 2B). As expected, increased lipid instability (Figure 2C) and severer bilayer curvature happened when 40 peptide chains self-assembled on the membranes, even some lipids inclined to liberate away (Figure 2E). Correspondingly, destabilized by increasing activity of aggregated peptides, the lipid tail gradually extended towards the membrane surface, reflecting the dramatically disorder of associated lipid and growing defects of the membrane structure (Figure S4). Molecular details investigation To reveal the molecular mechanism of PTP-7b in cytolysis, its parent peptide PTP-7 (FLGALFKALSKLL) with a weak self-assembling ability was subjected to simulations. Consistent with the mode in Figure 2D, simulations of PTP-7 at P/L of 15/168 and 20/168 were built to examine their dynamic behaviors. Similar with PTP-7b (Figure 3A), PTP-7 at the lower P/L ratio did not aggregate, and the situation maintained at the higher P/L ratio (Figure S3). The simulation results were consistent with the previous data that showed the distinct self-assembling capabilities between PTP-7 and PTP-7b25. However, the peptide orientation of PTP-7 and PTP-7b at the lower concentration exhibited a common mode: the N-portion was concealed in the bilayer, retaining the C-portion outward (Figure 3B and S5). The specific peptide arrangement is interrelated with the fact that the N-portion of the peptides is relative hydrophobic (Figure 3C and 3D), which is favorable for minimizing the system energy. The likewise structural character is also found by previous experimental data, which suggests that PTP-7 adopts the “carpet” like model in killing cancer cells 26.

8


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Figure 3. Peptide arrangement details relative with membrane surface of PTP-7b. (A) Top view of the dispersive

distribution of the peptide individuals and (B) lateral view of one random peptide inserting in the membrane. The peptide backbone was presented in blue. Phe, Lys and His were presented in red, green and olive, respectively. The solvent molecules were presented by iceblue beads. (C) Density distributions of the lipid head, tail, Phe and Lys&His residues along the membrane normal. (D) Respective distance involvements of the C-potion and N-portion to the upper membrane surface. The blue dotted line was used to connect the horizontal level of the upper leaflet surface in B and D.

In order to explain the different routines of PTP-7b and PTP-7 behavior following on membranes, the influence of the unique 11th histidine was taken into consideration. It was interesting that the histidine residues situated on the top of peptide aggregates, arranged linearly and exposed to the water phase (Figure 4A). By calculating the residue mean smallest distance between the neighboring peptides, the histidine residues presented remarkable on the interacting matrix (Figure S6). As a better comparison, the distance between the 11th histidine backbones on the neighboring peptide pair was calculated, and it was obviously lower than the distance between the neighboring peptides (Figure 4B). The result therefore revealed the importance of the histidine in peptide association.

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Figure 4. (A) The arrangement of the histidine residues in the peptide self-assembly. The histidine residues were

presented by yellow beads and the presentation methods of the bilayer were consistent with Figure 2. Top and lateral views were shown respectively. (B) The distance evolvements of the two interacting peptides and the two histidine residues. Only the backbone beads were considered in the calculations. (C) Number of peptide clusters (NC) evolvements of PTP-7, PTP-7b, H11A and H11Y as a function of time. (D) Lipid disorder parameters of the upper leaflets subjected by PTP-7, PTP-7b, H11A and H11Y.

Furthermore, the 11th histidine was substituted by alanine (H11A) to remain the same charge amount and by tyrosine (H11Y) to keep a comparable size of the sidechain. The peptide variants were subjected to simulation at the ratio of 20/168. As a result, the clustering of H11A exhibited low ability as weak as PTP-7 (Figure 4C). Despite H11Y showed a propensity to clustering, the lipid disorder of the upper leaflet affected by the two mutants were slighter than that caused by PTP-7b (Figure 4D). PTP-7b is an amphipathic peptide. However, an asymmetrical contact distribution of the peptide hydrophobic interface with the lipid bilayer showed that only the 1-F, 5-L, 9-L and 12-L predominantly residing in the hydrophobic bilayer (Figure 5A). In contrast, the 6-F exhibited weaker affinity with the lipid bilayer. The abnormal residue alignment with bilayer not only favored for the peptide hydrophilic surface distributing in the polar solvent, it also allowed for the interaction occurrence between the histidine residues (Figure 5B). To quantitatively describe the association energy of PTP-7b, PMF of PTP-7b and the two mutants along with the separation distance between the paired peptides were estimated, respectively (Figure 5C). The association energy of PTP-7b showed two minimum regions with energy of -23.0 kJ/mol (0.9 nm) and -12.9 kJ/mol (~1.4 nm). In comparison, the binding energy of H11A reached to only one lowest valley at the distance of ~1.4 nm, and it showed a shallow energy valley of -6.8 kJ/mol. Despite the association energy profile of H11Y presented a similar shape with PTP-7b, the typical well depth was -8.5 kJ/mol which was similar with that of H11A. The value was much lower than that of PTP-7b, implying the easier dissociation of the two histidine mutations. Therefore, the 10


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results confirmed the gift of the 11th histidine in stabilizing PTP-7b self-assembling on membrane interface.

Figure 5. (A) The contact intensity between the respective residues on PTP-7b and the upper bilayer lipids. The data

was generated from the system with P/L ratio of 20/168. All the peptides were taken into account and then averaged. (B) Presentation of one interacting peptide pair on the membrane surface. The assembling-involved histidine residues were denoted by red dotted circle and the bilayer were shown in red square. (C) PMFs of PTP-7b-WT, H11A and H11Y calculated by umbrella sampling calculations at a sampling generation of 1.0 µs per window (configure samplings were shown in Figure S7). The association energy was set to zero at the largest pulling distance.

Moreover, calculated from the non-bonded potentials of PTP-7b and the histidine mutations, the Coulomb potential evolvements of them were similar and changed little (Figure 6A). The Lennard-Jones (L-J) potential of H11A also remained unchanged, corresponding to the dispersive peptide distribution on membrane (Figure S3). H11Y showed evident L-J potential decrease, which acted as the motivation for the obvious aggregation of H11Y (Figure 4C). In comparison, wild type (WT) of PTP-7b generated the most dramatic decline of the L-J potential (Figure 6B), reflecting the remarkable molecular interaction derived from the histidine residues. The results revealed that the high affinity between PTP-7b peptides did not merely depend on the π-π stacking of the histidine aromatic sidechains 58. Study shows that the nitrogen atoms of the imidazole ring can act as a hydrogen bond acceptor and/or donor in the neutral state 59. It is speculated that interaction of the N-H···N type formed between histidine imidazole and the lysine sidechain of the paired peptide. Despite the explicit hydrogen bond is not available in Martini CG model, the interaction between lysine sidechain and histidine imidazole is at the “attractive”, ranking the second strongest level of the L-J interaction (cgmartini.nl). In fact, obvious crossed interaction between the 11-histidine and 7'-lysine was observed in the residue contact distribution (Figure S6). Therefore, the histidine enabled PTP-7b to tightly bind together relying on the π-π stacking and additive hydrogen bonds with the lysine on the paired peptide. Because PTP-7b adopted alpha-helical structure, the lysine and histidine locate at the same surface of the peptide helical projection, which is favorable for the crossed interaction between them (Figure 5B).

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Figure 6. (A) The Coulomb potentials and (B) L-J potentials of PTP-7b, H11A, H11Y as a function of time. (C) NC

evolvements of the three peptides on membranes. (D) Disorder level of acyl carbon bonds of upper lipids impacted by three different peptides.

The histidine residue can be protonated when the pH is below 5.5. The cell surface of cancer especially the carcinoma is more acidic than the normal cells. Our previous study shows that the human lung carcinoma cell A549 has a more acidic surface (pH = 7.15) than that of the normal human lung cell CCD-13Lu (pH = 7.4) under physiological condition60. However, the pH level of A549 did not reach the pH threshold to protonate the histidine residue of PTP-7b 6. When the histidine residue was protonated, PTP-7b could not behave the biphasic cell lysis because of the loss of peptide self-assembling, resulting the PTP-7b in an identical pathway to that of PTP-7 25. The histidine-protonated PTP-7b at the P/L ratio of 20/168 was conducted in simulation. In consistence with the experiments, the protonated PTP-7b could not aggregate on the membrane surface (Figure 6C) and the membrane structure kept normal (Figure 6D). Evidence shows that the acidic periplasmic space of tumor tissues mainly generated from the glycolytic metabolism61. In addition to the pH is changeable in different periods of cancer cells, the protons transport from the cytosol and anionic lipids can be obstructed by the membrane-anchored glycocalyx, and then form an acid layer outside of the bilayer62. The bilayer interface thus forms negative net potential that is responsible for attracting the cationic peptides48, 63. Because PTP-7b destabilizes the bilayer primarily relying on the assembling of the membrane-inserted peptides, the protonation state is hard to maintain because of the negative net potential of the membrane interface. However, as the protons of anionic lipids such as the phosphatidylserines transfer toward the periplasmic space to form the acidic layer, PTP-7b is possibly protonated during its approaching to the lipid membrane while passing the acidic layer. The protonated PTP-7b shows a weak self-assembling in periplasmic space (Figure S8), which is favorable for its selective binding to cancer cells. Modeling details of PTP-7b aggregates to induce bilayer leakage 12


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Enlighted by the dynamics evidence of some lipids extending up in the system at the highest P/L ratio of 40/168 (Figure 2E), further cell lysis is believed to realize by the continued peptide-lipid interplay. Considering the successive adhesion of the periplasmic peptides toward the target cells, which resulted in a concentration gradient of peptides distributing in vicinity of membranes64, the peptide-bilayer system with the P/L ratio of 40/168 was performed again but to divide the peptide pool in two classes which were positioned outside and inserted into the model membrane, respectively. As a result, accompanying with the clustering of peptides, some individual lipids were completely abstracted from the upper leaflet (Figure 7A, 7B-black line), which was considered as the directive phenomenon of cell leakage25, 65. By calculating the distance of the translocated lipids from the membrane, the separated lipids reached to a high level of 2.5 nm from the bilayer center (Figure 7B, blue line). Moreover, the peptides from the membrane outside encircled the liberated lipids by neatly parallel arrangement, which was consistent with that on the membrane surface shown in Figure 4A. The arrangement pattern is structurally favorable since it is able to tie the neighboring peptides in the most efficient way to block the lipids. In addition, it was favorable for the stability of the system by coordinating the peptide amphipathicity and histidine interaction.

Figure 7. (A) The membrane-outside and -inserted peptides were distinguished with yellow and blue without the

sidechains. Acyl tails of POPC and POPS were colored as green and purple respectively. The images were captured at the final frame in lateral and top view. The initial state can be seen in Figure 9. (B) The peptide self-assembling evolvement (black line) and breaking away of the peptide-associated lipids from the membrane (blue line).

Meanwhile, segregated subdomain of POPS was observed to be enriched below the peptide assembly (Figure S9), which has been considered as a benchmark of membrane degradation66-67. Note that the liberated lipids were mainly POPC. It was speculated that the leakage of the POPC lipids increased the positional possibility of POPS lipids for concentrative translocation. Moreover, the enrichment of POPS could be additionally guided by the electrostatic attraction derived from the growing positive PTP-7b aggregate. The continuous peptide aggregation produced the lipid-peptide mixed cluster that extended to the water phase. In order to define the structure character of the mixture, the compositions on the shell and core of the mixed cluster were analyzed. An obvious increase of the contact intensity between the liberated lipids and the peptides was observed during the peptide-bilayer interplay (Figure 8A, red line), which implied the enhanced integration of peptide-lipid mixture. Meanwhile, the solvent accessible surface area of the liberated lipids declined evidently during the dynamic evolution (Figure 8A, green line). The results indicated that though the liberated lipids moved away from the 13



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bilayer, they were kept from the solvent environment. In other words, during the out-leakage of the bilayer lipids, they were sealed inside the peptide shell that performed as the “cage-like” structure. Comparing the distance evolvements of the membrane-inserted peptides from bilayer center with and without the periplasmic peptides, the periplasmic peptides could assist the membrane-inserted peptides to extend increasingly out to the solvent environment (Figure 8B). Taken together, the results indicated that the bilayer destruction induced by aggregated peptides also relied on the additive contribution of the periplasmic peptides.

Figure 8. (A) The contacts between peptide and liberated lipids (red line) and solvent accessible surface area (SASA,

green line) of the liberated lipids along with the time. (B) Distances of the membrane-inserted peptide with or without the peptides outside of membrane from the bilayer center.

DISCUSSION Peptide self-assembling has been extensively studied for years in drug delivery68 and sustained release25. Antibiotic assessment of natural and artificial peptides by forming ordered aggregates have been extensively explored recently 69-70. As the majority class of AMPs, CPs in assembling action attracts increasing attentions and the interrelated cytolytic mechanism is different from the existing pore-forming models. It is important to establish molecular knowledge of the modeling details, structure relevance and dynamics underlying the membrane distortion followed by ultimate leakage. In this study, the molecular details of membrane disruption induced by aggregated CP of PTP-7b were revealed combining experiments and extended CG dynamic simulations, which is hopeful to provide referential information in the development of new CP drugs. The membrane disruption process shares several hallmarks similar with previously proposed models. For example, when peptides bind on membrane surface, CPs adopt an orientation with the hydrophobic terminal inserted into the membrane and distribute as dispersive individuals at low concentration. This phase is generally considered as the preparation for the following membrane insertion or pore formation 71. PTP-7b in assembled and non-assembled cases do not present “pore” style that is suitable for Magainin-2 and Melittin studied in CG modeling40. It is suspected that PTP-7b in the initial phase of binding membrane surface conforms to the description of the “carpet” model, which has been used to explain the mechanism of the cationic lytic peptides72, such as PTP-7 73. The membrane coated by “carpet” peptides is often followed by membrane disruption alternatively realizing in a “detergents” manner, or forming transient pores to disintegrate membranes. One review describes that the “detergents” model derives from the “carpet” model and 14


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gradually generates micelle-like mixtures of lipids and peptides 74. It is likewise with the active processing of aggregated PTP-7b when exceeds the critical concentration. However, a controversial report describes that onset of “detergents” model starts from the attachment of peptide clusters concomitant with pore formation4, 75. In this regard, more efforts are required to depict the principles of the “detergents” style. In this study, the preassembled PTP-7b is proved showing lower efficiency to disintegrate membrane without formation of transmembrane pore, and PTP-7b mostly adopts another variant of “carpet” model to extend out the localized membrane by aggregation. In a previous study exploring the modeling of maculatin, a plenty of disputed evidence is reported that maculatin adopts pore-like model76-77 or carpet-like model78-79 depending on giant unilamellar vesicles and NMR spectroscopy etc. It indicates that the modeling presentation is affected by the methods and conditions. The CG simulations were hence executed and membrane insertion of maculatin aggregates was observed at a higher P/L ratio39, which compensated the molecular details and explanation for experiments. To explore the cytolysis of PTP-7b, we built lines of simulations based on known experimental data to explore the substantial molecular details. As shown in Figure 9, peptides initially bind to the anionic membrane as individuals relying on the electrostatic interaction. As continued accretion of peptides attach on the localized membrane, the membrane suffers unfavorable stress and the membrane integrity starts to lose. Furthermore, the membrane distortion becomes more dramatic by addition of the peripheral peptides. Relying on the traction of the volume-increased peptide aggregate towards the water phase, the associated lipids gradually migrate from the hydrophobic membrane and were concealed in peptides (Movie S1). The resulting mixture of lipids and peptides might act as the preliminary micelle that cracks away from the membrane, followed by the membrane disruption and cell lysis. The phenomenon is likewise with the report of another CP 80, which highlights the importance of peptide behaving at the water-lipid interface.

Figure 9. The evolvement of the bilayer lysis triggered by the spatiotemporal aggregation of PTP-7b. The periplasmic

and the membrane-inserted peptides were respectively rendered with orange and blue to clarify their initial positions and the subsequent cooperation. The POPC and POPS lipids were represented with green and purple beads respectively.

During the process, the completed lipid migration caused by aggregated peptides incorporates the membrane-inserted as well as the periplasmic peptides, indicating the significance of the periplasmic peptides in membrane disruption. The likewise phenomenon is also observed in the formation of “pre-pore” by melittin, which requires a cooperative response of periplasmic melittin molecules to lower the free energy in creating small transmembrane-pore81. Note that in the delivery and uptake of peptide drugs, the situations of soluble and assembled peptides usually coexist. Moreover, affected by environment factors like ion buffering and changeable local pH, the 15



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conversion between the soluble and assembled peptides therefore changes occasionally, especially on the drug targets of membrane surface. Based on the above results, we suggest some propositions underlying the short CP in associating with lipid bilayer. First, the cationic PTP-7b shows propensity to binding the anionic cancer cell membranes, which renders for its anticancer selectivity. To this point, PTP-7 possesses a high cytolysis efficacy probably relying on the enhanced electrostatic interaction with cancer cells5. However, PTP-7 loses the ability to form aggregates because of electrostatic repulsion. In contrast, a neutral peptide variant can forms ordered nanofibers in pure water in previous study 24. Note that aggregation of PTP-7b occurred at membrane interface. On the one hand, it ensures for the peptide stability depending on the hydrophobic residues of 9th-Phe, 1st-Leu and 12th-Leu anchoring on membrane. In fact, the amphipathic distribution of polar and hydrophobic residues acts as a benchmark for membrane-active peptides in disturbing bilayer integrity, either by creation of lipid disruption or pore formation4. On the other hand, the residue arrangement is responsible for the interaction between the 11th histidine residue that is proved stabilizing the peptide assembly in membrane82-83. Moreover, the histidine residues can form hydrogen bonds with the opposite lysine on the paired peptide. It therefore compensates the availability for further fusion between the peptide assemblies on membranes and the outside counterpart. The peptide arrangement makes the positive lysine outward to water instead of interacting to the lipid phosphates. The phenomenon has been considered as one of “carpet” model principles84, which supports that PTP-7b acts as a derivative “carpet” model. Note that the peptide aggregation ability and bioactivity was sensitive to histidine position, since the other variant sequence of FLGALFHALSKL (exchange the lysine and histidine of PTP-7b) shows degraded aggregation and membrane-lysis 25. In a previous CG simulation study42, an aggregated cyclic peptide induces bilayer lipids to form micelle-like mixtures. Note that the preassembled cyclic peptides show higher membrane destruction than the membrane-inserted peptides, which is different from the linear helical PTP-7b in this study. Given the difference on structure and sequence, they would adopt distinct mechanism during their bilayer leakage processes. For example, for the cyclic peptide, no evidence shows the cooperation between the membrane-inserted and periplasmic peptides at high peptide concentration. However, the linear helical PTP-7b needs extra assistance of the periplasmic peptides for lipid further migrating outwards. In addition, the generated micelle-like mixture formed by the cyclic peptide is disordered except for the limited peptide inter-stacking. In contrast, PTP-7b forms “cage-like” structure where the peptides display a parallel arrangement to better pack together. Therefore, PTP-7b assembles on membrane surface and induces cell lysis suggesting it adopts a special pathway. It attributes to the specific residue alignments in balancing the amphipathic property and interaction based on the 11th histidine residues. Moreover, it is important for the anticancer activity of PTP-7b since the balance between charges and hydrophobicity is important for selectivity of amphipathic helical peptides85-86. The phenomenon-based models, such as “carpet” model and “membrane-pore” model, give limited understanding of short helical CPs to predict the activity and design novel peptide drugs9. The modeling of action described in this study represents a new type of aggregated CP, which comprises the peptide distribution between solvent and hydrophobic membranes by specific peptide interaction mode. Taken account of increasing demand of antibiotic agents with improving properties and practicability, molecular details involved the modeling and driving forces of the modified CPs with enhancing stability, efficiency and simplification requires for continued efforts. 16


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Despite the successfulness of CG simulations in presenting AMPs pore-forming model39-40, deficiency of the CG simulation by a reduction of atom resolution and screening limitation of the electrostatic interaction should be carefully regarded. A more accurate definition is promising for the future research of the bioactive aggregated peptide upon membrane association. In addition to anticancer activity and sustainable release of the short helical CPs, the unknown clinical applications like drug delivery and drug modification deserve to be further exploited. CONCLUSIONS In summary, we employed CG molecular dynamic simulations combining experiments to explore the molecular basis of cytolysis mechanism of the self-assembling peptide PTP-7b. (1) Efficient membrane perturbation is induced by lateral aggregation of the membrane-inserted peptides, rather than the preassembled peptides. (2) The self-assembling and membrane-lytic abilities of PTP-7b on membranes depend on the amphipathic property and peptide interaction based on the histidine residues. (3) The processing of lipid leakage triggered by peptide aggregation requires for a cooperation of the membrane-inserted and the periplasmic peptides. Taken together, the molecular perspective of this study is favorable for the understanding about action modeling of short helical CPs and development of novel clinical peptides.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. * Corresponding Author E-mail: [email protected]; [email protected] Tel: 86-10-64438015

Notes The authors declare no competing financial interest.

Acknowledgment This work was supported by the National Basic Research Program of China (973 program) (2013CB910700), the National Natural Science Foundation of China (21672019, 21372026, 21402006, 61304147), the Fundamental Research Funds for the Central Universities (Grant No. YS1407, 2050205, 20130801, PT1613-08 and PYBZ1711), and Special Program for Applied Research on Super Computation of the NSFC-Guangdong Joint Fund (the second phase).

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Figure 1. (A) The AFM images of fresh (non-assembled) and overnight-incubated (assembled) PTP-7b dissolved in pure water. (B) PTP-7b aggregation on cell membranes induced lysis of Hela cells. The cell membranes were labeled by di-8-ANEPPS (red) and the peptide was labeled by FITC (green). Peptide concentration was fixed at 40 µM. (C) The cytotoxicity of non-assembled and assembled PTP-7b measured on Hela cells. Three independent measurements were conducted. (D) Peptide-induced surface pressure changes of lipid monolayer with a composition of DPPC-cholesterol-DPPS (50: 10: 2.5 in molar ratio) mimicking mammalian cell membranes. The initial surface pressure of the lipid monolayer was set at about 30 mN/m. The final concentration of loaded peptides was 10 µΜ. 283x275mm (300 x 300 DPI)



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Figure 2. Presentations of membrane deformations affected by association of PTP-7b in different situations. Peptides were respectively placed outside (A) and inserting (B, D, E) the membrane. Only backbones of peptides were shown with blue chains. Tails of POPC and POPS were distinguished in green and purple, and the head groups were presented in red. For clarification, the water solvents were omitted. The upper images in A, B, D and E represented the final states of the four lipid-bilayer systems at different concentrations. The lower images showed the mass density profiles 55 of the upper lipid leaflet incorporating peptides within the last 1.0 µs, which were generated in the same view to the upper snapshots. (C) Disorder parameters (Sz) of the acyl carbon bonds of the lipids in the upper leaflet of the four systems. 353x265mm (300 x 300 DPI)


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Figure 3. Peptide arrangement details relative with membrane surface of PTP-7b. (A) Top view of the dispersive distribution of the peptide individuals and (B) lateral view of one random peptide inserting in the membrane. The peptide backbone was presented in blue. Phe, Lys and His were presented in red, green and olive, respectively. The solvent molecules were presented by iceblue beads. (C) Density distributions of the lipid head, tail, Phe and Lys&His residues along the membrane normal. (D) Respective distance involvements of the C-potion and N-portion to the upper membrane surface. The blue dotted line was used to connect the horizontal level of the upper leaflet surface in B and D. 241x203mm (300 x 300 DPI)



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Figure 4. (A) The arrangement of the histidine residues in the peptide self-assembly. The histidine residues were presented by yellow beads and the presentation methods of the bilayer were consistent with Figure 2. Top and lateral views were shown respectively. (B) The distance evolvements of the two interacting peptides and the two histidine residues. Only the backbone beads were considered in the calculations. (C) Number of peptide clusters (NC) evolvements of PTP-7, PTP-7b, H11A and H11Y as a function of time. (D) Lipid disorder parameters of the upper leaflets subjected by PTP-7, PTP-7b, H11A and H11Y. 229x183mm (300 x 300 DPI)


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Figure 5. (A) The contact intensity between the respective residues on PTP-7b and the upper bilayer lipids. The data was generated from the system with P/L ratio of 20/168. All the peptides were taken into account and then averaged. (B) Presentation of one interacting peptide pair on the membrane surface. The assembling-involved histidine residues were denoted by red dotted circle and the bilayer were shown in red square. (C) PMFs of PTP-7b, H11A and H11Y calculated by umbrella sampling calculations at a sampling generation of 1.0 µs per window (configure samplings were shown in Figure S7). The association energy was set to zero at the largest pulling distance. 309x82mm (300 x 300 DPI)



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Figure 6. (A) The Coulomb potentials and (B) L-J potentials of PTP-7b, H11A, H11Y as a function of time. (C) NC evolvements of the three peptides on membranes. (D) Disorder level of acyl carbon bonds of upper lipids impacted by three different peptides. 276x209mm (300 x 300 DPI)


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Figure 7. (A) The membrane-outside and -inserted peptides were distinguished with yellow and blue without the sidechains. Acyl tails of POPC and POPS were colored as green and purple respectively. The images were captured at the final frame in lateral and top view. The initial state can be seen in Figure 9. (B) The peptide self-assembling evolvement (black line) and breaking away of the peptide-associated lipids from the membrane (blue line). 348x120mm (300 x 300 DPI)



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Figure 8. (A) The contacts between peptide and liberated lipids (red line) and solvent accessible surface area (SASA, green line) of the liberated lipids along with the time. (B) Distances of the membrane-inserted peptide with or without the peptides outside of membrane from the bilayer center. 257x91mm (300 x 300 DPI)


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Figure 9. The evolvement of the bilayer lysis triggered by the spatiotemporal aggregation of PTP-7b. The periplasmic and the membrane-inserted peptides were respectively rendered with orange and blue to clarify their initial positions and the subsequent cooperation. The POPC and POPS lipids were represented with green and purple beads respectively. 453x121mm (300 x 300 DPI)



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A Molecular Dynamics Study of the Short-Helical ... - ACS Publications

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