Design, Synthesis, Antibacterial Potential, and Structural

Article pubs.acs.org/bc

Cite This: Bioconjugate Chem. 2019, 30, 1998−2010

Design, Synthesis, Antibacterial Potential, and Structural Characterization of N‑Acylated Derivatives of the Human Autophagy 16 Polypeptide Kyriakos Gabriel Varnava,† Sk. Abdul Mohid,‡ Paolo Calligari,§ Lorenzo Stella,§ Jóhannes Reynison,†,∥ Anirban Bhunia,*,‡ and Vijayalekshmi Sarojini*,†,⊥ †

School of Chemical Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata 700054, India § Department of Chemical Science and Technologies, University of Rome Tor Vergata, 00133 Rome, Italy ∥ School of Pharmacy, Hornbeam Building, Keele University, Staffordshire ST5 5BG, United Kingdom ⊥ The MacDiarmid Institute for Advanced Materials and Nanotechnology, Wellington 6140, New Zealand

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‡

S Supporting Information *

ABSTRACT: A synthetic antimicrobial peptide library based on the human autophagy 16 polypeptide has been developed. Designed acetylated peptides bearing lipids of different chain lengths resulted in peptides with enhanced potency compared to the parent Atg16. A 21-residue fragment of Atg16 conjugated to 4-methylhexanoic acid (K30) emerged as the most potent antibacterial, with negligible hemolysis. Several studies, including microscopy, dye leakage, and ITC, were conducted to gain insight into the antibacterial mechanism of action of the peptide. Visual inspection using both SEM and TEM revealed the membranolytic effect of the peptide on bacterial cells. The selectivity of the peptide against bacterial cell membranes was also proven using dye leakage assays. ITC analysis revealed the exothermic nature of the binding interaction of the peptide to D8PG micelles. The three-dimensional solution NMR structure of K30 in complex with dioctanoylphosphatidylglycerol (D8PG) micelles revealed that the peptide adopts a helix−loop−helix structure in the presence of anionic membrane lipids mimicking bacterial membranes. Intermolecular NOEs between the peptide and lipid deciphered the location of the peptide in the bound state, which was subsequently supported by the paramagnetic relaxation enhancement (PRE) NMR experiment. Collectively, these results describe the structure−function relationship of the peptide in the bacterial membrane.

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teins.10,14−16 The overall net positive charge possessed by many AMPs facilitates the interactions with the negatively charged bacterial membrane.17 The design of a library of peptide antibiotics is usually done using a parent peptide (naturally occurring or designed in the laboratory), followed by structure−activity relationship (SAR) studies and fine-tuning the activity of the peptide.18 The conjugation of fatty acids is often used to enhance the activity and serum stability of AMPs and is a strategy inspired by lipopeptide antibiotics such as polymyxin.19 N-Acylated derivatives of other AMPs have been reported in the literature and have generally led to enhancements in antibacterial activity.20−23 Our ongoing work in this area has led to fine-tuning the structure and length of the fatty acid component of the antimicrobial lipopeptide battacin.24 The work reported in this article continues on from our recent report on the antibacterial activity of the human

INTRODUCTION Increased morbidity and mortality from bacterial infections1 and the ability of bacteria to mutate and become resistant to successive generations of antibiotics2 necessitate the development of novel antibacterial drugs. Novel platforms that help to deepen our current understanding of antibacterial drug discovery and create drugs that can be used as weapons against lethal bacterial infections are urgently needed. Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli are bacterial pathogens implicated in numerous severe health conditions and infections.3−8 Antimicrobial peptides (AMPs) are widely distributed in humans, plants, and animals and play crucial roles in protecting the host from bacterial infections.9−13 AMPs primarily target the bacterial cell membrane and either create pores or cover the membrane like a carpet, ultimately causing bacterial cell lysis. Pore-forming AMPs insert part of the peptide into bacterial phospholipid membranes, bind to a component of the membrane causing membrane lysis, or enter the cells and bind to intracellular targets such as DNA, enzymes, or pro© 2019 American Chemical Society

Received: April 20, 2019 Revised: May 15, 2019 Published: May 30, 2019 1998

DOI: 10.1021/acs.bioconjchem.9b00290 Bioconjugate Chem. 2019, 30, 1998−2010

Article

Bioconjugate Chemistry Table 1. Antibacterial Activity of Peptides and Peptide Conjugatesa MIC (μM) C. albicans code

sequence

K17 RWKRHISEQLRRRDRLQR K18 RWKRHISEQLRRRDRLQRQAF K22 RWKRHISEQLRRRDRLQRQAJ K22.2 RWKRHISEQLRRRDRLQRQAJ-NH2 K30 R1 −RWKRHISEQLRRRDRLQRQAJ-NH2 K31 R2 −RWKRHISEQLRRRDRLQRQAJ-NH2 K33 R3 −RWKRHISEQLRRRDRLQRQAJ-NH2 K36 R5 −RWKRHISEQLRRRDRLQRQAJ-NH2 K46 R4−RWKRHISEQLRRRDRLQRQAJ-NH2 streptomycin polymyxin amphotericin

24 h

48 h

P. aeruginosa

E. coli

S. aureus

net charge at pH 7

>120.0 >120.0 60.0−120.0 60.0−120.0 30.0−60.0 3.2−6.4 1.8−3.7 15.0−30.0 30.0−60.0 NT NT 0.9−1.8

>120.0 >120.0 >120.0 >120.0 60.0−120.0 7.5−15.0 3.7−7.5 15.0−30.0 30.0−60.0 NT NT 0.9−1.8

>120.0 60.0−120.0 60.0−120.0 17.5−35.0 13.5−27 3.2−6.4 6.4−12.8 6.4−12.8 3.2−6.4 NT 120 60−120 30−60 17.5−35.0 6.4−12.8 3.2−6.4 3.2−6.4 3.2−6.4 3.2−6.4 NT i + 5) NOEs between the aliphatic side chains of I6, L10, L16, and A20 with the aromatic ring protons of W2. Surprisingly, the indole ring proton (NεH) of W2 did not show any long-range NOEs with the aliphatic side chain of any amino acids (data not shown). In contrast, we observed a larger number of NOEs between aromatic ring protons of W2 and aliphatic CβHs of A20 and CγHs/CδHs of L16, depicting that these long-range NOEs could be instrumental in forming a hydrophobic hub in the central region to stabilize K30 in D8PG micelles. To further understand the role of acylation and amidation, two-dimensional TOCSY and NOESY experiments were also performed using K22 and K22.2 in D8PG micelles. However, because of the strong precipitation of the peptides under these conditions, we could not observe any NOEs (Figures S10 and S11), and hence no structure could be determined. The three-dimensional solution structure of K30 bound to D8PG was first calculated using the CYANA program with 132 NOE-derived distance restrains and 18 backbone-angle restraints (details in Materials and Methods). Backboneangle restraints were derived from PREDITOR,43 based on Hα protons of the amino acids. An ensemble of 20 backbone structures of K30 bound to D8PG micelles is shown in Figure 5. The average backbone RMSD of the 20 lowest-energy structures (excluding the N-terminal 4-methyl hexanoyl and Cterminal 2-Nal because they are very flexible) is 0.74 ± 0.22 Å, whereas the RMSD values for heavy atoms was found to be 1.74 ± 0.34 Å (Table 3). PROCHECK analysis of the peptide shows that 95% of the residues are in favored regions of the Ramachandran plot. Table 3 shows detailed statistics of the structures. Subsequently, an MD simulation in water was performed including 4-methylhexanoyl and 3-(2-naphthyl)-Lalanine) at the N- and C-termini of K30, respectively. The backbone RMSD was 0.89 Å in the absence and presence of capping at both terminals. The solution structure of K30 bound to D8PG was characterized by a helical hairpin or helix−loop−helix conformation, resembling several other antimicrobial peptides such as MSI-594,44 paradaxin,45 and KYE28.46 The architecture of the D8PG-bound K30 is tuned by N-terminal helix W2R11, followed by a loop involving residues R12−R15 and then a short helix L16−A20 (Figure 5B). Close inspection suggests that the peptide adopts an amphipathic structure. The folded conformation was stabilized by the hydrophobic interactions among W2, I6, L10, L16, A20, and 2-Nal residues, which created a compact hydrophobic hub opposite to the charged

Figure 5. (A) Ensemble showing the superposition of the 20 lowestenergy backbone (N, Cα, and C′) structures of the K30 peptide in D8PG micelles. The PDB acquisition code is 5ZYX. (B) A representative structure of K30 peptide bound to D8PG, showing the orientation of the side chains. The peptide K30 adopts amphipathic structure in D8PG micelles. (C) Hydrophobic hub of the K30 peptide comprising aromatic (W2) and nonpolar residues such as I6, L10, L16, and A20. (D) The black dotted line represents the plausible electrostatic interactions and/or hydrogen bonds for further stabilization of K30 in the micelle bound form. (E) Molecular surface of the structure attained by K30 in D8PG micelles, colored according to residue type. Blue, cationic; red, anionic; yellow, polar; and white, apolar. (F) Molecular surface of the structure attained by K30 in D8PG micelles, colored according to the local values of the electrostatic potential. The color palette ranges from −83 kcal/(mol· e) (red) to +83 kcal/(mol·e) (blue). The images were prepared by MOLMOL, PyMOL, and Chimera software.

residues (Figure 5C). The interesting feature of this peptide sequence is that it has only 25% hydrophobic/aromatic amino acid residues and all of them are involved in forming the hydrophobic hub, governing the structural stabilization. In addition, the N-terminal helix is stabilized either by a salt bridge or by hydrogen bond interactions between side chains of R4-E8, E8-R11, or S7-R11 (Figure 5D). The amphipathic composition of K30 helps in stabilizing the loop region to a great extent. Because D14 is juxtaposed with R13 and R18, it can easily form triad unit R13-D14-R18 by side-chain/side2003


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Bioconjugate Chemistry Table 3. Summary of Statistical Analysis of the 20 LowestEnergy Structures of Peptide K30 in D8PG Micelles Distance Restraints intraresidue (i − j = 0) sequential (|i − j| = 1) medium range (2 ≤ |i − j| ≤ 4) long range (|i − j| ≥ 5) total

46 59 19 8 132

Angular Restraints Φ Ψ

20 20

Distance Restraints from Violation (≥0.4 Å)

0

Deviation from Mean Structure (Å) average backbone to mean structure average heavy atom to mean structure

0.74 ± 0.22 1.74 ± 0.34

% % % %

residues residues residues residues

Ramachandran Plota in the most favored regions in the additional allowed region in the generously allowed region in the disallowed region

88.90 11.10 0 0

a

Obtained from PROCHECK NMR.

chain electrostatic interactions (Figure 5D). Additionally, the backbone/side-chain hydrogen bond was also observed between L16 and D14, which subsequently stabilizes the folded loop structure of K30 (data not shown). The hydrophobicity and charge distribution on the surface of the peptide are also shown in Figure 5E, which clearly shows that the cationic residues are present on one side and hydrophobic residues are present on the opposite side, which makes the peptide amphipathic in nature. The presence of a positively charged microenvironment throughout the peptide sequence, supported by a potential electrostatic map (Figure 5E), helps in the initial interaction with negatively charged bacterial membranes, which, we believe, in turn aids the membranelytic property of the peptide with respect to microbial cells. Orientation of the K30 Peptide in D8PG Micelles. The orientation of K30 in D8PG micelles was further established by the investigation of the intermolecular NOEs between the peptide and lipid from the NOESY spectra. Because peptide K30 and D8PG possess unique chemical shifts, it is possible to probe the intermolecular NOEs between the two different molecules.26 Figure 6A shows the chemical structure of the D8PG and Figures 6B−E and S12 (Supporting Information) show several 1D slices taken from the 2D spectral regions, corresponding to D8PG signals (e.g., 1.23 ppm (C3−C7 H), 2.34 ppm (C2H), and 5.25 ppm (Hβ)). These unique chemical shifts of the D8PG lipid molecule do not mix with the peptide signals. They also cover the entire peptide signals, providing crucial intermolecular NOEs, which help us to map the location of the peptide in the lipid environment. The observed distinct sharp peaks shown in Figure 6C reveal that a majority of the hydrophobic and aromatic residues are interacting with the C3−C7 acyl chain of the lipid molecule. In contrast, charged and polar residues such as E8, Q9, and Q17/Q19 are in close proximity to C2 of D8PG (Figure 6D), suggesting that the hydrophobic and aromatic residues are in close proximity to the hydrophobic acyl chain and the charged

Figure 6. (A) Chemical structure of the D8PG molecule. (B) Partial NOESY spectrum (mixing time 150 ms) of K30 in complex with D8PG. The dotted lines show the unique chemical shifts of D8PG, where the 1D slice analysis was performed. One-dimensional slices taken at (C) 1.23, (D) 2.34, and (E) 5.25 ppm (well-resolved lipid signals) of the indirect dimension of the 2D NOESY spectrum. The residues that are in close proximity to micelles are shown (R′−CH3 stands for 4-methyl hexanoyl). The experiment was performed at 310 K on a Bruker Avance III 600 MHz NMR spectrometer.

residues are in closer proximity to the more polar C2 carbon. Surprisingly, the aromatic ring protons of W2 did not show any intermolecular NOEs with the backbone glycerol CβH proton of D8PG, whereas the aliphatic side chain of I6 and L10/L16 showed strong peaks with the CβH proton. Taken together, these data prove that K30 binds and interacts at the interface of the lipid molecule. This finding was further supported by the paramagnetic relaxation enhancement (PRE) experiment. To obtain atomistic information about the depth of insertion and to probe the position of the peptide within the D8PG lipid micelle, two different paramagnetic probes (MnCl2 and 16-doxyl stearic acid (16-DSA)) were used.27,28 MnCl2 was used as a surface quencher because Mn2+ ions possess paramagnetic properties and thus are able to quench the diamagnetic signals coming from the residues that are exposed at the surface of the peptide. The 2D TOCSY spectra of K30 in the presence of D8PG micelles titrated with MnCl2 (Figure 7) revealed a signal 2004


Bioconjugate Chemistry

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Article

CONCLUSIONS

N-Acylation of a promising fragment from the autophagy 16 polypeptide (Atg 16) using 4-methyl hexanoic acid led to a lipopeptide that exhibited nano- to low micromolar activity against an S. aureus strain and low micromolar activity against opportunistic pathogenic Gram negative strains P. aeruginosa and E. coli. Biophysical studies indicate that the peptide adopts a random coil conformation in aqueous buffer but folds into an α helix in the presence of D8PG micelles. Membrane perturbation causing cell wall detachment and cell lysis has been established as the mode of action of this peptide using SEM and TEM experiments. The ability of the peptide to insert into and destabilize the membrane has been established using dye leakage experiments. ITC experiments identified the crucial thermodynamic forces underlying the binding of K30 to the D8PG and DPC micelles. ITC data showed that the binding of the peptide is stronger in D8PG micelles (and therefore negatively charged membranes) than in DPC micelles (zwitterionic membranes). The standard enthalpy values also suggest that the K30−D8PG interaction is mainly stabilized by hydrophobic and van der Waals interactions. The solution NMR structure of K30 in D8PG micelles shows that the peptide adopts a helix−loop−helix structure and maintains the crucial amphipathicity in the PG-rich membrane. The probing of the localization of K30 in the micelles by 1D slice analysis from the NOESY spectrum as well as in the PRE NMR experiment proves that the peptide fits into the interface area of the membrane. Moreover, 2D TOCSY experiments using two different paramagnetic probes (MnCl2 and 16-DSA) in the presence of D8PG lipid revealed the exact orientation of the peptide upon interaction with the lipid and showed the side chains of the charged residues exposed to the surface. These findings are helpful in correlating the membrane-active property of the K30 peptide with the bacterial membranes. On the basis of these observations, it is clear that the negatively charged membrane becomes an anchor point for the peptide. The subsequent interactions with the hydrophobic residues of the peptide force the peptide to fold. The folded structure is able to penetrate the nonpolar acyl chain of the lipid molecules, where it subsequently causes membrane disintegration leading to cell lysis.

Figure 7. (A) Comparison of the 2D TOCSY NMR profile of the K30 peptide in D8PG. Control (without any paramagnetic quencher) shows all of the intraresidual peaks. The addition of 0.2 mM MnCl2 and 16-DSA shows the immediate diamagnetic signal exchange of the residues that are present in the “solvent-exposed region” or “in depth”, respectively. (B) Schematic diagram of K30 in D8PG, showing the residues affected by MnCl2 (red) and 16-DSA (blue). The residues that are not affected are marked green. The cartoon representation (B) has not been prepared according to the lipid−peptide ratio but more clearly describes the location of the peptide in the D8PG micelle. The area of action of the quencher molecules is shown in the box. The flipping of the acyl chain of 16-DSA is shown by an arrow.

perturbation of the side chains of charged residues such as R1, H5, S7, R11, R13, and D14. Additionally, significant resonance broadening was observed for all of the charged residues including Q17 and Q19 in the presence of 0.4 mM MnCl2 (Figure S13). In contrast, the number of residues affected by 16-DSA is comparatively smaller because the paramagnetic doxyl moiety is present at the 16th carbon position of 16-DSA and therefore the residues which are inserted deeply inside the micelle are expected to observe higher quenching of the NMR signal intensity (Figure 7). We observed the broadening of signals for H5β, I6γ, L16β, and R18α/β of K30 in the context of 0.4 mM 16-DSA. It is worth mentioning that H5 and R18 are on two sides of the same structure; hence, the result suggests that the acyl groups of 16-DSA can flip and be exposed to the doxyl group in such a way that it can quench the residues both in the hydrophobic core and at the interface of the micelle (Figures 7 and S13). Collectively, the PRE data suggests that the peptide is oriented at the interface of the micelles, where the side chains of the charged residues are exposed to the surface of the micelle, keeping the backbone and mostly Hα inaccessible to both quencher molecules. A schematic representation of these observations is shown in Figure 7B, where the residues affected by MnCl2 and 16-DSA are colored red and blue, respectively. Thus, we can hypothesize that the peptide initially anchors on the negatively charged membrane and then folds because of the interactions with hydrophobic residues. Subsequently, the folded structure inserts into the nonpolar acyl chain of the lipid molecules and destabilizes the integrity of the membrane.

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EXPERIMENTAL SECTION Materials and Methods. All Fmoc-protected amino acids and coupling reagents were purchased from Protein Technologies Inc. 2-Nal (3-(2-naphthyl)-L-alanine) was purchased from ChemPep and OxymaPure, piperidine, trifluoroacetic acid (TFA), triisopropyl silane (TIS), 2,2′-(ethylenedioxy)diethanethiol (EDDT), and N,N-diisopropylethylamine (DIPEA) were purchased from Sigma-Aldrich. Synthesis and Purification of Peptides. All peptides were manually synthesized by solid-phase peptide synthesis following standard Fmoc protocols using capping steps with acetic anhydride.47 The peptides were assembled on 2chlorotrityl chloride resin, on either a 0.1 or 0.2 mmol scale in N,N-dimethylformamide (DMF) as C-terminal acids. The C-terminal amide peptides were assembled on Tentagel S NH2 resin (substitution level of 0.29 mmol/g) using the Rink amide linker on either the 0.1 or 0.2 mmol scale. A 4-fold excess of each amino acid, 3.9 equiv of coupling reagent (HCTU), and OxymaPure (3.9 equiv) as a suppressor of racemization were used. COMU (3.9 equiv) was used as the coupling reagent for 2005


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were incubated for 48 h at 37 °C. The minimal inhibitory concentration (MIC) was defined as the lowest concentration that inhibited the growth of the fungus as confirmed by visual inspection. The MICs were determined in triplicate, and results were taken at both 24 and 48 h. Hemolysis of Mouse Blood Cells.24 The Vernon Jensen Unit, Faculty of Medical and Health Sciences, University of Auckland provided us with mouse blood cell that were used fresh for this assay. Several washes of the cells with Tris buffer (10 mM Tris, 150 mM NaCl, pH 7.4) upon centrifugation at 1000× for 5 min were performed, and finally the cells were resuspended in 2% (v/v) Tris buffer. The same buffer was used to dissolve the peptides, which were then serially diluted in 96well plates. The resuspended blood cells were added to each well, and the plates were left to incubate for 1 h at 37 °C without agitation. Neat buffer was used as the negative control, and 0.25% Triton X-100 was used as the positive control. The assay was done in triplicate. After incubation, the plates were centrifuged (3500g for 10 min) and the supernatant from each well (100 μL) was transferred to new 96-well plates and absorbance at 540 nm was measured (Enspire Plate Reader). The percentage of hemolysis, at each peptide concentration, was calculated using the following equation:

the conjugation of caprylic and palmitic acids only. N,NDiisopropylethylamine (DIPEA) was used as the base for couplings. Fmoc deprotection was performed using 20% piperidine in DMF. Cleavage from the resin was accomplished using 10 mL of the trifluoroacetic acid (TFA) cocktail mixture (TFA−TIS−H2O 95:2.5:2.5 v/v/v) per gram of the resin. Upon evaporation of TFA, diethyl ether was added to precipitate the crude peptides. The crude peptides were lyophilized and purified using reverse-phase high-performance liquid chromatography (RP-HPLC) on a GE Pharmacia Ä KTA purifier 10 system or a Thermo Scientific Dionex VWD 3x00 system using a Phenomenex Luna 5 μm C18 100 Å (250 mm × 10 mm) column. Solvent A was 0.1% TFA in water, and solvent B was 0.1% TFA and 0.09% water in 99% acetonitrile at a flow rate of 10 mL per min. Analytical RP-HPLC was performed using a Phenomenex Luna 5 μm C18 100 Å (250 mm × 4.6 mm) column with the same solvent system as above at a flow rate of 1 mL per min. Electrospray ionization mass spectrometry (ESI-MS) recorded on a Bruker micrOTOF-Q mass spectrometer and matrix-assisted laser desorption/ ionization−time-of-flight mass spectrometry (MALDI-TOF MS) recorded on a Bruker Ultraftlextreme MALDI/TOF were used to characterize the peptides. Antibacterial Assays. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined following previously published protocols.48 Bacterial strains (P. aeuriginosa, E. coli DH5α, and S. aureus 16207) were plated in Mueller Hinton (MH) plates and grown overnight. From the overnight cultures, single colonies were diluted in fresh MH broth and further incubated to grow to an OD625 of 0.1 (approximately 1 × 108 CFU mL−1). To prepare the bacterial inoculum, further dilutions according to the above-mentioned protocol were made. The peptide stock solutions were dissolved in type 1 water and serially diluted in the 96-well plates. Then bacterial inoculum was added to each well, and the plates were incubated at 37 °C for 24 h and the absorbance at 625 nm was measured using an EnSpire Multimode Multiskan Go (Thermo). The MIC was considered to be the lowest concentration that showed growth inhibition after 24 h, as observed visually and through absorbance. For MBC determination, 20 μL from each well of the MIC experiment containing peptide K30 and bacteria were plated on Mueller Hinton agar plates in triplicate, and the plates were incubated at 37 °C overnight. MBC was determined to be the concentration where no bacterial growth occurred on the plates after this overnight incubation. Antifungal Assays. The antifungal activity of the designed peptides was determined according to a standardized broth microdilution method (Clinical and Laboratory Standards Institute (CLSI) document M27-A2).49 Briefly, the yeast colonies from 24 h cultures of Candida albicans were picked and resuspended in 5 mL of sterile 0.145 mol/L saline and adjusted to a cell density of 1 × 106 to 5 × 106 cells/mL. The yeast stock suspension was then diluted to obtain a starting inoculum of 5.0 × 102 to 2.5 × 103 cells/mL. Conventional antifungal drug amphotericin B (Sigma, USA) was included in this study as the control and to compare the antifungal activity with the designed peptides. Peptide and amphotericin B stock solutions were serially diluted in RPMI medium, which was further supplemented with glucose to a final volume of 50 μL per well, giving final concentrations ranging from 120 to 0.235 μM in sterile 96-well polypropylene microplates. Standardized yeast suspensions (50 μL) were then added to each well. Plates

ij Aexp − A Tris yz zz × 100 % hemolysis = jjj j A100% − A Tris zz k {

(1)

Aexp is the experimental absorbance at 540 nm measurement, ATris is the absorbance of the negative control, where only Tris buffer was added to mouse blood cells, and A100% is the absorbance of the positive control, where 0.25% Triton X-100 was used to cause the lysis of 100% mouse blood cells present. Scanning Electron Microscopy.50 The preparation of the slides that were used for SEM was done using the previously described protocols.50 Briefly, the bacterial strains (P. aeuriginosa, E. coli DH5α, and S. aureus 16207) were regrown as above from overnight cultures to an OD of 0.3 in MHB with coverslips (10 mm). The slides were washed with sodium phosphate buffer (10 mM; pH 7.4) and treated with the peptide (at twice its respective MICs) for 30 min. Slides were washed and postfixed with a 4% solution of glutaraldehyde in the buffer for 1 h and then washed with water. Before platinum coating, the slides were dehydrated with a graded ethanol series (25−100%) and left to dry at 60 °C for 10 min. The slides were coated with platinum for 2 min at 20 mA before viewing under high vacuum with a Hitachi SU-70 FESEM at 10 kV. Transmission Electron Microscopy (TEM).51 The preparation of the bacterial pellets that were used for TEM was done using protocols previously described.51 As described above, the bacterial suspensions were prepared to an OD of 0.4 in MHB using the overnight cultures. After treatment with the peptide (at 4 times its respective MICs) for 1 h at 37 °C in centrifuge tubes, the tubes were centrifuged and the pellets were washed three times at 5000g for 3 min with Tris buffer (10 mM Tris, 150 mM NaCl, pH 7.4) and fixed in 4% gluteraldehyde solution overnight at −4 °C. The pellets were washed again using buffer, further fixed with 1% osmium tetroxide in PBS for 1 h, and dehydrated through a graded ethanol series (50, 90, and 100%). Then acetone was added to the pellets, followed by a 1:1 mixture of absolute acetone and epoxy resin, and the samples were left in pure epoxy resin overnight. The pellets were incubated at 60 °C for 48 h. The 2006


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Isothermal Titration Calorimetry (ITC).28 ITC was performed to determine the thermodynamic parameters of binding of K30, K22, and K22.2 with D8PG or DPC micelles using either a VP-ITC microcal calorimeter (Malvern PANalytical Inc., Westborough, MA, USA) or a TA-affinity ITC (TA Instruments, Lukens Drive, New Castle, DE, USA). All detergents and peptides were dissolved in 10 mM phosphate buffer at pH 7.4 and degassed. A sample cell (volume 200 μL) containing 0.5 mM K30 was titrated against D8PG and DPC from a stock solution of 25 mM at 308 K. A total of 20 injections were carried out at an interval of 3 min with 2 μL of detergent aliquots per injection. The raw data were plotted using Origin v9 software supplied with the instrument. The data was fitted using a single site binding model. The fitting was done with the same software to obtain the change in the heat of enthalpy of reaction (ΔH°). NMR Spectroscopic Analysis.26,56 All NMR experiments were performed at 310 K on a Bruker Avance III 500 MHz equipped with a 5 mm SMART probe spectrometer and a 600 MHz instrument equipped with an RT probe. NMR samples were prepared in 10% deuterated water (pH 4.5), and 3(trimethylsilyl)propanoic acid (TSP) was used as an internal standard (0.00 ppm). Two-dimensional (2D) 1H−1H total correlation spectroscopy (2D TOCSY) and 2D 1H−1H nuclear Overhauser effect spectroscopy (2D NOESY) were recorded for the peptide with mixing times of 80 and 150 ms, respectively, keeping a spectral width of 12 ppm in both directions. Next, the interaction of the K30/K22/K22.2 peptide (1 mM) upon successive titration with D8PG and predeuterated d25-SDS (200 mM) was monitored by 1D proton NMR acquired using an excitation-sculpting scheme for water suppression and the States-TPPI for quadrature detection in the t1 dimension. Consequently, 2D NOESY spectra of the peptide in the context of D8PG and SDS were acquired with a 150 ms mixing time. Paramagnetic relaxation enhancement (PRE) experiments were performed to measure the depth of insertion of the K30 peptide in D8PG/SDS micelles. The experiment was performed by titrating with a surface paramagnetic quencher, manganese chloride (MnCl2) dissolved in water, and in-depth paramagnetic quencher 16-doxyl-stearic acid (16-DSA) dissolved in deuterated methanol (d4-MeOH) into the NMR samples containing peptide and micelles. The sample was allowed to equilibrate for few minutes before the acquisition of 2D TOCSY spectra with the same acquisition parameters as mentioned above. NMR data processing and analysis were carried out using the Topspin v3.1 (Bruker Biospin, Switzerland) and SPARKY (Goddard, T. D., and Kneller, D. G. University of California, San Francisco) programs, respectively. NMR-Derived Structure Calculations. The three-dimensional NMR-derived structures were first calculated using the CYANA program (version 2.1) as reported previously46,57 without the N- and C- terminal 4-methayl hexanoyl and 3-(2naphthyl)-L-alanine residues, respectively. Briefly, on the basis of cross-peak intensities obtained from the NOESY spectra recorded in the presence of D8PG and SDS micelles at a mixing time of 150 ms, the NOE intensities were qualitatively categorized into strong, medium, and weak and then translated to upper bound distance limits to 2.5, 3.5, and 5.0 Å, respectively. The lower-bound distance constraint was fixed at 2.0 Å. Backbone dihedral angles φ and ψ were restrained (−30 to −120° and 120 to −120°, respectively) for all nonglycine residues of the peptide to minimize the conformational space.

samples were sectioned and stained with uranyl acetate and lead citrate. Images were taken using a transmission electron microscope (FEI Technai 12). Circular Dichroism (CD) in SDS, D8PG, and DPC Micelles. CD spectra of K30 were recorded in negatively charged (SDS and D8PG) micelles following a previously reported protocol.52 The spectra were recorded in a Jasco J815 spectrophotometer (Jasco International Co., Ltd. Tokyo, Japan) equipped with a Peltier cell holder and a temperature controller unit accessory. Stock solutions of the peptide and detergents were prepared in 10 mM phosphate buffer (pH 7.4). The CD data was obtained at 37 °C in a 0.2 cm quartz cuvette at a final peptide concentration of 25 μM and by titrating with increasing concentrations of SDS (2−50 mM) and D8PG (2−10 mM) micelles. The far-UV spectral range was set at 190 to 260 nm with a 1 nm data pitch and an average of five accumulations. The buffer-subtracted spectral data obtained in millidegrees was converted to molar ellipticity (θ) (deg cm2 dmol−1) using eq 2, where m0 is millidegrees, M is the molecular weight (g mol−1), L is the path length of the cuvette (cm), and C is the concentration (g L−1). molar ellipticity (θ) =

m0M ×L×C 10

(2)

Calcein Leakage Assay.53 All fluorescence experiments were performed in a Jasco FP-8500 spectrophotometer using the path length of a 0.1 cm quartz cuvette at 37 °C. The anionic bacterial membrane and zwitterionic mammalian membrane mimicking model was prepared by using 1,2dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol lipids in 3:1 DOPE/DOPG, 7:3 DOPE/DOPG, and 6:4 DOPC/cholesterol, respectively, at a final concentration of 2 mg/mL.53−55 The lipid mixture was dissolved in chloroform, and then a lipid film was prepared by passing N2 gas over it and lyophilizing overnight. The films was subsequently hydrated with 70 mM calcein prepared in 10 mM Tris-HCl buffer (pH 7.4) and then subjected to five freeze−thaw cycles by using liquid nitrogen for freezing and hot water (60 °C) for thawing. Large unilamellar vesicles (LUVs) were obtained by extruding the calcein-containing solution 27 times through a polycarbonate filter (Avanti Polar Lipids, Inc.) of 100 nm diameter. Free calcein was removed via gel filtration using a Sephadex G50 column (GE Healthcare, Uppsala, Sweden) previously saturated with the same buffer. A calcein leakage experiment was conducted by adding K30 peptide using a series of concentrations starting from 1 to 20 μM to the fixed concentration of LUVs (20 μM) suspended in 10 mM TrisHCl buffer with 100 mM NaCl. Measurement were made using the emitted fluorescence of calcein (515 nm) that leaked from LUVs via the interaction of the K30 peptide. Triton X100 (0.1%, Sigma-Aldrich, St. Louis, MO, USA) was added as a positive control, and only LUVs were added as a negative control. The calcein leakage (C) was calculated using eq 3 C=

F − F0 × 100 FT − F0

(3)

where F0 is the initial calcein fluorescence intensity, F is the final fluorescence intensity 5 min after addition of the peptide, and FT is the highest fluorescence intensity after the addition of Triton X 100. 2007



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ACKNOWLEDGMENTS K.G.V. thanks the University of Auckland for a doctoral scholarship. We are grateful to Dr. Adrian Peter Turner for help with TEM microscopy. This research was partially supported by the Science and Engineering Research Board (EMR/2017/003457 to A.B.), Government of India and partially by the “Indo-Sweden” research collaboration fund (DST/INT/SWD/VR/P-02/2016 to A.B.).

No hydrogen bond constraints were used in the structure calculation. Several rounds of structure calculations were carried out, and depending on NOE violations, the distance constraints were adjusted. Out of the 100 structures, the 20 lowest-energy structures were used for further analysis. The peptide structure obtained from CYANA was completed with the N-terminal 4-methylhaxanol and the Cterminal 3-(2-naphthyl)-L-alanine (2Nal). The resulting structure was then refined by all-atom molecular dynamics (MD) simulation with NOE-derived distance restraints. The AMBER03 force field was used to model all atomic interactions.58 Calculation of the partial atomic charges for the terminal groups was performed by calculating the electrostatic potential at the HF/6-31G (d) level of theory using R.E.D. Server Development.59,60 MD was performed with GROMACS software61 applying ensemble-averaged NOE distance restraints62 as flat-bottomed harmonic potential terms with a force constant of 2000 kJ/mol. The initial structure was first refined with energy minimization and a short simulated annealing run (temperature range 300−500 K) of 500 ps in a periodic simulation box filled with 1600 explicit water molecules (modeled by the TIP3P force field). The particle mesh Ewald algorithm was applied to calculate electrostatic forces, while range-limited nonbonded interactions were calculated with a 10 Å cutoff. During these steps, only the terminal residues were free to move without additional position restraints. After equilibration, four replica production runs of 30 ns were performed and a set of 20 lowest-energy structures were extracted from the resulting trajectories. For comparison, the peptide structure was also modeled by in vacuo MD simulations; the resulting structures were very similar to the solvated ones (backbone RMSD ∼1 Å between the lowest-energy conformers obtained by the two approaches).

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ABBREVIATIONS 16-DSA, 16-doxyl stearic acid; 2-Nal, 2-naphthyl alanine; AMPs, antimicrobial peptides; Atg16, autophagy 16 polypeptide; C. albicans, Candida albicans; CD, circular dichroism; COMU, (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate; CSI, chemical shift index; D8PG, dioctanoylphosphatidylglycerol; DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOPG, 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol); DPC, dodecylphosphatidylcholine; DPH, 1,6-diphenyl-1,3,5hexatriene; E. coli, Escherichia coli; EDDT, 2,2′(ethylenedioxy)diethanethiol; HCTU, O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N-tetramethyluronium hexafluorophosphate; ITC, isothermal titration calorimetry; KD, dissociation constant; LUV, large unilamellar vesicle; MD, molecular dynamics; MHB, Mueller Hinton broth; MIC, minimal inhibitory concentration; NOESY, nuclear Overhauser effect spectroscopy; OxymaPure, ethyl cyano(hydroxyimino)acetate; P. aeruginosa, Pseudomonas aeruginosa; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PRE-NMR, paramagnetic relaxation enhancement NMR; RP-HPLC, reversed-phase high-performance liquid chromatography; S. aureus, Staphylococcus aureus; SDS, sodium dodecyl sulfate; SEM, scanning electron microscopy; TEM, transmission electron microscopy; TFA, trifluoroacetic acid; TIS, triisopropyl silane; TOCSY, total correlation spectroscopy; TSP, 3-(trimethylsilyl)propanoic acid; ΔH°, change of enthalpy; ΔS°, change of entropy

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.9b00290.

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All peptides and sequence and motif information for Atg-16; physical characteristics of peptides; analytical HPLC traces; ESI-MS spectra; hemolysis graph and relevant NMR spectra (PDF)

REFERENCES

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Accession Codes

The structures of K30 bound to D8PG have been deposited in the Protein Data Bank (PDB) with PDB accession code 5ZYX.

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Article

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +91 33 25693336. *E-mail: [email protected]. Phone: + 64 9 923 3387. Fax: + 64 9 373 7422. ORCID

Lorenzo Stella: 0000-0002-5489-7381 Anirban Bhunia: 0000-0002-8752-2842 Vijayalekshmi Sarojini: 0000-0002-5593-8851 Notes

The authors declare no competing financial interest. 2008


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Design, Synthesis, Antibacterial Potential, and Structural

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