Differentiating interactions of antimicrobials with Gram-negative and Gram-positive bacterial cell walls using molecular dynamics simulations

INTRODUCTION

Section:

ChooseTop of pageABSTRACTINTRODUCTION <<MOLECULAR STRUCTURES OF P...SIMULATION METHODOLOGYSTRUCTURAL PROPERTIESINTERACTIONS OF PEPTIDOGL...FREE ENERGY BARRIER FOR T...CONCLUSIONSREFERENCESPrevious sectionNext sectionUnderstanding the structure–function relationship of bacterial cell envelopes enables development of potentially effective antimicrobial agents and therapies to curb virulent bacterial infections. Having structural diversity, the cell envelopes of Gram-positive bacteria are quite distinct from those of Gram-negative strains. The former possesses a 20–40 nm thick cell wall of peptidoglycan surrounding the cytoplasmic bilayer membrane,11. S. J. Kim, J. Chang, and M. Singh, Biochim. Biophys. Acta Biomembr. 1848, 350 (2015). https://doi.org/10.1016/j.bbamem.2014.05.031 while the latter comprises of a more complex envelope with an asymmetric outer membrane of lipopolysaccharides and a thin periplasmic space protecting the cytoplasm.22. B. Lugtenberg, Trends Biochem. Sci. 6, 262 (1981). https://doi.org/10.1016/0968-0004(81)90095-5 Due to the structural diversity, the cell response and protective mechanisms are also distinct among the bacterial strains.Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) are the typical strains that are representative of Gram-negative and Gram-positive bacteria, respectively. S. aureus is an opportunistic pathogen, causing nosocomial infections. With the emergence of multidrug-resistant strains, such as Methicillin-resistant Staphylococcus aureus (MRSA), there is a pressing need to develop alternate treatment protocols. The cell wall plays a crucial role in infectivity and pathogenicity.33. J. van Heijenoort and L. Gutmann, Proc. Natl. Acad. Sci. U.S.A. 97, 5028 (2000). https://doi.org/10.1073/pnas.97.10.5028 Therefore, the Staphylococcal cell wall is a representative system of interest in clinical medicine for infections caused by Gram-positive strains.Peptidoglycan, an essential constituent of the cell wall of bacteria, is an important target for antibiotics and antimicrobials. With its unique role as an exoskeleton, peptidoglycan resists turgor pressure within cells, dictates cell shape, and is primarily responsible for structural rigidity of the cells.44. J.-M. Ghuysen and R. Hakenbeck, Bacterial Cell Wall (Elsevier, New York, 1994). Peptidoglycan is a mesh-like macromolecule and a heteropolymer consisting of amino-polysaccharides. Peptidoglycan precursors contain glycans, namely, N-acetyl glucosamine (NAG) and N-acetylmuramic acid (NAM), found in uridine nucleotides as well as in several amino acids.5,65. J. T. Park, J. Biol. Chem. 194, 877 (1952). https://doi.org/10.1016/S0021-9258(18)55843-96. J. T. Park and J. L. Strominger, Science 125, 99 (1957). https://doi.org/10.1126/science.125.3238.99 Diamino acids such as diaminopimelic acid (m-A2pm) and lysine (Lys) are building blocks detected in the cell walls.7–97. E. S. Holdsworth, Biochim. Biophys. Acta 9, 19 (1952). https://doi.org/10.1016/0006-3002(52)90115-78. M. Salton, Biochim. Biophys. Acta 22, 495 (1956). https://doi.org/10.1016/0006-3002(56)90060-99. C. Cummins and H. Harris, Biochem. J. 57, xxxii (1954). https://doi.org/10.1042/bj057xxvii Furthermore, the biochemistry of peptidoglycan confirms the presence of D-isomers of alanine (D-Ala) and glutamic acid (D-Glu), which are structurally distinct from the standard amino acids.1010. M. Ikawa and E. E. Snell, Biochim. Biophys. Acta 19, 576 (1956). https://doi.org/10.1016/0006-3002(56)90499-1Peptidoglycan is composed of a two- or three-dimensional polymer network comprising of glycan strands of NAG and NAM (Fig. 1). A disaccharide unit of NAG-NAM occupies ∼1 nm spacing along the axis of the glycan helix,1111. R. Burge, A. Fowler, and D. Reaveley, J. Mol. Biol. 117, 927 (1977). https://doi.org/10.1016/S0022-2836(77)80006-5 while the separation between the adjacent glycan strands in the network is ∼2 nm.11,1211. R. Burge, A. Fowler, and D. Reaveley, J. Mol. Biol. 117, 927 (1977). https://doi.org/10.1016/S0022-2836(77)80006-512. L. Pasquina-Lemonche, J. Burns, R. D. Turner, S. Kumar, R. Tank, N. Mullin, J. S. Wilson, B. Chakrabarti, P. A. Bullough, S. J. Foster, and J. K. Hobbs, Nature 582, 294 (2020). https://doi.org/10.1038/s41586-020-2236-6 A short stem of pentapeptide consisting of L- and D-isomers of alanine, glutamic acid, and diamino acid is covalently bound to the D-lactyl group of each muramic acid residue. The cross-linking among the glycan strands occurs through side chains in peptides, either directly via an amide linkage between the amino group of diamino acid and carboxyl group of D-Ala in Gram-negative strains or through an interpeptide pentaglycine bridge in Gram-positive strains, as indicated in Fig. 1. We review some of the findings pertaining to the structure, architecture, and dynamical interactions of the cell wall of Gram-positive bacterium, specifically peptidoglycan of S. aureus.An experimental study1313. I. G. Boneca, Z.-H. Huang, D. A. Gage, and A. Tomasz, J. Biol. Chem. 275, 9910 (2000). https://doi.org/10.1074/jbc.275.14.9910 using reverse-phase high-pressure liquid chromatography and mass spectrometry revealed that a major fraction of glycan strands of S. aureus comprises of only 3–10 disaccharide units of NAG-NAM, with an average length of six disaccharides, while 10%–15% of the strands are longer than 25 disaccharides. X-ray diffraction study of isolated peptidoglycan and whole-cells S. aureus showed a fourfold helical orientational symmetry of the peptide stems.11,1411. R. Burge, A. Fowler, and D. Reaveley, J. Mol. Biol. 117, 927 (1977). https://doi.org/10.1016/S0022-2836(77)80006-514. H. Labischinski, G. Barnickel, H. Bradaczek, and P. Giesbrecht, Eur. J. Biochem. 95, 147 (1979). https://doi.org/10.1111/j.1432-1033.1979.tb12949.x The peptide stems, thus, enable cross-linking of glycans in two orthogonal directions. The interpeptide cross-link density was reported to be 67% using a solid state NMR study.1515. S. J. Kim, L. Cegelski, D. R. Studelska, R. D. O’Connor, A. K. Mehta, and J. Schaefer, Biochemistry 41, 6967 (2002). https://doi.org/10.1021/bi0121407 A study using cryo-electron microscopy confirmed an outer wall zone in cell envelope of S. aureus, consisting of an ∼19 nm thick and uniformly dense peptidoglycan-teichoic acid network.1616. V. R. Matias and T. J. Beveridge, J. Bacteriol. 188, 1011 (2006). https://doi.org/10.1128/JB.188.3.1011-1021.2006 The isolated cell walls can expand and contract in response to salt and pH.1717. L.-T. Ou and R. E. Marquis, J. Bacteriol. 101, 92 (1970). https://doi.org/10.1128/jb.101.1.92-101.1970Corroborating the biosynthesis of interpeptide bridging in S. aureus, Kamiryo and Matsuhashi1818. T. Kamiryo and M. Matsuhashi, J. Biol. Chem. 247, 6306 (1972). https://doi.org/10.1016/S0021-9258(19)44798-4 identified that addition of a pentaglycine spacer to ϵ-amino group in Lys residues takes place sequentially from glycyl-tRNA, which acts as a glycine (Gly) donor. In a three-stage bridging process, the transferase FemX initiates incorporation of a Gly to Lys, followed by two additional Gly units using FemA. While the transferase FemB is essential for adding the remaining two glycines to complete the bridge.1919. U. Kopp, M. Roos, J. Wecke, and H. Labischinski, Microb. Drug Resist. 2, 29 (1996). https://doi.org/10.1089/mdr.1996.2.29 The measured bridge-link density is 85%; this includes the cross-link density as well as the bridge-links that have C-termini of pentaglycines bonded to the Lys residues, while its N-terminal is not bonded to any other peptides1515. S. J. Kim, L. Cegelski, D. R. Studelska, R. D. O’Connor, A. K. Mehta, and J. Schaefer, Biochemistry 41, 6967 (2002). https://doi.org/10.1021/bi0121407 [Fig. 1(b)]. A tertiary cell wall structure of S. aureus was characterized in an experimental study of Sharif et al.2020. S. Sharif, M. Singh, S. J. Kim, and J. Schaefer, J. Am. Chem. Soc. 131, 7023 (2009). https://doi.org/10.1021/ja808971c using CODEX spin diffusion of carbon-13. Accordingly, the peptide stems are in a plane perpendicular to the glycan chain, and the glycyl-carbonyl carbon of the pentaglycine bridge lies within 5 Å from the anomeric carbon of the disaccharide. The three-dimensional molecular models for peptidoglycan were designed in the work of Kelemen and Rogers.2121. M. Kelemen and H. Rogers, Proc. Natl. Acad. Sci. U.S.A. 68, 992 (1971). https://doi.org/10.1073/pnas.68.5.992The contrasting views on orientation of cross-linked peptides were discussed in literature; these are parallel, anti-parallel, and perpendicular orientations.11. S. J. Kim, J. Chang, and M. Singh, Biochim. Biophys. Acta Biomembr. 1848, 350 (2015). https://doi.org/10.1016/j.bbamem.2014.05.031 The high degree of cross-linking, shorter glycan strands, and longer bridges of pentaglycine in S. aureus support the parallel orientation viewpoint, where the cross-linked peptides are parallel to each other. The cross-linking is restricted to only 50% in anti-parallel orientation. The cell walls with long glycan strands without interpeptide bridges (in E. coli) favor the cross-linked peptides orienting in the opposite directions. An architecture with cross-linked peptides orchestrated in perpendicular orientation is most suitable for the cell walls having intermediate bridge length and glycan chain length.11. S. J. Kim, J. Chang, and M. Singh, Biochim. Biophys. Acta Biomembr. 1848, 350 (2015). https://doi.org/10.1016/j.bbamem.2014.05.031A fragment of murein with disaccharide attached to decapeptide L-Ala–D-iso-Gln–L-Lys–(Gly)5–D-Ala–D-Ala was optimized in a computer simulation using a molecular mechanics force field.2222. B. A. Dmitriev, F. V. Toukach, O. Holst, E. Rietschel, and S. Ehlers, J. Bacteriol. 186, 7141 (2004). https://doi.org/10.1128/JB.186.21.7141-7148.2004 In the proposed scaffold model, the glycan strands and oligopeptides take up the planar orientation, which is orthogonal to the cytoplasmic membrane. The peptides adopted helical conformations around the glycan strands. The simulated murein matrix resulted into 83% cross-linking, accommodating the experimental evidence of high degree of cross-linking in S. aureus.To elucidate the architecture of cell wall of Gram-positive bacteria, electron cryo-tomography of murein of Bacillus subtilis showed a uniformly dense matrix with no internal structure.2323. M. Beeby, J. C. Gumbart, B. Roux, and G. J. Jensen, Mol. Microbiol. 88, 664 (2013). https://doi.org/10.1111/mmi.12203 The study proposed a circumferential architecture of the peptidoglycan strands around the cell sacculus for rod-shaped Gram-positive bacteria and provided an evidence using atomistic molecular dynamics simulations to support the proposed architecture.The binding of antimicrobials with peptidoglycan inhibits transpeptidation, thereby impeding the peptidoglycan biosynthesis. In a simulation study on interactions of glycopeptides (vancomycin and its derivatives) with D-Ala–D-Ala termini of peptidoglycan precursors, the simplified models of S. aureus were developed, and the energetic conformations of the glycopeptides were examined.2424. R. Ślusarz, M. Szulc, and J. Madaj, Carbohydr. Res. 389, 154 (2014). https://doi.org/10.1016/j.carres.2014.02.002 Elucidating the structural insights into interactions of pentapeptide-pentaglycine stem of S. aureus with glycopeptide antibiotics, the studies using solid state NMR and molecular dynamics simulations revealed that the binding of Eremomycin to non-(D-Ala–D-Ala) segment of the peptide stem is facilitated by strong interactions of carboxyl terminus of the glycopeptide, while the N-methyl-leucine in the vancomycin is essential for strong binding to D-Ala–D-Ala termini.25,2625. J. Chang, H. Zhou, M. Preobrazhenskaya, P. Tao, and S. J. Kim, Biochemistry 55, 3383 (2016). https://doi.org/10.1021/acs.biochem.6b0018826. F. Wang, H. Zhou, O. P. Olademehin, S. J. Kim, and P. Tao, ACS Omega 3, 37 (2018). https://doi.org/10.1021/acsomega.7b01483To unravel the molecular mechanism of lysozyme-mediated inhibition of S. aureus, a model lysozyme was docked with the tetrasaccharide chain of NAG and NAM.2727. A. C. Pushkaran, N. Nataraj, N. Nair, F. Gotz, R. Biswas, and C. G. Mohan, J. Chem. Inf. Model. 55, 760 (2015). https://doi.org/10.1021/ci500734k The latter was modified by O-acetylation at O6 in muramic residues. The structure of tetrasaccharide with O-acetylated modification was highly distorted, indicating high strain and instability in comparison to the chemically unmodified tetrasaccharide. Such structurally high-energy conformations resulted in steric hindrances at the active cleavage site of lysozyme, leading to escape the sugar residues from lysing the glycosidic linkage. Perhaps the first detailed atomistic peptidoglycan model for E. coli cell wall was developed by Gumbart et al.2828. J. C. Gumbart, M. Beeby, G. J. Jensen, and B. Roux, PLoS Comput. Biol. 10, e1003475 (2014). https://doi.org/10.1371/journal.pcbi.1003475 where a single-layered peptidoglycan structure was studied. More recently, we developed a MARTINI-based model for the E. coli cell wall, and the free energy of interactions with thymol was assessed using umbrella sampling calculations.2929. R. Vaiwala, P. Sharma, M. Puranik, and K. G. Ayappa, J. Chem. Theory Comput. 16, 5369 (2020). https://doi.org/10.1021/acs.jctc.0c00539 The interactions of surfactant molecules with a model peptidoglycan that mimics the cell wall of E. coli has also been studied using molecular dynamics simulations.3030. P. Sharma, R. K. Vaiwala, S. Parthasarathi, N. Patil, M. Waskar, J. S. Raut, J. K. Basu, and K. G. Ayappa, “Interactions of surfactants with the bacterial cell wall and inner membrane: Revealing the link between aggregation and antimicrobial activity,” Langmuir (published online 2022). https://doi.org/10.1021/acs.langmuir.2c02520 The study reveals a correspondence between surfactant aggregation and their antimicrobial activity. Models for peptidoglycan with united atom descriptions have also been employed in simulation studies of the outer membranes of E. coli.31,3231. A. T. Boags, F. Samsudin, and S. Khalid, Structure 27, 713 (2019). https://doi.org/10.1016/j.str.2019.01.00132. F. Samsudin, A. Boags, T. J. Piggot, and S. Khalid, Biophys. J. 113, 1496 (2017). https://doi.org/10.1016/j.bpj.2017.08.011It is clear from the above review that although the peptidoglycan precursors and simplified peptidoglycan models have been investigated in MD simulations for Gram-positive bacteria,24–2724. R. Ślusarz, M. Szulc, and J. Madaj, Carbohydr. Res. 389, 154 (2014). https://doi.org/10.1016/j.carres.2014.02.00225. J. Chang, H. Zhou, M. Preobrazhenskaya, P. Tao, and S. J. Kim, Biochemistry 55, 3383 (2016). https://doi.org/10.1021/acs.biochem.6b0018826. F. Wang, H. Zhou, O. P. Olademehin, S. J. Kim, and P. Tao, ACS Omega 3, 37 (2018). https://doi.org/10.1021/acsomega.7b0148327. A. C. Pushkaran, N. Nataraj, N. Nair, F. Gotz, R. Biswas, and C. G. Mohan, J. Chem. Inf. Model. 55, 760 (2015). https://doi.org/10.1021/ci500734k a model that incorporates the naturally occurring three-dimensional multilayered topology for the Staphylococcal cell wall with its high degree of cross-linking is yet to be developed. In this work, we develop an all-atom model of the Staphylococcal peptidoglycan cell wall with the CHARMM36 compatible force field. The multilayered model constructed in this work incorporates the structural features of the S. aureus cell wall. In addition to the differences in cell wall thickness, differences in the amino acids in peptide stems as well as the manner in which peptides are cross-linked in Gram-positive and Gram-negative peptidoglycan models differentiate between the Gram-positive and Gram-negative strains. We make a comparative assessment between the model cell wall structures of S. aureus and E. coli. We, therefore, also constructed single-layered structures using peptidoglycan precursors of S. aureus and E. coli, and the structural properties were evaluated. Binding affinity of these structures with melittin peptides is assessed in the present study. The free energy for translocation of antimicrobial thymol molecules through the quadrilayered peptidoglycan structure is also computed using the densities of thymol molecules in a restraint-free simulation.

INTERACTIONS OF PEPTIDOGLYCAN WITH MELITTIN

Section:

ChooseTop of pageABSTRACTINTRODUCTIONMOLECULAR STRUCTURES OF P...SIMULATION METHODOLOGYSTRUCTURAL PROPERTIESINTERACTIONS OF PEPTIDOGL... <<FREE ENERGY BARRIER FOR T...CONCLUSIONSREFERENCESPrevious sectionNext sectionThe single-layered models of peptidoglycan discussed in previous sections have been employed to investigate the interactions of peptidoglycan precursors with melittin peptides. Melittin is a naturally occurring antimicrobial peptide (PDB entry: 2MLT). Melittin is comprised of 26 residues, with polar and nonpolar amino acids unevenly distributed. It is a major component in the venom of honey bees (Apis mellifera) and has been widely investigated as an antimicrobial agent against bacteria and fungi.53–5553. S. E. Blondelle and R. A. Houghten, Biochemistry 30, 4671 (1991). https://doi.org/10.1021/bi00233a00654. L. Pan, J. Na, Z. Xing, H. Fang, and G. Wang, Chin. Sci. Bull. 52, 639 (2007). https://doi.org/10.1007/s11434-007-0117-055. S. Dosler and A. A. Gerceker, J. Chemother. 24, 137 (2012). https://doi.org/10.1179/1973947812Y.0000000007 Melittin is recognized as a promising antibacterial agent in healing MRSA-infected skin wounds.56,5756. J. H. Choi, A. Y. Jang, S. Lin, S. Lim, D. Kim, K. Park, S.-M. Han, J.-H. Yeo, and H. S. Seo, Mol. Med. Rep. 12, 6483 (2015). https://doi.org/10.3892/mmr.2015.427557. W. G. Lima, J. C. M. de Brito, V. N. Cardoso, and S. O. A. Fernandes, Eur. J. Pharm. Sci. 156, 105592 (2021). https://doi.org/10.1016/j.ejps.2020.105592 Melittin tends to lose its native helical structure in water (Fig. S1 in SM).3333. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002087 for molecular topology and structure files, secondary structure of melittin in water, free energy profiles for melittin interactions with S. aureus and E. coli model cell walls, histograms for helix angles, and secondary structure analysis for melittin interacting with model cell walls. This unfolding of melittin in an aqueous environment has been reported previously in molecular dynamics simulation studies.5858. A. Glättli, I. Chandrasekhar, and W. F. van Gunsteren, Eur. Biophys. J. 35, 255 (2006). https://doi.org/10.1007/s00249-005-0033-7 Using transmission electron microscopy, a study on melittin-treated S. aureus cells showed the morphological changes with cytoplasmic disintegration.5959. A. F. Marques Pereira et al., Microb. Pathogen 141, 104011 (2020). https://doi.org/10.1016/j.micpath.2020.104011 Our recent study reports the free energy landscapes for CM15 peptide (cecropin A and melittin residues) for its translocation across the complex outer membrane of E. coli.6060. P. Sharma and K. G. Ayappa, J. Membr. Biol. 255, 665 (2022). https://doi.org/10.1007/s00232-022-00258-6Although the interactions of melittin with lipid membranes have been extensively studied and several molecular insights have emerged,61–6561. C. E. Dempsey, Biochim. Biophys. Acta Rev. Biomembr. 1031, 143 (1990). https://doi.org/10.1016/0304-4157(90)90006-X62. A. K. Ghosh, R. Rukmini, and A. Chattopadhyay, Biochemistry 36, 14291 (1997). https://doi.org/10.1021/bi971933j63. H. Raghuraman and A. Chattopadhyay, Biosci. Rep. 27, 189 (2007). https://doi.org/10.1007/s10540-006-9030-z64. C. Xu, W. Ma, K. Wang, K. He, Z. Chen, J. Liu, K. Yang, and B. Yuan, J. Phys. Chem. Lett. 11, 4834 (2020). https://doi.org/10.1021/acs.jpclett.0c0116965. I. Brand and B. Khairalla, Faraday Discuss. 232, 68 (2021). https://doi.org/10.1039/D0FD00039F only few studies have reported the interaction with the more complex components of the bacterial cell wall. Using single-molecule fluorescence microscopy, the electrostatic interactions of melittin with lipid A containing supported bilayers have been investigated, and melittin showed a much slower diffusion when bound to lipid A moieties compared to that of phospholipid-bound melittin.6666. N. Nelson and D. K. Schwartz, Biophys. J. 114, 2606 (2018). https://doi.org/10.1016/j.bpj.2018.04.019 The efficacy of melittin in disruption of biofilms formed by S. aureus and E. coli has been investigated using experimental assays, and it has been found that the peptide is more effective against biofilm formation by S. aureus than E. coli.6767. T. Picoli et al., Microb. Pathogen 112, 57 (2017). https://doi.org/10.1016/j.micpath.2017.09.046A molecular understanding of cell wall interactions with antimicrobial peptides remains elusive.6868. O. P. Neelay, C. A. Peterson, M. E. Snavely, T. C. Brown, A. F. TecleMariam, J. A. Campbell, A. M. Blake, S. C. Schneider, and M. E. Cremeens, J. Mol. Struct. 1146, 329 (2017). https://doi.org/10.1016/j.molstruc.2017.06.018 To bring out detailed insights on binding of melittin peptide with model peptidoglycan structures, we have simulated the single-layered peptidoglycan models with eight melittin peptides (helix-turn-helix structure) initially placed in bulk water. We also carried out simulations with unfolded melittins interacting with peptidoglycan models to study the possibility of structural transitions, if any, within 500 ns of simulation runs. The system details are given in Table II. The lateral dimensions of the model cell walls were ∼12.5nm×12.5 nm and ∼17.5nm×11.5 nm for S. aureus and E. coli structures, respectively.Table icon

TABLE II. Model cell wall interactions with small actives.

SystemsPeptidoglycanTIP3/IonsAntimicrobialSingle-layered model (S. aureus)a12 63753 851/40 (Cl−)8 (Melittin)Single-layered model (E. coli)a11 62273 487/142 (K+)8 (Melittin)Single-layered model (S. aureus)b12 63752 558/20 (Cl−)4 (Melittin)Single-layered model (E. coli)b11 62271 736/162 (K+)4 (Melittin)Four-layered model (S. aureus)26 86139 07630 (Thymol)

Melittin binds strongly with E. coli

Initially, we studied the interactions of folded peptides with the peptidoglycan layer. Since the center-of-mass of the peptidoglycan layer showed small variations in the z-direction during simulation, we evaluated the z-distance between the center-of-mass of the peptide and the center-of-mass of the membrane after shifting the distances to reposition the membrane center-of-mass at z=0. The actual center-of-mass distances are illustrated in Figs. S2(a) and S2(b) in SM.3333. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002087 for molecular topology and structure files, secondary structure of melittin in water, free energy profiles for melittin interactions with S. aureus and E. coli model cell walls, histograms for helix angles, and secondary structure analysis for melittin interacting with model cell walls. Figures 5(a) and 5(d) show the time evolution of the distance heat maps for S. aureus and E. coli structures, respectively. Prone to interact favorably with peptidoglycan, melittin eventually approaches the peptidoglycan layer during the course of 1 μs simulation (Fig. 6). The depth of peptide penetration is stronger for the E. coli model when compared with interactions with the S. aureus peptidoglycan. This increased propensity toward the peptidoglycan of E. coli can be seen in Figs. 5(b) and 5(e) where a relatively higher density of melittins in the core of the E. coli structure are observed and all the melittin molecules are well within the peptidoglycan layer of E. coli. In the case of S. aureus, the melittin molecules are more spread out across the membrane and we observe a greater number of surface-bound peptides.We also computed the free energy landscapes [Fig. S2(c) in SM3333. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002087 for molecular topology and structure files, secondary structure of melittin in water, free energy profiles for melittin interactions with S. aureus and E. coli model cell walls, histograms for helix angles, and secondary structure analysis for melittin interacting with model cell walls.] from the melittin density profiles. The energy landscapes indicate that melittin prefers to be located in the central regions of E. coli peptidoglycan. However, as the density profiles reveal in the case of S. aureus, the energy minima are present toward the surface of peptidoglycan with a small barrier of ∼1 kT present in the central regions of the membrane. These differential binding propensities for melittin between the S. aureus and E. coli membranes are also observed in the time-averaged z-coordinates of the center-of-mass of individual amino acid residues of melittins [Figs. 5(c) and 5(f)]. Snapshots from the molecular dynamics simulations (Fig. 6) clearly illustrate the surface bound propensity of melittin for the S. aureus membrane. Although we did observe some tendency for aggregation of melittin on the peptidoglycan layers, we did not systematically investigate this property.The electrostatic interactions of cationic melittins with the negatively charged E. coli structure is expected to be stronger in comparison to the binding with the electrostatically neutral structure of the model S. aureus. In both the models of peptidoglycan, the melittin peptides are found to be preferentially oriented with majority of the residues interacting with peptidoglycan structures [Figs. 5(c) and 5(f)]. The orientation was quantified by computing the helix axes according to the algorithm described elsewhere.6969. P. C. Kahn, Comput. Chem. 13, 185 (1989). https://doi.org/10.1016/0097-8485(89)85005-3 The distributions for angles that the helix axes make with z-axis (normal to peptidoglycan) are given in Figs. S3 and S5 of SM, which indicate the preferential membrane parallel orientation adopted by melittin. We also observed that the melittin peptides retain their helicity; however, some loss of helicity is observed for few peptides interacting with the peptidoglycan models [Figs. S4(g), S6(b), and S6(d) in SM]. In order to compare the influence of the secondary structure on melittin binding, we also carried out simulations with the unfolded state. From the simulations with unfolded peptides, we did not observe any structural transition over the 500 ns long simulations (Fig. S7 in SM).3333. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002087 for molecular topology and structure files, secondary structure of melittin in water, free energy profiles for melittin interactions with S. aureus and E. coli model cell walls, histograms for helix angles, and secondary structure analysis for melittin interacting with model cell walls. The melittin peptides remained unfolded. Additional enhanced sampling free energy computations would be required to completely understand the favored secondary structure of bound melittin on peptidoglycan.6060. P. Sharma and K. G. Ayappa, J. Membr. Biol. 255, 665 (2022). https://doi.org/10.1007/s00232-022-00258-6

Melittin binds preferentially with D-Ala residues

The interactions of melittin with peptidoglycan are quantified by calculating the contacts that the melittin peptides make with peptidoglycan precursors, namely, glycans (NAG and NAM) and amino acid residues of peptidoglycan. We used a distance criterion with a spherical cutoff of 0.5 nm to calculate the contacts between a melittin and a given peptidoglycan unit. At a given instant, an atom of melittin making multiple contacts with a given unit of peptidoglycan is counted as unity. The contact counts are time averaged over the last 500 ns of the trajectory in each case. The counts are prominent for sugars and D-Ala residues (Fig. 7). The contacts with sugars are greater in E. coli as a consequence of stronger binding with E. coli compared to S. aureus. Moreover, melittin shows preferential binding with higher contacts for D-Ala residues among amino acids in peptidoglycan due to the negative charge of D-Ala. This observation is in agreement with a recent experimental work on antimicrobial peptides binding to peptidoglycan, wherein melittin was shown to interact with peptidoglycan to an extent similar to vancomycin.6868. O. P. Neelay, C. A. Peterson, M. E. Snavely, T. C. Brown, A. F. TecleMariam, J. A. Campbell, A. M. Blake, S. C. Schneider, and M. E. Cremeens, J. Mol. Struct. 1146, 329 (2017). https://doi.org/10.1016/j.molstruc.2017.06.018 The latter is known to bind to D-Ala moieties in peptidoglycan.7070. H. R. Perkins and M. Nieto, “The significance of d-alanyl-d-alanine termini in the biosynthesis of bacterial cell walls and the action of penicillin, vancomycin and ristocetin,” in Medicinal Chemistry III (Elsevier, London, 1973), pp. 371–381. Binding of melittin with D-Ala residues is a signature of inhibitory action of melittin for transpeptidation in the biosynthesis pathway of peptidoglycan.

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