Mechanistic and structural basis of bioengineered bovine Cathelicidin-5 with optimized therapeutic activity

Peptide-drug discovery using host-defense peptides becomes promising against antibiotic-resistant pathogens and cancer cells. Here, we customized the therapeutic activity of bovine cathelicidin-5 targeting to bacteria, protozoa, and tumor cells. The membrane dependent conformational adaptability and plasticity of cathelicidin-5 is revealed by biophysical analysis and atomistic simulations over 200 μs in thymocytes, leukemia, and E. coli cell-membranes. Our understanding of energy-dependent cathelicidin-5 intrusion in heterogeneous membranes aided in designing novel loss/gain-of-function analogues. In vitro findings identified leucine-zipper to phenylalanine substitution in cathelicidin-5 (1–18) significantly enhance the antimicrobial and anticancer activity with trivial hemolytic activity. Targeted mutants of cathelicidin-5 at kink region and N-terminal truncation revealed loss-of-function. We ensured the existence of a bimodal mechanism of peptide action (membranolytic and non-membranolytic) in vitro. The melanoma mouse model in vivo study further supports the in vitro findings. This is the first structural report on cathelicidin-5 and our findings revealed potent therapeutic application of designed cathelicidin-5 analogues.


Results
Insights from atomistic simulations. The amino acid sequences of all synthesized peptides by reference to our structural findings in different model membrane studies are shown in Fig. 1a. The simulated BMAP-28 helical structure in aqueous solution generated an extended conformation with the commencement of β -sheet structures at each terminus and is consistent with the sequence based structure prediction ( Supplementary Fig. 1a-c). In presence of bio-membranes the structural folding may vary as seen in the homologous BMAP-27 protein 24 . Our restrained molecular dynamics (MD) simulation on microsecond scale showed a helical conformation (residues [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] in DOPS membrane for the first 400 ns followed by the induction of a central helix kink ( Supplementary Fig. 2a,b). The central kink (I10-L11) facilitate a desirable orientation for BMAP-28 penetration by stimulating an amphipathic environment at the peptide-membrane interface. In contrast, partial unfolding (residues 10-18) and aqueous phase peptide distribution was identified in DPPC (Fig. 1b, Supplementary Fig. 2b). The eukaryotic model leukemia-like membrane (LLM) system portrayed a relatively high order helix and kink formation as compared to the thymocytes-like membrane (TLM) (Fig. 1b,c). A peptide penetration of ~6 Å and relatively small ~2 Å was calculated in the anionic and zwitterionic systems, respectively ( Supplementary Fig. 2c). The W14 residue was oriented towards the aqueous and membrane core regions in the zwitterionic and anionic systems, respectively (Supplementary Fig. 3a-d). At a higher peptide to lipid (P/L = 1:25) ratio, both folded and unfolded monomers in the membrane interface and water phase regions, respectively, were revealed. No stable oligomerization or membrane pore formation was observed during the 1 μ s time period (Supplementary Fig. 3e). The all-atom atomistic simulation on microsecond scale could identify the peptide N-terminal association, C-terminal insertion and kink formation in the model membrane systems.
The coarse-grained (CG) MD simulation of BMAP-28 (P/L = 1:40) showed a membrane dependent peptide binding, oligomerization and dissemination. In anionic systems, the BMAP-28 peptides were identified to interact as monomers and bordered by the anionic lipid heads (Fig. 2a). Unstable intermediate dimers/trimers were observed in the LLM system. Interestingly, acceleration of peptide self-association by C-terminal hydrophobic residues (tetramer/hexamer) was observed in the DPPC and TLM systems (Fig. 2a). The C-terminal truncated BMAP-28 1-18 also showed a membrane dependent self-association. However, unlike the parent type, BMAP-28 1-18 rendered a stable dimerization mediated by alanine and tryptophan residues and an unstable oligomer in the zwitterionic systems (Fig. 2b). A prominent dimeric association mode of interaction was also observed in Syn1 containing the phenylalanine zipper sequence. During the 15-30 μ s, we did not observe any spontaneous pore on bilayer membrane at a concentration above the threshold reported for the homologous human cathelicidin 25 . To this end, our 100 μ s MD simulation analysis of BMAP-28 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] and E. coli membrane interaction further ensured the selective peptide distribution around the anionic (15% POPG and 5% cardiolipin) lipids. No spontaneous membrane pore formation was revealed on this time scale. But, the bilayer thickness was significantly affected as illustrated in Fig. 2c. In contrast, the effect of peptides on the membrane thickness was very limited in zwitterionic and TLM systems (Fig. 2d). This suggested in addition to P/L concentration, the peptide action is time dependent and the membrane disruption is beyond the chosen simulated time scale.
The comparative analysis also suggested the peptide aggregation and binding orientation is highly modulated by the lipid composition. The tendency of peptide oligomerization was also revealed in the aqueous solution. The 1 H-NMR spectra of BMAP-28 and BMAP-28 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] in phosphate buffer and TFE solution revealed sharp spectral peaks with small line broadening in the methyl and amide regions. No significant line broadening at the imidazole proton peak in tryptophan was observed ( Supplementary Fig. 4). This suggested small oligomers (dimers/ trimers) may be expected in both aqueous and hydrophobic environments; however, large oligomers are unanticipated. Taken together, we speculated the peptide aggregation in zwitterionic systems may be due to the weak membrane attachment and aqueous phase distribution. The differential penetration efficacy and self-assembly in heterogeneous membranes also indicated their distinguished cell and species specific cytotoxicity.
Scientific RepoRts | 7:44781 | DOI: 10.1038/srep44781 Structure and energy based peptide screening. Establishing an interplay between the chemical properties of the peptide (cationicity and hydrophobicity) and the plasma membrane is crucial for toxicity modulation. To this end, we studied a chimera peptide armed with elevated amphipathicity and hydrophobicity (Fig. 1a). Interestingly, a very fast and significant loss in peptide secondary structure was observed during the 2 μ s MD simulation ( Supplementary Fig. 2d). This suggested a balanced cationicity and hydrophobicity is required to optimize the BMAP-28 peptide activity 26 . The identified N-terminal association, C-terminal insertion and kink regions in BMAP-28 were thus targeted with variable hydrophobicity and hydrophobic movement to create a loss-/gain-of-function peptide library ( Supplementary Fig. 1c). As shown in Fig. 1a, four different BMAP-28 derivatives were designed by terminal residue truncation and kink residue mutation (Fig. 1a). To accelerate the self-association and structural stability 27,28 , the leucine-phenylalanine zipper derivative was designed.
The free energy (Δ G) profiling in the DOPS system calculated substantial Δ G at the water/membrane interface with energy minima of − 7.4 kcal mol −1 for BMAP-28 intrusion. The peptide transition from the water phase to lipid core revealed an oblique peptide orientation with maximum tilt angle ranging from ~45° to 60°. Transition of BMAP-28 from the water phase to membrane phase showed a negative energy minimum for all  GGLRSLGRKILRAWKKYGPIIVPIIRI-NH2 BMAP-28 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] GGLRSLGRKILRAWKKYG-NH2  Syn1  GGFRSFGRKIFRAWKKYG-NH2  Syn2  GGLRSLGRKAARAWKKYG-NH2  Syn3  GRKILRAWKKYG-NH2  Chimera  GRFKRFRKKFKKLFKKLGPIIVPIIRI- studied model membrane systems (Fig. 3). However, the Δ G minima were relatively strong in the LLM and E. coli systems (~− 6 kcal mol −1 ) as compared to the TLM (~− 2 kcal mol −1 ). The favorable Δ G in anionic systems at the water/membrane interface suggested a low energy barrier for the peptide intrusion. By contrast, the relatively weak Δ G in the TLM system suggested peptide inability to cross the rigid membrane barrier enriched in cholesterol (Fig. 3a). In E. coli membrane, the BMAP-28 and Syn1 peptide yielded the highest Δ G with an average value of − 4.73 ± 0.24 and − 4.04 ± 0.04 kcal mol −1 , respectively; while the average Δ G for BMAP-28 1-18 was marginally small. Remarkably, the Δ G was significantly affected for Syn2 and Syn3 peptides with an average value of − 2.73 ± 0.14, and − 2.20 ± 0.09 kcal mol −1 , respectively (Fig. 3b). This suggested the onset of a potential energy barrier for the Syn2 and Syn3 peptides permeabilization into the E. coli membrane. Taken together, our atomistic findings correlate with the free energy results and highlights the importance of peptide kink and N-terminal residues towards the membrane intrusion and cytotoxic activity.
Biophysical analysis of membrane binding. The CD spectrum of BMAP-28 in sodium phosphate buffer showed an extended BMAP-28 conformation; however α -helical peptide folding was ascertained with an increasing concentration of TFE solution (Fig. 3c). The CD spectrum analysis using BeStSel 29 presented an increasing percentage of helical content from 4.7 to 38.7% and decreasing percentage of β -sheet conformation from 25.3 to 7.1% in 0 and 45% TFE solutions, respectively. The BMAP-28 derivatives also exhibited a well restrained α -helix conformation in 45% TFE solution with respect to their unfolded conformation in the aqueous buffer ( Supplementary Fig. 5a). This specified the structural similarity under our selective peptide modifications. The structural folding were further analyzed by adding different concentration of 100 nm size LUVs ( Table 1). Addition of anionic LUVs shifted the CD spectrum of BMAP-28 from an unfolded to folded state with negative peaks displacement from 195 nm to 208 nm (Fig. 3d). In E. coli LUV, a profound α -helical BMAP-28 was revealed with 38.2% helix and 7.5% β -sheet (similar to 45% TFE) conformation. Similarly, the unfolded BMAP-28 in the   buffer yielded a partial folded conformation with 11.9/14.3 and 11.1/21.5% of α /β content in the heterogeneous LLM and TLM LUVs, respectively (Fig. 3d). In contrast, the CD spectra of target peptide in DPPC and TLM LUVs showed an increase percentage of random coil like conformation. The membrane mediated BMAP-28 folding that combines MD simulation and CD spectroscopy correlates one another. The fluorescence spectroscopy of tryptophan in BMAP-28 is a suitable probe for the membrane mediated folding, penetration and self-assembly analysis. Fluorescence spectra of W14 showed a blue shift (331-335 nm) and comparatively high yield of fluorescence intensity for all anionic lipid containing LUVs (Fig. 3e). By contrast, the zwitterionic DPPC and TLM LUVs displayed relatively small quantum yield of fluorescence and the emission band at 345 and 348 nm, respectively (Fig. 3e). The blue shift and fluorescence intensity comparison ensured the polar and non-polar environment of W14 in zwitterionic and anionic LUVs, respectively. The membrane dependent distinct fluorescence emission bands proposes two major possibilities i.e. (i) The non-polar environment in anionic LUVs may be due to the substantial peptide binding and insertion, or (ii) may arise due to a relatively high order self-assembly on the membrane surface. To investigate this, the fluorescent probe 8-anilinonapthalene-1-sulfonic acid (ANS) binding assay was performed. Results showed a shift of the ANS fluorescence peak from 507 nm to 465 nm, from 493 nm to 471 nm and from 483 nm to 464 nm in DOPS, LLM and E. coli LUV systems, respectively ( Supplementary Fig. 5b). In contrast, negligible peak shift (from 473 nm to 467 nm) and significant increase in fluorescence yield was revealed for the TLM and DPPC systems, respectively ( Supplementary Fig. 5b). The ANS favorably binds to the hydrophobic residue cluster and has been reported to be accelerated in unfolded/partial folded and protein aggregation states 30,31 . The significant increase in the fluorescence intensity in DPPC indicated peptide aggregation or unfolding that generated a non-polar cavity for ANS binding. On the other hand, the low fluorescence yields in DOPS and E. coli suggested a folded or non-aggregated state of BMAP-28 binding ( Supplementary Fig. 5b). The truncated BMAP-28 1-18 also revealed similar ANS fluorescence spectra in the anionic and zwitterionic LUV systems.
The thermodynamic properties of BMAP-28 binding to LUVs using ITC measurement at 37 °C were probed. An endothermic reaction was revealed for the homogeneous DPPC LUV (Fig. 4a) with a binding dissociation constant (K D ) of 74.0 μ M (Table 2). Similarly, the K D for the anionic systems DOPS, LLM and E. coli were estimated to be 1.5 μ M, 16.1 and 24.8 μ M, respectively. A negative change in enthalpy in these systems indicated an exothermic reaction with small favorable enthalpic contribution ( Fig. 4b-d). In contrast, the enthalpic contribution disfavors the peptide binding to the DPPC liposomes. The binding of BMAP-28 to all target LUVs except TLM was strongly driven by the entropic contribution (TΔS). Interestingly, as shown in the ITC thermograms in Fig. 4e, the TLM LUVs demonstrated a non-specific binding kinetics for BMAP-28. The heat generated was comparatively low and the binding reaction did not reach saturation resulting in an uncertainty in fitting. A fast and slow saturation in anionic and zwitterionic LUVs (P/L = 1:50), respectively, can be correlated to the peptide binding efficacy and absorption. In our titration approach, the strong electrostatic binding of anionic liposomes rendered fast peptide absorption. In contrast, the low binding efficacy of BMAP-28 to TLM signified its non-specific interaction and/or weak peptide attachment/absorption. The thermodynamic parameters for each LUV systems were detailed in Table 2.
Biological applications of target peptides. Antimicrobial and antimalarial efficacy. The antimicrobial assay showed low antimicrobial potency of BMAP-28 in comparison to BMAP-28 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] and Syn1 with a minimum inhibition concentration (MIC) of ~2 μ M for both Gram-positive and Gram-negative strains. Interestingly, the Syn2 and Syn3 peptides with kink residue mutation and N-terminal truncation, respectively, depicted a loss of activity even at one order higher concentration (Fig. 5a). The BMAP-28 1-18 and Syn1 peptides depicted equipotent       Fig. 6a); whereas the membrane integrity was restrained in the untreated E. coli BL21 cells. On the contrary, the membrane abnormalities or disruption of E. coli DH5α strain was not observed even at 4 μ M of Syn1 (Supplementary Fig. 6b). The flow cytometry test using propidium iodide (PI) dye in Syn1 treated E. coli BL21 cells displayed ~100% PI influx at peptide MIC. In contrast, the E. coli BL21 strains showed < 5% PI influx treated with 4 μ M BMAP-28 ( Fig. 6a; left panel). As observed in the cell assay, the E. coli DH5α strains depicted no significant PI influx for both peptides (Fig. 6a; right panel). This suggested the peptide resistance of E. coli DH5α strains can be correlated to their membrane integrity as observed by TEM analysis. Taken together, the findings suggested that the toxicity of Syn1 to E. coli BL21 strain was mediated by the disruption of membrane integrity. Nonetheless, alternation in the cytoplasmic concentration was also perceived in the BL21 cells. This anticipated a possible intracellular binding of the target peptide after transient membrane pore formation. Our analysis revealed the mechanism of the peptide action; however, the key molecules that mediate E. coli DH5α resistance remains unclear. We speculated that the resistive characteristic may accelerate through membrane surface modification and/or proteolytic enzyme secretion.
Anticancer efficacy. The BMAP-28 exhibited a significant anticancer activity at 2-4 μ M against T-and B-cell lymphoma cells (Fig. 5e). The B-cell lymphoma were more susceptible to BMAP-28 (at ~2 μ M; cell viability = 38%) as compared to T-cell lymphoma (Fig. 5f). Interestingly, the potent BMAP-28 analogues also possessed a substantial anticancer activity both in T-and B-cell lymphoma at a comparatively little higher concentration. The T-cell lymphoma was relatively more and less resistant to Syn1 and BMAP-28 1-18 , respectively. The Syn1 depicted a nearly similar potential anticancer activity like BMAP-28 in B-cell lymphoma (Fig. 5e,f). As conceived, the Syn2 and Syn3 peptides presented loss-of activity at a concentration higher than 30 μ M. The anticancer efficacy of BMAP-28 and its derivatives against the melanoma B16-F1 cells were relatively small at 2 to 5 μ M. A cell viability loss of > 70% was revealed by BMAP-28 at 10 μ M (Fig. 7a); whereas the BMAP-28 1-18 and Syn1 exhibited fairly low anticancer activity at this concentration and found to be active at ~30 μ M.
The BMAP-28 analogues presented low cytotoxic activity against healthy bone marrow cells at ~10 μ M. In contrast, the BMAP-28 molecule was significantly cytotoxic at ~5 μ M (Fig. 7b). At this concentration, a substantial loss of cell viability was ensured for macrophages, whereas no effect was seen for Syn1. The flow cytometry study ensured the binding of annexin V and PI to the lymphoma cells treated with the potential peptides at 2-10 μ M as shown in Fig. 6b. This makes certain the membranolytic and peptide mediated cell proliferation activity of the target peptides. Further study of BMAP-28 analogues in RANKL-induced osteoclastogenesis showed significant activity of Syn1 at 3 μ M, and a complete abolition was revealed at 10 μ M ( Supplementary Fig. 8). Taken together, the cell assay indicated the potential anticancer activity of Syn1 and BMAP-28 1-18 with low hemolytic and cytotoxic activity towards the healthy cells (Figs 5 and 6). By contrast, the significant hemolytic and cytotoxic activity of BMAP-28 in healthy cells dampen its therapeutic interest.
The efficacy of target peptide Syn1 was further reveled from the in vivo analysis. By way of illustration, Fig. 7c shows a distinct tumor development in control and Syn1 treated mice on the day 14 and 20. The average tumor volume in control group mice was measured to be 233 and 1031 mm 3 on day 14 and 20, respectively (Fig. 7d). In contrast, the Syn1 treated mice showed no tumor formation and were alive. The significant difference in the tumor volume correlates to the in vitro efficacy of Syn1 and highlights its potent anticancer activity.

Discussion
Peptide-based rational drug designing against drug-resistant pathogens is advancing in recent years. However, the inadequate structural and functional information challenges the successful therapeutic optimization 32 . Here, we focused on modulating the inordinate cytotoxicity for a successful remedial design of the target bovine peptide whose human homologous protein is under clinical development 17,22,23,33 . The mechanistic and structural basis of bovine BMAP-28 activity explored here described its membrane dependent folding, kinetics and specificity through selective phospholipid binding and reorganization. Structural analysis in other AMPs using partial lipid distribution that imitates bacteria and eukaryotic cells 33,34 have been amply discussed. To our knowledge, this is the first structural report that provides a comparative study of cathelicidin action on eukaryotic cancer and normal cell membrane that significantly varied on net surface charge and phospholipids distribution. The BMAP-28 binding and penetration relies on the anionic lipid distribution as reveled from our calorimetry and spectroscopy measurements. Despite of a likely membrane composition in TLM and LLM, the trivial anionic PS phospholipid variation defines the membrane attachment and binding kinetics (Fig. 8).
The atomic simulation and biophysical results ensured the lipid heterogeneity dependent BMAP-28 structural transition, reorientation and membrane intrusion. The coulombic forces coupled with the substantial hydrophobic moment reorient the BMAP-28 molecule by aligning its polar and non-polar surfaces to the membrane hydrophilic and hydrophobic surfaces, respectively. The computational findings obtained from structural and kinetic interpretation closely resemblance to the biophysical results. Thus, centering to the optimization of BMAP-28, this study proposes a synergistic combinatorial chemistry and biophysical approach for the successful therapeutic advancement. The central helix-kink identified in BMAP-28 could be crucial to provide an improved structural rearrangement on the water/membrane interface for insertion. Structural kinks also have seen in other AMPs that generates an optimized amphipathic environment for an oblique membrane permeation 35 . In addition to peptide reorientation, the CG-MD studies showed a distinguished peptide self-assembly in the vicinity of model membranes.
The membrane mediated folded and partial folded helical states of BMAP-28 correlates other class of AMPs 26,36 . The leucine to phenylalanine zipper substitution ascertained the differential behavior of the synthetic BMAP-28 analogue where the former facilitates membrane fusion 37 ; and the latter accelerates structural integrity through dimerization 27 . The potential of mean force (PMF) profiling based screening, identified the gain/ loss-of-function peptides by anticipating their Δ G barrier (Fig. 8) at the water/membrane interface. The smaller Δ G minima (Fig. 3b) and unaffected Δ G profile upon C-terminal truncation supported our assumption that the helix-kink and N-terminal regions are crucial for the peptide activity 14,16 . The compressive computational and experimental peptide screening procedure presented here displayed a promising approach to rationally design more efficient host-defense peptide analogues.
The monomeric binding and relatively high membrane penetration of BMAP-28 in anionic system driven by both enthalpic and entropic contributions proposed a toroidal pore mechanism of action 25 . By contrast, the peptide molecules are tightly confined to the water/membrane surface through substantial entropic influence in zwitterionic membranes. This weak binding and self-assembly could resist the peptide diffusion unless an optimum concentration is reached. As revealed from our CG-MD study, the substantial binding could obtained in the aqueous phase followed by membrane attachment. Despite of the membrane heterogeneity, these observations could be correlated to the P/L dependent cytotoxic activity of BMAP-28. At a high peptide concentration that showed hemolysis (10-30 μ M), the self-assembly propensity will be much stronger in comparison to their antimicrobial and anticancer MIC. Taken together, we hypothesize that in bacteria and tumor cells, the peptide mediated lipid reorganization and substantial membrane intrusion could commence cell lysis in relatively low concentration. The proposed arguments based on peptide-membrane study could also be limited in terms of natural cell-membrane composition that contains ~50% proteins. In addition, the bio-membranes of eukaryotic and E. coli cells are reinforced by cytoskeletons and peptidoglycans, respectively, to provide cell resistance to environmental stress. The present study performed using artificial LUVs and lipid-bilayer lacks such consideration and is vulnerable to stress.
Although, the peptide penetration could be directly correlated to their cytotoxic action, an interconnections between the peptide folding/unfolding and active/inactive states are still confrontational. The challenge has been observed in the transient structural switching of AMPs on or within the target cell membrane. The α -helical BMAP-28 shown to be unstructured in the aqueous phase and folded upon selective target cell binding. In alliance, the folded conformation shows active cytotoxic activity at low P/L in bacteria and cancer cells in vitro. Thus,  taken together, an assumption can be made that the bioactive conformation of BMAP-28 centers on the dynamic structural transition from extended/β -sheet to α -helix conformational state. The aim to track the time-dependent pore formation by BMAP-28 or its potential analogues was unsuccessful in the present study at 100 μ s. This suggests the structural insights into a spontaneous pore formation is far beyond the selected time scale of simulation. However, the electron microscopy, flow cytometry and in vitro transcription/translation assay evidenced cell pore formation and protein synthesis inhibition (Fig. 8). Thus, we proposed a bimodal mechanism of antimicrobial and anticancer action for the target class of peptides (Fig. 8).
The membrane model based rational designing of potent anticancer and antimicrobial BMAP-28 analogues through combined computational and biophysical techniques supports our cell assay results. The truncated BMAP-28 1-18 and Syn1 were more potent antimicrobial and anticancer agents which may be due to their less self-assembly propensity and high amphipathic movement. Their variable cytotoxic activity to T-and B-cells could be related to the differential membrane composition and/or enzymatic inhibition. In absence of the lipidomics information for the lymphoma, melanoma and bone marrow cells, it is difficult to correlate the peptide cytotoxicity solely based on membrane heterogeneity. However, assuming the cell-growth properties and membrane composition of the target cancer and normal cells are different, a constructive conclusion can be proposed in correlation to the biophysical and computational results. Furthermore, the membrane integrity in E. coli DH5α strains also ascertained existence of indirect membrane disruption pathway such as enzyme binding like penicillin, lysozyme mediated cleavage of glucosamine etc. The membrane integrity in DH5α also could be due to the host membrane surface modification and/or protease activity. These findings challenge the previous results 14 , but the cause of resistance at the molecular level remains unopened and is beyond the scope of this study.
In summary, the combined computational and experimental approach open the gateway to advance the research in host-defense peptide optimization. This holistic approach can be used to rationally design potential AMPs by studying their membrane penetration potentiality and/or intracellular activity. The optimized BMAP-28 analogues displayed an enhanced antimicrobial, anticancer and antiprotozoal activity. Their low hemolytic activity and cytotoxity to healthy cells further claimed the potent therapeutic activity. The energetically favorable and unfavorable binding of BMAP-28 suggests its cell/species specificity and is proposed to be relying on the membrane heterogeneity. In summation, the intracellular binding affinity of our target peptides that inhibits the target cell transcription/translation process propose a bimodal antimicrobial/anticancer action as illustrated in Fig. 8 (right panel). The membrane disruption and intracellular activities has also been seen in a well-studied α -helical peptide MSI-78 [38][39][40] . On the whole, the projected approach shade lights on the mechanistic and structural basis of bovine cathelicidin-5 interaction at the atomic level to develop therapies against multiple diseases.
Molecular mechanics and molecular dynamics simulation. The three dimensional structure of BMAP-28 was built using ab-initio modeling method 41 in reference to the circular dichroism, helical wheel and secondary structure information. A peptide chimera comprised of high cationic N-terminus of BMAP-27 (PBD ID: 2KET) and strong hydrophobic (19)(20)(21)(22)(23)(24)(25)(26)(27) C-terminus was prepared. Unbiased and biased all-atom and coarse-grained (CG) MD simulations were performed on GROMACS 5.0.1 42 using amber99sb-ildn 43 , charmm36 44 and Martini 45 force fields as described in our previous report 24 . The MD simulations were performed in homogeneous/heterogeneous lipid-bilayer systems as listed in Supplementary Table 1. The lipid composition of heterogeneous membrane systems i.e. thymocytes-like (TLM), leukemia-like (LLM) and E. coli like membranes were directly referred from the experimental molar concentration as listed in Table 1 46,47 . A total of 100 windows with different peptide initial position was selected and each window was simulated for 100 ns to enable adequate sampling configuration.
Antimicrobial assay. The Escherichia coli strains BL21 and DH5α were obtained from Novagen and Takara Clontech, respectively. The Bacillus subtilis (3009) and Staphylococcus epidermis (12993) were purchased from NITE Biological Resource Center (NBRC, Japan). The bacterial growth procedures followed in this study were described elsewhere 38 . The mid-log phase microbial growth (~4 × 10 7 cells/mL, 5 μ L) was used for growth assay in a 96-well plate (5 replicates) at a variable peptide concentration incubated at 37 °C for 12-48 h.
The Plasmodium lactate dehydrogenase (pLDH) assay was performed to measure the parasitic growth inhibition as described elsewhere 49 with BMAP-28 and its analogues treatment. In brief, malstat reagent was added to a 96-well plate (100 μ L in each well). A Plasmodium infected RBC mixture of 20 μ L in each well was mixed thoroughly using a plate shaker. And then 10 μ L of each 1 mg/ml diaphorase and Nitroblue tetrozolium (NBT) were added to each well. The solution in the 96-well plate was mixed properly until a brown color appears and the absorbance was measured at 650 nm under a plate reader (PowerWaveHT, BioTek Instruments, USA). The hemolytic assay was carried out as described earlier 50 . Briefly, RBCs prepared from fresh blood (Hematocrit ~5%) were incubated for 1 h at 37 °C after addition of test peptides. Relative hemoglobin concentration in supernatants after centrifugation at 2000× g for 5 min was monitored by measuring the absorbance at 540 nm by the plate reader (PowerWaveHT, BioTek Instruments, USA). As positive control, 100% hemolysis was taken from samples in which 2% Triton X-100 was added.
Cell-culture and MTT assay. The T-cell lymphoma (Jurkat) and B-cell lymphoma (Ramos) were cultured using RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 100 U ml −1 penicillin and 100 μ g ml −1 streptomycin. An initial cell densities of 5 × 10 3 cells/well was uniformly maintained in a 96 well plate. The B16-F1 melanoma cells were grown in DMEM medium containing 10% FCS, 100 U ml −1 penicillin and 100 μ g ml −1 streptomycin and 55 μ M 2-mercaptoethanol. The cells were trypsinized and plated in a 96-well plate at a density of 5 × 10 3 cells/well. The cells were grown overnight at 37 °C using a 5% CO 2 incubator. The cells were treated with different peptide concentration followed by cell proliferation assay. The MTT assay kit (Cayman chemicals, USA) was used to measure the cell viability at a peptide concentration varying from 2 to 15 μ M (in triplicate). Fresh culture medium and cells in medium without peptide were considered as negative and positive controls for viability assay and was monitored at 24, 48 and 72 h. The Cayman MTT cell proliferation assay protocol was followed, and the absorbance were recorded at 570 nm using a microplate reader (PowerWaveHT, BioTek Instruments, USA).
Mice and in vitro osteoclast culture. The C57BL/6 mice (8 weeks old male) were generated and maintained in a pathogen-free environment as described elsewhere 51 46,47 . The detailed procedure of liposome preparation was described elsewhere 52 . In brief, phosphate buffer assay was performed to calculate the stock concentration of different lipids suspended in chloroform. Lipid films were generated under a stream of nitrogen gas followed by drying under vacuum for 1 h. The lipid films were rehydrated using sodium phosphate buffer, and were disrupted by several freeze-thaw cycle followed by 10 times extrusions through 100 nm polycarbonate membrane. The integrity of extruded LUVs and effect of peptide on LUV size was studied by dynamic light scattering (DLS) method (Malvern Zetasizer Nano; model: ZMV2000).
Fluorescence spectroscopy. The fluorescence spectra of BMAP-28 and its derivative in different LUV systems were obtained using an F-2500 (Hitachi, Japan) spectrometer with emission and excitation slits each set at 2.5 nm. The tryptophan residue was excited at 290 nm and emission spectra was scanned between 300 and 370 nm at 37 °C. A peptide concentration of 2.5 μ M and LUV concentration was increased of 100 μ M was used to analyze the membrane dependent peptide orientation and binding. The membrane mediated self-assembling potentiality of both peptides was studied by employing ANS binding assays. A final concentration of 2 μ M ANS was added to different LUV systems (100 μ M) at 2.5 μ M peptide concentration. The fluorescence spectra were assessed at 350 nm excitation and emission wavelengths ranging from 430 to 600 nm.

Circular dichroism (CD).
The secondary structure analysis of wild and mutated synthesized peptides in different 2,2,2-trifluoroethanol (TFE) and LUV concentrations was conducted by CD on a JASCO CD-J-820 spectropolarimeter with a path length of 0.1 cm. Constant peptide solution (0.15 mg/ml) in sodium phosphate buffer with an increasing TFE concentration (range from 15 to 75%) was used to analyze the BMAP-28 and its derivatives folding properties and effect of mutation and terminal truncation. Thereafter, the BMAP-28 folding was also studied with and without LUVs (250 μ M) at 50 μ M peptide concentration.

Isothermal titration calorimetry (ITC).
Binding affinities of peptide for LUV in sodium phosphate buffer were measured through ITC experiments at 37 °C on a VP-ITC instrument (GE Healthcare). A peptide at a concentration of 50 μ M in the cell was titrated with liposome solution at a concentration of 2.5 mM in the syringe. Experimental procedures included a reference power of 10 μ cal/s, a filter period of 2 s, stirring speed of 307 rpm, initial delay of 1800 s, 28 injections of volume 10 μ L each, and time spacing of 300 s. ITC data peak integration with automated baseline adjustment was performed using Microcal program.
Flow cytometry test. The effect of peptides on the cell-membrane integrity of E. coli BL21 and DH5α and lymphoma cells was studied using the flow cytometer analysis as described elsewhere 53 . The cells were cultured and washed three times using PBS buffer and incubated for 30 minutes with peptides at different concentration. The incubated lymphoma cells were further processed referring to the annexin V-FITC apoptosis kit. Peptide treated and untreated cells (E. coli/lymphoma) were stained with 10 μ g ml −1 propidium iodide (PI) and incubated for additional 30 min. The incubated cells were washed using PBS to remove unbound dye and flow cytometry analysis was carried out using the FACSCalibur and analyzed by CellQuest Pro Version 5.2.1 software (BD Biosciences, NJ, USA).

Transmission Electron Microscopy (TEM). E. coli
DH5α and BL21 strains were subjected to TEM analysis. E. coli strains were cultured at 37°C in LB broth to mid-log phase and harvested by centrifugation at 5000 g for 5 minutes. The cell pellets were re-suspended in fresh LB media with an OD 600 = 0.18. A peptide (Syn1) solution at a concentration of 2 μ M (MIC) was added to E. coli DH5α and BL21 strains and the samples were incubated at 37°C for 1 hour. The culture was centrifuged at 5000 g for 5 minutes and the cell pellets were collected. The cell pellets were washed twice and re-suspended in 1 mL fixation buffer that contains 2% formaldehyde, 2.5% glutaryldehide in 0.1 M phosphate buffer (pH = 7.4). The fixed bacterial cells were observed under the microscope, JEM-1011 (JEOL, Tokyo, Japan).
Luciferase synthesis assay. Luciferase synthesis from Luciferase T7 Control DNA plasmid (L482B, Promega) was examined using E. coli T7 S30 Extract System (L1130, Promega). The complete protocol for reaction mixture and final solution preparation was followed as described elsewhere 38 . Peptide concentration of 2, 10, 30, 60 and 100 μ M were used for our analysis. Kanamycin (500 μ g/mL) and RNase free distilled water was taken as a positive and negative control, respectively. Luciferase Assay Reagent (E1500, Promega) was used to monitor the luciferase luminescence and the samples were measured using a spectrophotometer (Powerscan HT, Biotek Instruments, USA).
In vivo. Healthy C57BL/6 mice (n = 6) were administered with 30 μ M Syn1 peptide mixed with 1 × 10 6 B16-F1 melanoma cells in 0.1 ml of saline via subcutaneous injections. As a control, six mice were implanted with 1 × 10 6 B16-F1 melanoma cells in 0.1 ml of saline. The growth size of tumors were monitored in every two days and the tumor volume was measured by a dial-caliper on day 14 and 20 using the formula volume = width 2 × length × 0.52.