Critical Evaluation and Compilation of Physicochemical Determinants

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Critical Evaluation and Compilation of Physicochemical Determinants and Membrane Interactions of MMGP1 Antifungal Peptide Pushpanathan Muthuirulan, Pooja Sharma, Paramasamy Gunasekaran, and Jeyaprakash Rajendhran Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00086 • Publication Date (Web): 17 Mar 2016 Downloaded from http://pubs.acs.org on March 20, 2016

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Molecular Pharmaceutics

Critical Evaluation and Compilation of Physicochemical Determinants and Membrane Interactions of MMGP1 Antifungal Peptide

Muthuirulan

Pushpanathan+,

Sharma

Pooja++,

Paramasamy

Gunasekaran#

and

Jeyaprakash

Rajendhran#* +

Laboratory of Gene Regulation and Development, National Institutes of Child Health and Human

Development, National Institutes of Health, Bethesda, Maryland-20892, USA. ++

Department of Animal and Avian Sciences, University of Maryland, College Park, Maryland-20740,

USA.

# Department of Genetics, School of Biological Sciences, Madurai Kamaraj University, Madurai- 625 021, India

Running title: Physicochemical and Biological Properties of MMGP1

*Corresponding author: Mailing address: Department of Genetics, School of Biological Sciences, Madurai Kamaraj University, Madurai-625021, India Email: [email protected] Tel: ++91-452-2458478 Fax: ++91-452-2459873

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Abstract

A growing issue of pathogen resistant to antibiotics has fostered the development of innovative approaches for novel drug development. Here, we report the physicochemical and biological properties of an antifungal peptide, MMGP1 based on computational analysis. Computation of physicochemical properties has revealed that the natural biological activities of MMGP1 are coordinated by its intrinsic properties such as net positive charge (+5.04), amphipathicity, high hydrophobicity, low hydrophobic moment and higher isoelectric point (11.915). Prediction of aggregation hot spots in MMGP1 had revealed the presence of potentially aggregation-prone segments that can nucleate in vivo aggregation (on the membrane), whereas, no aggregating regions were predicted for in vitro aggregation (in solutions) of MMGP1. This ability of MMGP1 to form oligomeric aggregates on membrane further substantiates it's direct–cell penetrating potency. Monte Carlo simulation of the interactions of MMGP1 in the aqueous phase and different membrane environments revealed that increasing the proportion of acidic lipids on membrane had led to increase in the peptide helicity. Furthermore, the peptide adopts energetically favorable transmembrane configuration, by inserting peptide loop and helix termini into the membrane containing >60 % of anionic lipids. The charged lipid-based insertion of MMGP1 into membrane might be responsible for the selectivity of peptide toward fungal cells. Additionally, MMGP1 possessed DNA-binding property. Computational docking has identified DNA-binding residues (TRP3, SER4, MET7, ARG8, PHE10, ALA11, GLY20, THR21, ARG22, MET23, TRP34 and LYS36) in MMGP1 crucial for its DNA-binding property. Furthermore, computational mutation analysis revealed that aromatic amino acids are crucial for in vivo aggregation, membrane insertion and DNA binding property of MMGP1. These data provide new insight into the molecular determinants of MMGP1 antifungal activity and also serves as the template for the design of novel peptide antibiotics.

Keywords: antifungal peptide; peptide aggregation; amphipathicity; membrane‒peptide interaction; DNA‒ binding property.


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Introduction

Antimicrobial peptides (AMPs) are essential components of host defense system that fights against infections by exerting cytotoxicity on the invading pathogenic microorganisms. Several AMPs have been identified and characterized from different sources such as plants, mammals, insects, marine invertebrates, and environmental libraries.1 Although AMPs have diverse sequences owing to a wide range of structures and functions, they have been classified into relatively a few conformational paradigms. The existence of a high degree of degeneracy within structural conformation affords unique functional properties among AMPs.2 AMPs are considered as in-discriminant membrane detergents, which exhibit different mechanisms of action based on structure‒activity relationship.3,4 Antimicrobial activities of AMPs are highly associated with their physicochemical parameters and target cell properties, as well as the biological conditions at which AMPs and target cells interacts with each other. 5 Most AMPs are unstructured before interaction with the target cells and may undergo significant conformational dynamics to helical or other structures upon binding to the target cell membrane.6 Further, AMPs differentiates pathogen and host cells based on the cell-type specific discriminating features such as membrane composition, energetics (such as transmembrane potential and polarization), structural components (sterols, lipopolysaccharide, and peptidoglycan) of the pathogen and the host cell membranes. The ability of AMPs to discriminate and exhibit selective toxicity against pathogen enables them to serve as potential candidates for the pharmacological application3.

Bacterial cell membranes are highly electronegative composed predominantly of hydroxylated phospholipids phosphatidylglycerol (PG), cardiolipin (CL) or phosphatidylserine (PS). On contrary, eukaryotic cell membranes are enriched with both anionic (PS) and zwitterionic phospholipids [phosphatidylethanolamine (PE) & phosphatidylcholine (PC)]. PS represents the major anionic lipid in plasma membranes of eukaryotes.7 Additionally, the plasma membranes of human cells and fungi contain sphingolipids and sterols, which is absent in bacteria. Sphingolipids in yeast are characterized by their inositol moiety and are located primarily in the plasma membrane, which accounts for ̴7–8% of the total mass of the membrane (30% of the plasma membrane phospholipids). The most abundant sterol in yeast plasma membrane is ergosterol, while the plasma membrane of human cells instead contains cholesterol. The cholesterol containing membranes of human cells are more resistant to the disrupting activities by AMPs. All these differences in the membrane composition make AMPs to discriminate between host and bacterial/fungal cells.8, 9 ACS Paragon Plus Environment


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Especially Candida spp. have a complex cell wall consisting of a plasma membrane and a cell envelope consisting of β-glucan, chitin and mannoprotein, all these resulting in a highly negatively charged membrane surface that could attract AMPs.10 All bacteria have at least 15 % anionic lipid, which can be either PG or CL or both. The exposure of anionic lipids on the membrane surfaces affords cationic AMPs to recognize and exhibit selective toxicity against bacteria or fungal cells.11 Also, net charge and hydrophobicity play a crucial role in the cellular association of AMPs to the target cell membranes in exerting antimicrobial activity.12 AMPs share numerous features that attribute for their antimicrobial actions, including small size (typically 60%), might explain the selectivity of peptide towards the fungal cells, which display a higher abundance of anionic lipids in their membrane compared to bacterial membranes. Moreover, the simulation of MMGP1 with membrane containing 70 % to 90 % charged lipids showed helix distortion at N-termini compared to the 3D-modelled structure. However, the peptide possessed α-helical conformation with significantly lower free energy of membrane-association in 100 % of charged lipids Further, MMGP1 possessed less deep transmembrane configuration and decreased helical content with 100% of charged lipids, which



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might be due to the increased surface charge density and tail group saturation of anionic lipids in the membrane that causes transition in the free energy of membrane-association of MMGP1 “inner” conformations (∆Gtotal) from -2.11 kT±036 kT (90 % charged lipids) to -2.42 ±037 kT (100% charged lipids). Additionally, there was also significant decrease in the Coulombic interactions between titratable residues of the peptide and charge of the membrane surface (∆Gcoul) from -24.73±0.04 kT (90 % charged lipids) to -27.22±0.04 kT (100% charged lipids) that entails the peptide to attain α-helical conformation with significantly lower free energy of membrane-association (i.e., more stable). These significant decrease in the free energy of membrane-association and Coulombic interactions might be the reason for less deep transmembrane configuration and decreased helical content of MMGP1 in association with the membrane containing 100% of charged lipids. Also, the peptide did show neither surface nor TM orientation on interacting with the membrane containing < 60 % of anionic lipids, which is the characteristic of most bacterial membranes. All these results were in good agreement with our experimental data that the MMGP1 initially localized on the fungal cell membrane and subsequently enters the fungal cells through energy independent direct-cell penetration mechanism. Additionally, MMGP1 also did not show antibacterial activity.15

According to our previous studies, MMGP1 also known to possess DNA−binding property, by which it inhibits the cellular transcription and induce cell death. Our previous experimental data had shown that the intrinsic DNA−binding ability of MMGP1 corresponds to the stacking between indole ring of tryptophyl residue and nucleotide bases.16 As we expected, the molecular docking studies have shown the favorable interaction of MMGP1 with DNA and also the involvement of two tryptophyl residues (TRP3 and TRP34) along with other amino acid residues crucial for its DNA−binding function. Computational mutation analysis has uncovered the crucial role of aromatic amino acids in aggregation, membrane insertion and DNA binding property of MMGP1. This is consistent with earlier report on antimicrobial peptides belonging to the pediocin-like family of bacteriocins, which utilizes the unique role of aromatic amino acid, tryptophan in membrane interaction and positioning of peptide in membrane-water interface.33 Furthermore, bovine antimicrobial peptide, indolicidin also evidenced the involvement of basic and aromatic amino acids in DNA-binding mechanism to exert antimicrobial action.34 It was interesting to note that, besides DNA−binding property, MMGP1 also possessed chitin−binding and proteolytic property. The amino acid residues contributing to different biological properties are summarized in Fig. 9. Overall, the present study demonstrates the physicochemical features and the membrane interactions of


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MMGP1. Initially, MMGP1 get adsorbed onto the target cell membrane, which is coordinated by its sophisticated intrinsic physicochemical properties. Upon interacting with the membranes, the peptide forms oligomeric aggregates and undergo significant conformational dynamics to the helical structure. Furthermore, the peptide utilizes its amphipathic nature and transmembrane properties to penetrate into target cell membrane. Within the target cells the peptide binds with DNA and interferes cellular transcription processes that triggered cascades of events and causes cell death (Fig. 10).

Conflict of Interests The authors declare no conflict of interest.

Acknowledgements Authors gratefully acknowledge the central facilities, UGC-CAS, UGC-NRCBS, DBT-IPLS, DSTFIST and DST-PURSE at MKU.

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23. Ben-Tal, N.; Ben-Shaul, A.; Nicholls, A.; Honig, B.; Free-energy determinants of alpha-helix insertion into lipid bilayers. Biophys. J. 1996, 70, 1803–1812. 24. Kessel, A., Ben-Tal, N. Free energy determinants of peptide association with lipid bilayers. Curr. Top. Membr. 2002, 52, 205–253. 25. Shental-Bechor, D.; Kirca, S.; Ben-Tal, N.; Haliloglu, T. Monte Carlo studies of folding, dynamics, and stability in α-helices. Biophy. J. 2005, 88, 2391–2402. 26. Frishman, D.; Argos, P. Knowledge‐based protein secondary structure assignment. Proteins: Struct. Funct. and Bioinf. 1995, 23, 566–579. 27. Gao, M.; Skolnick, J. From nonspecific DNA-protein encounter complexes to the prediction of DNA-protein interactions. PLoS Comput. Biol. 2009, 5, e1000341. 28. Gao, M.; Skolnick, J. DBD-Hunter: a knowledge-based method for the prediction of DNA– protein interactions. Nucleic Acids Res. 2008, 36, 3978-3992. 29. Shai, Y. Mode of action of membrane active antimicrobial peptides. Biopolymers. 2002, 66, 236– 248. 30. de Groot, N.S.; Ventura, S. Protein aggregation profile of the bacterial cytosol. PLoS One. 2010, 5, e9383. 31. Belli, M.; Ramazzotti, M.; Chiti, F. Prediction of amyloid aggregation in vivo. EMBO rep. 2011, 12, 657–663. 32. Berg, J. M.; Tymoczko, J. L.; Stryer, L.; Biochemistry, 5th edition. New York: W. H. Freeman. 2002. 33. Fimland, G.; Eijsink, V. G.; Nissen-Meyer, J. Mutational analysis of the role of tryptophan residues in an antimicrobial peptide. Biochem. 2002, 41, 9508–9515. 34. Hsu, C. H.; Chen, C.; Jou, M. L.; Lee, A. Y. L.; Lin, Y. C.; Yu, Y. P.; Huang, W. T.; Wu, S. H. Structural and DNA-binding studies on the bovine antimicrobial peptide, indolicidin: evidence for multiple conformations involved in binding to membranes and DNA. Nucleic Acids Res. 2005, 33, 4053–4064.



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Figure Legends

Fig. 1. Physicochemical properties of MMGP1 (a) Physicochemical properties of MMGP1 predicted using HeliQuest server. The α-helical segment of MMGP1 (1−18 aa) showing relatively polarized hydrophobic (Red dotted circle) and hydrophilic domains (blue asterisk), when the peptide was aligned in a α-helical configuration as a helical wheel diagram. The numbers in the helical wheel diagram represent the amino acid position in the given input sequence of MMGP1 α-helical segment (shown at the top of helical wheel diagram) (b) Amino acid residues forming hydrophobic face within the α-helical region of MMGP1. (c) The Isoelectric point and net charge of MMGP1 predicted using Prot pi. The theoretical isoelectric point and a net charge of MMGP1 were predicted to be 11.915 and +5.04, respectively.

Fig. 2. Aggregation propensity of MMGP1 (a) In vitro aggregation of MMGP1 predicted using TANGO software. No aggregation hot spots were predicted within MMGP1 for in vitro aggregation (in solutions) (b) In vivo aggregation of MMGP1 predicted using AGGRESCAN. Three aggregation‒ prone segments (1−5, 7−15, 21−29 residues) were predicted within MMGP1 for in vivo aggregation (in the presence of cell materials).

Fig. 3. The average location of the amino acids of MMGP1 in the membrane (a) Surface and (b) TM orientations- The membrane includes 10 to 100 % of anionic lipids. The horizontal dashed line in (a) designates the surface of the lipid bilayer. The horizontal dashed lines in (b) designate the location of the phosphate groups of the lipid polar heads. The hydrophobic residues are in orange, polar residues are in green and charged amino acids in blue. The peptide did show neither surface nor TM orientation with membranes containing anionic lipids ranging from 10 to 60 %. The peptide showed energetically favorable TM orientation with membranes containing 70 % anionic lipids.


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Fig. 4. The centroid conformation of the largest cluster of MMGP1 in TM orientation Centroid conformation of MMGP1 in TM orientation within membrane environment containing different proportions of anionic lipid- (a), (b), (c) & (d) and (a’), (b’), (c’) & (d) showing ribbon & mesh surface representation of MMGP1 in TM-orientation within different membrane environment, respectively. The colored rectangles represent the average location of the phosphate heads of the two leaflets of the lipid bilayer. Red- membrane with 70% anionic lipid, Green- membrane with 80% anionic lipid, Yellow- membrane with 80 % anionic lipid and Cyan- membrane with 100 % anionic lipid. The peptide shows exceptionally favorable (∆Gtotal= -2.42±0.37 kT) TM-orientation within membrane containing 100 % of anionic lipid.

Fig. 5. The average helical content of MMGP1 versus residue number as predicted by the MCPep server (A) MMGP1 in aqueous phase (a-e). (B) MMGP1 in membrane environment containing different proportions of charged lipids. Membrane with 10 % of anionic lipids (a) showed no significant increase in peptide α-helical content, membrane with 50 % of anionic lipids (b) showed moderate increase in peptide α-helical content, whereas membrane with >60% (c-e) charged lipid showed significant increase in peptide α-helical content, at which the peptide displayed TM configuration.

Fig. 6. Secondary structure ‘inner’ conformation of MMGP1 in association with membrane. (a) 3D modelled structure of MMGP1 constructed using I-TASSER server. (b) & (c) Representative secondary structure ‘inner’ conformation of MMGP1 obtained from MC simulations in the membrane containing 70 % and 100 % of charged lipids, respectively. The peptide showed completely distorted αhelix in association with membrane containing 70 % of charged lipids, whereas the helical conformation of MMGP1 was compatible with that of predicted 3D modelled structure in membrane containing 100 % of charged lipids.



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Fig. 7. Molecular docking of MMGP1 with DNA Molecular docking of MMGP1 with DNA performed using DP dock web server. (a) & (b) showing front (helix facing outwards and loop facing inward) and back (loop facing outwards and helix facing inward) view of MMGP1-DNA docked structure, respectively. (c) surface view of the MMGP1-DNA interactions (d)The amino acid residues of MMGP1 crucial for DNA binding function: TRP3, SER4, MET7, ARG8, PHE10, ALA11, GLY20, THR21, ARG22, MET23, TRP34 and LYS36.

Fig. 8. Effect of mutation on in vivo aggregation, membrane insertion and DNA binding propensity of MMGP1. (A)

MMGP1_a (substitution of polar basic amino acid residues with polar uncharged residues in

native MMGP1 i.e., R to S and L to T) showing no significant effect on in vivo aggregation (i) and transmembrane configuration (ii). However, MMGP1_a mutant showed lesser ability to bind with BDNA (iii). (B) MMGP1_b (substitution of aromatic amino acid residues with non-aromatic polar acidic amino acid residues in native MMGP1 i.e., W to D, F to E and Y to D) showing drastic decrease in number of in vivo aggregation hot spot and hot spot area (i), lacking both surface & transmembrane configuration (ii) and possessed lesser DNA binding property (iii).

Fig. 9. Amino acid residues of MMGP1 contributing for different biological properties MMGP1 possessed chitin-binding, proteolytic and DNA binding properties. [Chitin-binding14, proteolytic residues 15 information of MMGP1 has been adopted from our previous publications]

Fig. 10. Schematic representation of antifungal action of MMGP1 MMGP1 remains unstructured in membrane free environment (i.e., in vitro condition). MMGP1 get adsorbed onto the fungal cell membrane through its sophisticated intrinsic physicochemical properties. Upon interacting with the fungal membrane, the peptide forms oligomeric aggregates and undergoes conformational dynamics to the helical structure. Furthermore, MMGP1 penetrates the target cell membrane by utilizing it amphipathic nature and transmembrane potential. Within the target cells, the peptide binds to DNA and interferes with cellular transcription that leads to hyperproduction of reactive oxygen species, which triggered cascade of the events to cause cell death.


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Supplementary Fig.1. Hot spot area graphic for MMGP1 generated by AGGRESCAN. AGGRESCAN server predicted three putative hot spot regions (HSA1, HSA2 and HSA3) in MMGP1 for in vivo aggregation based on aggregation-propensity scale for natural amino acids derived from previous experimental data. AA in the graphic represents the amino acids sequence of MMGP1. a4v is the average aggregation-propensity values per amino acid derived previously from experimental data. HSA (hot spot area) is the area of peak profile. NHSA is the normalized HSA for 100-residue ‘hot spot’. a4vAHS is the average a4v in each "hot spot".



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Table. 1 Characterization of physicochemical features of MMGP1 α-helical region by HELIQUEST analysis

Physicochemical properties of α-helical segment of MMGP1 Hydrophobic face

FWFMGA

Hydrophobicity

0.600

Hydrophobic moment

0.251

Net charge, z

0.2

Polar residues (n/%)

8/44.44

Non-polar residues (n/%)

10/55.56

Uncharged residues

SER 4, THR 1, GLY 1

Charged residues

ARG2

Aromatic residues

TRP 1 and PHE 2


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Table 2. Thermodynamic characteristics of MMGP1 in TM and surface configuration within membrane having 70% and 100 % of anionic lipids

Quality

TM orientation

Surface orientation

70% charged 100% charged 70% charged 100% charged lipids lipids lipids lipids ∆Gtotal (kT)

-0.70 ± 0.47

-2.42 ± 0.37

-28.39 ± 0.41

-29.19± 0.39

∆Gcon (kT)

13.75 ± 0.48

9.56 ± 0.32

-6.55 ± 0.39

-3.50 ± 0.37

Τ∆S (kT)

-33.89 ± 0.00

-32.48± 0.02

-9.68 ± 0.03

-10.88 ± 0.06

∆Eint (kT)

-20.14 ± 0.48

-22.92 ± 0.33

-16.23 ± 0.39

-14.38 ± 0.38

∆GSIL (kT)

7.67 ± 0.12

14.32 ± 0.20

-0.47 ± 0.04

-0.01 ± 0.04

∆Gdef (kT)

0.92 ± 0.00

0.91 ± 0.00

0.25 ± 0.00

0.25 ± 0.00

∆GCoul (kT)

-23.04 ± 0.06

-27.22 ± 0.04

-21.62 ± 0.05

-25.92 ± 0.05

Μwidth (Å)

25.90 ± 0.01

25.94 ± 0.01

30.02 ± 0.04

30.02 ± 0.04

Zcenter (Å)

7.30 ± 0.03

7.95 ± 0.05

23.38 ± 0.05

23.18 ± 0.05

Tilt (º)

21.75 ± 0.31

23.47± 0.18

76.61 ± 0.21

77.39 ± 0.22

All values are reported as means ± standard error. The free energy terms are defined in Equation 1. Mwidth: the width of the membrane hydrophobic core. Zcenter: The average distance of the peptide’s center of mass from the membrane midplane. The Z-axis is the membrane normal, and the origin coincides with the membrane midplane. Tilt: the angle between the N’-to-C’ vector of the peptide’s helical core and membrane normal.



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Fig. 1. Physicochemical properties of MMGP1 (a) Physicochemical properties of MMGP1 predicted using HeliQuest server. The α-helical segment of MMGP1 (1−18 aa) showing relatively polarized hydrophobic (Red dotted circle) and hydrophilic domains (blue asterisk), when the peptide was aligned in a α-helical configuration as a helical wheel diagram. The numbers in the helical wheel diagram represent the amino acid position in the given input sequence of MMGP1 αhelical segment (shown at the top of helical wheel diagram) (b) Amino acid residues forming hydrophobic face within the α-helical region of MMGP1. (c) The Isoelectric point and net charge of MMGP1 predicted using Prot pi. The theoretical isoelectric point and a net charge of MMGP1 were predicted to be 11.915 and +5.04, respectively. 163x142mm (300 x 300 DPI)


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Fig. 2. Aggregation propensity of MMGP1 (a) In vitro aggregation of MMGP1 predicted using TANGO software. No aggregation hot spots were predicted within MMGP1 for in vitro aggregation (in solutions) (b) In vivo aggregation of MMGP1 predicted using AGGRESCAN. Three aggregation‒ prone segments (1−5, 7−15, 21−29 residues) were predicted within MMGP1 for in vivo aggregation (in the presence of cell materials). 135x86mm (300 x 300 DPI)



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Fig. 3. The average location of the amino acids of MMGP1 in the membrane (a) Surface and (b) TM orientations- The membrane includes 10 to 100 % of anionic lipids. The horizontal dashed line in (a) designates the surface of the lipid bilayer. The horizontal dashed lines in (b) designate the location of the phosphate groups of the lipid polar heads. The hydrophobic residues are in orange, polar residues are in green and charged amino acids in blue. The peptide did show neither surface nor TM orientation with membranes containing anionic lipids ranging from 10 to 60 %. The peptide showed energetically favorable TM orientation with membranes containing 70 % anionic lipids. 190x253mm (300 x 300 DPI)


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Fig. 4. The centroid conformation of the largest cluster of MMGP1 in TM orientation Centroid conformation of MMGP1 in TM orientation within membrane environment containing different proportions of anionic lipid- (a), (b), (c) & (d) and (a’), (b’), (c’) & (d) showing ribbon & mesh surface representation of MMGP1 in TM-orientation within different membrane environment, respectively. The colored rectangles represent the average location of the phosphate heads of the two leaflets of the lipid bilayer. Red- membrane with 70% anionic lipid, Green- membrane with 80% anionic lipid, Yellowmembrane with 80 % anionic lipid and Cyan- membrane with 100 % anionic lipid. The peptide shows exceptionally favorable (∆Gtotal= -2.42±0.37 kT) TM-orientation within membrane containing 100 % of anionic lipid. 100x71mm (300 x 300 DPI)



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Fig. 5. The average helical content of MMGP1 versus residue number as predicted by the MCPep server (A) MMGP1 in aqueous phase (a-e). (B) MMGP1 in membrane environment containing different proportions of charged lipids. Membrane with 10 % of anionic lipids (a) showed no significant increase in peptide αhelical content, membrane with 50 % of anionic lipids (b) showed moderate increase in peptide α-helical content, whereas membrane with >60% (c-e) charged lipid showed significant increase in peptide α-helical content, at which the peptide displayed TM configuration. 193x347mm (300 x 300 DPI)


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Fig. 6. Secondary structure ‘inner’ conformation of MMGP1 in association with membrane. (a) 3D modelled structure of MMGP1 constructed using I-TASSER server. (b) & (c) Representative secondary structure ‘inner’ conformation of MMGP1 obtained from MC simulations in the membrane containing 70 % and 100 % of charged lipids, respectively. The peptide showed completely distorted α- helix in association with membrane containing 70 % of charged lipids, whereas the helical conformation of MMGP1 was compatible with that of predicted 3D modelled structure in membrane containing 100 % of charged lipids. 192x443mm (300 x 300 DPI)



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Fig. 7. Molecular docking of MMGP1 with DNA Molecular docking of MMGP1 with DNA performed using DP dock web server. (a) & (b) showing front (helix facing outwards and loop facing inward) and back (loop facing outwards and helix facing inward) view of MMGP1-DNA docked structure, respectively. (c) surface view of the MMGP1-DNA interactions (d)The amino acid residues of MMGP1 crucial for DNA binding function: TRP3, SER4, MET7, ARG8, PHE10, ALA11, GLY20, THR21, ARG22, MET23, TRP34 and LYS36. 110x72mm (300 x 300 DPI)


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Fig. 8. Effect of mutation on in vivo aggregation, membrane insertion and DNA binding propensity of MMGP1. (A) MMGP1_a (substitution of polar basic amino acid residues with polar uncharged residues in native MMGP1 i.e., R to S and L to T) showing no significant effect on in vivo aggregation (i) and transmembrane configuration (ii). However, MMGP1_a mutant showed lesser ability to bind with B-DNA (iii). (B) MMGP1_b (substitution of aromatic amino acid residues with non-aromatic polar acidic amino acid residues in native MMGP1 i.e., W to D, F to E and Y to D) showing drastic decrease in number of in vivo aggregation hot spot and hot spot area (i), lacking both surface & transmembrane configuration (ii) and possessed lesser DNA binding property (iii). 189x200mm (300 x 300 DPI)



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Fig. 9. Amino acid residues of MMGP1 contributing for different biological properties MMGP1 possessed chitin-binding, proteolytic and DNA binding properties. [Chitin-binding14, proteolytic residues 15 information of MMGP1 has been adopted from our previous publications] 99x53mm (300 x 300 DPI)


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Fig. 10. Schematic representation of antifungal action of MMGP1 MMGP1 remains unstructured in membrane free environment (i.e., in vitro condition). MMGP1 get adsorbed onto the fungal cell membrane through its sophisticated intrinsic physicochemical properties. Upon interacting with the fungal membrane, the peptide forms oligomeric aggregates and undergoes conformational dynamics to the helical structure. Furthermore, MMGP1 penetrates the target cell membrane by utilizing it amphipathic nature and transmembrane potential. Within the target cells, the peptide binds to DNA and interferes with cellular transcription that leads to hyperproduction of reactive oxygen species, which triggered cascade of the events to cause cell death. 143x108mm (300 x 300 DPI)


Critical Evaluation and Compilation of Physicochemical Determinants

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