C-TERMINAL CLAW STABILISATION IN CORVUS AVIAN BETA DEFENSIN 7 AND ITS MUTATION STUDIESHTML Full Text
C-TERMINAL CLAW STABILISATION IN CORVUS AVIAN BETA DEFENSIN 7 AND ITS MUTATION STUDIES
Kannoth Shalini 1, Ganesh K. Prasanth 2, Kumar Arvind 1 and Tony Grace * 1
Department of Genomic Science 1, Department of Biochemistry and Molecular Biology 2, School of Biological Sciences, Central University of Kerala, Kasaragod - 671316, Kerala, India.
ABSTRACT: Antimicrobial peptides are considered as promising and potential next-generation antibiotics on account of their broad spectrum of antimicrobial activity and unique mode of microbial killing. For therapeutic application, it is essential to consider the hemolytic or cytotoxic property of AMPs along with antimicrobial function. In the current study, an avian beta-defensin 7 (AvBD7) gene was identified from Corvus splendens. The predicted amino acid sequence showed the presence of β defensin core motif and three disulphide bridges. We analysed the interaction of wild and mutant analogues of mature Corvus AvBD7 dimer with neutral 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) that mimics mammalian membranes using molecular dynamics simulation studies. Hydrophobic residues at the N-terminus of each monomer were found to provide stability in the dimeric form. In both monomer and dimer, Asp3 and Asp4 interact with Tyr40 and Ser41 thereby helping in maintaining a claw shape at the C-terminus. During simulation, we found that the C-terminus claw region of wild dimer remained intact than its mutant analogs, and also wild AvBD7 exhibited a greater propensity to bind POPC membrane than its mutant analogs. Based on the membrane-peptide distance and number of hydrogen bonds formed between peptide and membrane, we conclude that the alanine mutant might be least hemolytic as it showed reduced propensity to bind to POPC membrane.
Corvus splendens, Antimicrobial peptide, Avian beta defensin, Membrane, Dimer, Claw
INTRODUCTION: The alarming and rapid evolution of multidrug-resistant strains necessitates the development of novel therapeutic candidates possessing a reduced tendency to induce microbial resistance and least hemolytic and cytotoxic propensity. Antimicrobial peptides (AMPs) have been acknowledged as a promising target for prospective anti-infective agents due to their wide range of antimicrobial activity, low toxicity, and reduced susceptibility to gain drug resistance and multiple modes of killing 1-4.
They are, in general small cationic peptides (<100 amino acid residues) that possess a significant portion of hydrophobic amino acid residues (≥30% or more) and the capability to fold into amphipathic conformations on interaction with membrane 5-8. A major group of AMP found among vertebrates is defensin. They are characterized by the existence of a triple-stranded anti-parallel β-sheet structure with a less-conserved N-terminal helix of varying stability and three intramolecular disulfide bridges formed by 6-cysteine residues.
Out of the three different defensin subfamilies (α, β, and θ) that exist in vertebrates, only β-defensins have been reported in birds and are termed as avian beta-defensins (AvBDs). These classes of peptides are capable of killing bacteria, enveloped viruses, fungi, and eukaryotic parasites 9-12.
Though, the precise antimicrobial mechanisms of AMPs remain unclear, the cytoplasmic membrane is generally suggested as the primary target of antimicrobial peptides, and the accumulation of AMP on microbial membrane augment membrane permeability resulting in the release of cytoplasmic substances and cell death 13, 14. It has been reported that features such as cationicity and hydro-phobicity, and the potential for oligomerization play elementary roles in the microbicidal activity of these peptides by membrane disruption. Direct microbial killing is initiated by the electrostatic interactions between the peptides and the microbial membrane, and the hydrophobic residues of these peptides are responsible for the membrane permeabilization.
Although hydrophobicity and cationicity of the peptides are essential for their activity, beyond a limit these parameters do not improve antimicrobial activity 13, 15-22. Highly hydrophobic peptides target neutrally charged mammalian cell membrane leading to a reduction in the selectivity of target 18, 23-26. One of the major limitations in developing AMP as a therapeutic is peptide-mediated hemolysis or cytotoxicity against host cells 27.
Earlier studies have revealed that beta-defensins perform their activity in their most stable dimer form. Suresh and Verma recommended that in human β-defensin 2 (HBD-2), claw-shaped and positively charged tail region is important to hold on to the bacterial membrane for the initial interaction 28. MD simulation studies using Anas platyrhynchos avian beta-defensin 2 (Apl_AvBD2), wild type and their more cationic in-silico mutants (replacing hydrophobic residues by cationic) revealed that C-terminal ‘claw’ which is significant for antimicrobial activity remained intact in the wild type throughout the simulation, but the shape of the claw showed alteration in all the mutants during simulation 15.
The structure-activity relationship study of antimicrobial peptides helps us to design and develop many new analogs with improved activity, higher stability, and less toxicity to eukaryotic cells. Optimizing the natural AMP by insertion, deletion, and substitution of amino acid residues is one of the major approaches for peptide designing. Though the interactions of AMP with microbial membrane are of great concern, it is required to comprehend the effect of AMP on mammalian membranes as the AMP induced hemolysis of host cells restricts the use of these classes of defense peptides as potential therapeutics.
Production of antimicrobial peptides and proteins is one of the major non-oxidative antimicrobial mechanisms in birds as their heterophils lack superoxide ions and myeloperoxidase 29, 30. Avian scavengers offer a significant ecosystem service role, including carcass removal, without them getting affected. Hence these birds may be considered as an important source of antimicrobial peptides. Till now, the whole genomic information on defensin clusters has only been reported in very few avian species 31, 32.
In the current study, we identified and sequenced the AvBD7 gene of Corvus splendens and also attempted to understand the structure-activity relationship of the corresponding peptide. In order to recognize the role of hydrophobicity in mediating the interaction of the peptide with a mammalian membrane, we compared the interaction of wild and two in-silico mutants of AvBD7 dimer (W39F and W39A) with model membrane using Molecular Dynamic simulation analysis.
MATERIALS AND METHODS:
Ethics Statement: All experimental animal procedures were approved by the Institutional Animal Ethics Committee of College of Veterinary and Animal Sciences, Pookode, Kerala, India (Approval no. IAEC/COVAS/PKD/17/2019 dated 02.08.19).
Collection of Samples: Tissues/organs from live Corvus splendens were collected for this study. Birds were euthanized by intraperitoneal pentobarbitone sodium administration. The tissues were immediately dissected and placed in RNA later and stored at -20 °C or -80 °C until use.
RNA Extraction, Reverse Transcriptase Polymerase Chain Reaction Amplification, and Sequencing: Approximately 50-100 mg of tissue was homogenized in TRIzol reagent, and total cellular RNA was isolated using TRIzol (Invitrogen) method. The quality and concentration of RNA were assessed using a nanodrop spectrophotometer and were separated on 2% agarose gel. Reverse transcription of the extracted RNA was performed in a 20μl reaction using SuperScript™ III First-Strand System for RT-PCR from Invitrogen. The synthesized first-strand cDNA was amplified by polymerase chain reaction using Taq Polymerase with Corvus specific AvBD7 primer. The PCR amplification was performed in an Eppendorf Master cycler in 25 ul volume. PCR products generated were then run on a 1% agarose-TAE gel. The amplified product was purified using Wizard® SV PCR Clean-Up system Kit, and the purified samples were sequenced. The sequences obtained were aligned using SEQUENCHER 4.1.2 (Gene Codes Inc.) The sequence was analyzed for its identity using NCBI-BLAST and submitted to GenBank.
Computational Analysis: Newly identified avian beta-defensin 7 (AvBD7) gene of Corvus splendens was compared with other organisms using Blast at NCBI (www.ncbi.nlm.nih.gov/genome). Since the antimicrobial activity lies in the peptide, we translate the nucleotide sequence to its corresponding peptide, and further analysis was made on the peptide. The experimentally obtained nucleotide sequence of the gene was translated using the translate tool of ExPASy 33. The predicted sequence was compared with other protein sequences in the UniProt database to find out homologous sequence using Blastp at NCBI and was aligned with other organisms using ClustalW 34 using MEGA7 35. The signal peptide sequence of the identified AvBD7 sequence was predicted using SignalP 5.0 Server 36, and disulfide connectivity among six cysteine residues was determined by DISULFIND server 37.
Molecular Dynamics: Homology modeling of wild type and in-silico mutated AvBD7 peptides were predicted with NMR structure of Chicken AvBD7 as a template using Phyre2 38. The best probable template PDB ID, c5lcsA showed 66% similarity with the query sequence, and the modeling pipeline of Phyre2 returned a structure with 98% modeling confidence. High modeling confidence suggests that the model is likely to adopt the predicted 3D structure.
The dimeric state of the modeled peptide AvBD7 (wild type) and its mutant W39F and W39A were generated using ZDOCK server 39. Predicted 3D structures of AvBD7 monomer and dimers were visualized and analyzed using discovery studio visualizer Software (Accelrys Software Inc., 2007 Accelrys Discovery Studio Visualiser v 2.5.5. Accelrys Software Inc., San Diego). Hydrophobic Trp residue in the claw region of wild type protein was mutated to other hydrophobic residues such as Phenylalanine and Alanine, as expected there wasn’t much change in the predicted conformation of the claw regions located at the C-terminal end.
Molecular dynamic simulation studies of AvBD7 wild and mutant dimers with the mammalian membrane were performed in order to understand the difference in their interaction with the membrane. Modeled structures of wild and mutated AvBD7 dimers were loaded on to the VMD software, the system was embedded in a membrane environment containing POPC with membrane dimensions of 55, 56 Å. The peptide in each system was reoriented on the top of the lipid bilayer system with the claw region facing the head region of POPC at a distance of 12Å from C alpha atom of the terminal residue.
The system in the membrane environment was solvated in two steps, and the distance between the maximum and minimum Z-coordinates and the water box edges in the z-axis was set to 15 Å. To solvate the membrane system a total of 5767 water molecules TIP3 model were placed below the membrane plane with: a water box dimensions of 57.62, 57.00, 15.86 (x,y,z in Å), and a total of 5767 water molecules TIP3 model were placed above the plane with: a water box of the dimensions 57.62, 57.00, 33.17 (x,y,z in Å). The system was neutralized using 20 Chloride ions and 16 sodium ions, making up a salt concentration of 0.15 mol/L.
Molecular dynamics was performed on this system using NAMD molecular dynamics package 40, and CHARMM36 force field 41 was used in all MD simulations. Protein structure factors for the ionized structures were prepared using VMD 42 and the plugin QwikMD 43. Energy Minimization of the systems was done for 2000 steps. This and all further downstream process were performed under explicit solvent using the TIP3 water in the NpT ensemble, a distance cut-off of 12.0 Å was applied to short-range non-bonded interactions, 10.0 Å for the smothering functions, and Long-range electrostatic interactions were treated using the particle-mesh Ewald (PME) 44.
Annealing was performed with backbone restrained, explicit solvent in the NpT ensemble, for 0.29 ns of simulation, a temperature ramp from 60 K to 300 K was performed for 0.24 ns. The following parameters were used for further analysis. The pressure at 1 atm was maintained using Nosé-Hoover Langevin piston 45, 46. The equations of motion were integrated using the r-RESPA multiple time-step schemes 40 to update the short-range interactions every 1 step and long-range electrostatic interactions every 2 steps. The time step of integration was chosen to be 2 fs for all simulations.
The Equilibration steps were performed with backbone restrained under explicit solvent in the NpT ensemble, consisted of 1.00 ns of simulation. The temperature was maintained at 300 K using Langevin dynamics. The MD of the final systems were performed with a temperature maintained at 300 K using Langevin dynamics and consisted of 10.0 ns of simulation, no atoms were constrained.
RESULTS AND DISCUSSION:
Identification of AvBD7 of Corvus splendens: The entire nucleotide sequence of the Corvus AvBD7 cDNAs included open reading frames (ORFs) of 201bp and 66 amino acids. The results of the Blastp revealed that the sequence showed 100% sequence identity with AvBD7 of Corvus brachyrhynchos from residues 1 to 62. However, AvBD7 of C. splendens possessed additional 4 residues at the C-terminal in comparison to C. brachyrhynchos. Alignment of the AvBD7 protein with other related proteins was done by ClustalW; the presence of six cysteine residues is highlighted in red columns in Fig. 1.
The alignment showed the presence of a crucial structural component; β-Defensin core motif consists of six cysteine residues and GXC motifs that are conserved in all β-defensins 20, 47-50. This indicates that the newly identified protein belongs to the defensin group.
FIG. 1: ALIGNMENT RESULT OF CORVUS AVBD7
The signal peptide cleavage site of this gene was predicted between the positions 20 and 21 amino acid residues (between AAG-QH residues). Defensins are usually characterized by 3 disulphide bonds; disulphide bonding analysis of our peptide Fig. 2 also showed the presence of three disulphide bridges 51-53.
FIG. 2: DISULPHIDE CONNECTIVITY PREDICTION USING DISULPHIND
The AvBD7 of C. splendens identified in this study was found to possess 5 cationic residues (3Arg and 2 Lys) and 2 anionic residues (Asp). The overall surface charge of folded AvBD7 monomer was found to be +3.08, whereas folded dimer had an overall surface charge +5.9. Minimum cationicity required for AMPs to exhibit its activity has been reported as +2 1, 54, 55. As dimerization showed a significant increase in the cationicity, dimer-membrane interaction may be stronger than monomer-membrane interactions and probably more active than monomer. Oligomerization, which brings about an increase in cationicity may be carried out by augmentation in the local concentration of the peptides in response to several environmental factors 28, 56. Oligomeric structure of HBD-2 possesses uniform positively charged outer surface which is capable of eliciting electrostatic charge based permeabilization of the membrane rather than pore formation mechanism 57. It has been reported that human α-defensin hNP3 crystal structure revealed its dimeric state possessing 6 beta-strands 58.
Homology modeling of AvBD7 monomers (both wild and mutants) was carried out using Phyre2 software, and the dimeric forms were created using ZDOCK software. Preliminary analysis of the modeled monomeric structure revealed 4 distinct geometric faces Fig. 3A, out of which face 2 and 3 seems to have a role in membrane interaction and dimerization, respectively. Face1 and face2 harbour positively charged residues along with few hydrophobic residues, face1 is near to N-terminal region and contains positively charged Arg12 and a hydrophobic Phe14, the face2, which is adjacent to face1 encloses Arg19, Arg20, and Pro21 residue. Face3 in contrast, is distinct by the presence of several hydrophobic residues (Gly1, Leu2, Gly16, Ile17, Cys18, Trp24, Ile25, Gly31, Phe32, Cys34, and Trp39).
Earlier studies reveal that C-terminal claw-like structure in both Human beta-defensin 2 and avian b-defensin Apl_AvBD2 are highly cationic and is important for initial binding to the anionic bacterial membrane 15, 28. It is interesting to note that as in the case of Apl_AvBD2 both monomer and dimer of modeled AvBD7 of Corvus splendens also possessed the distinct C- terminal claw. In this peptide, claw region contained hydrophobic residues Tpr39, Tyr40, and Ser41, moreover, Tyr40 and Ser41 were found to be interacting with Asp3 and Asp4 on the N terminus of the same monomer Fig. 3B. This interaction seems to have a role in maintaining the shape of the hydrophobic claw on the C-terminus. This claw region is adjacent to face 2, the dimer was formed in such a way that the claw regions of both the monomer were positioned on one side of the dimer Fig. 3C. This claw region is reported to have a significant role in the initial interaction with bacterial membrane 15.
FIG. 3A: CORVUS AVBD7 SHOWING 4 DISTINCT FACES. B: MONOMER SHOWING INTERACTION BETWEEN ASP3 AND ASP4 IN THE N-TERMINUS WITH HYDROPHOBIC RESIDUES AT C-TERMINUS. C: AVBD7 DIMER
Monomeric human beta-defensin 2 (HBD2) exhibits amphiphilic nature having cationic and hydrophobic residues on either surface, and the hydrophobic residues are involved in dimerization 28. Interaction of hydrophobic residues on face 3 with the bulk aqueous solvent may be the driving force to form dimers. In the dimeric form, Face3 of two monomers interacted with each other by 1455 Van der Waals contacts (4A cutoff). Apart from the hydrophobic residues on Face3, Arg19 and Arg20 on Face2 and Trp39 present on the claw region were also found to be involved in interfacial bonding through Van der Waals forces. The first two residues at the N-terminus (Gly1 and Leu2) of one monomer clinged the residues present in beta-sheets of other monomers, thus imparting stability to AvBD7s dimeric form Fig. 4. Within 5A cut-off Gly1 of one monomer was seen interacting with Pro15, Gly16, and Phe32 of the second monomer by Van der Waals force, whereas Leu2 interacted with more number of residues, namely Pro15, Gly16, Ile17, Gly31, Phe32 and Ser33 within the same cut-off. The dimer which is devoid of Gly1, Leu2 and Asp3 at the N terminus was found to be less stable in the aqueous phase (data not given). Overall, N-terminal resides seen to have a significant role in maintaining the stability of the dimeric form.
FIG. 4: WILD DIMER SHOWING INTERACTION BETWEEN MONOMERS. RESIDUE GLY1 AND LEU2 (BLUE STICK) OF ONE MONOMER (RED TUBE) IS SEEN IN CLOSE CONTACT WITH SEVERAL RESIDUE (GREY STICKS) ON THE BETA SHEET OF THE SECOND MONOMER (YELLOW SOLID RIBBON)
TABLE 1: RESIDUES INVOLVED IN THE INTERFACIAL INTERACTIONS IN DIMERS
|Initial frame (before simulation)|
|Final frame (after simulation)|
After dimerization, face2, which carries more cationic residues compared to face1, was also seen to be exposed on the surface of the dimer. Positively charged residues and few hydrophobic residues are said to be the prerequisite for membrane interacting peptides 59-61. In concordance to this, similar residues were found to be present on both face1 and face2 of our peptide.
It is interesting to observe that these faces remained exposed even after dimerization. It was found that the accessible surface area for monomer and dimer are 3024.6 A2 and 4740.87 A2, respectively. Increased accessible surface area and masking of hydrophobic residues indicate the dimer resulted in the information of more hydrogen bonds with water.
To gain insights into the role of hydrophobicity of AvBD7 in mammalian membrane interaction, Trp39 at the C-terminal claw region was mutated to highly hydrophobic Phe (W39F) and mid-range hydrophobic Ala(W39A). The residues involved in the interfacial interaction of wild and mutant dimers are mentioned in Table 1. Gly16, Ile17, and Trp24 were found in the interfacial region before simulation in all three forms. In W39F mutant, five hydrogen bonds (between Trp24-Cys18, Gly1-Pro21, Gly1-Gly38, Arg19-Gly1, and Tyr22-Gly1) were also identified apart from Van der Waals interaction. As W39F, W39A dimer also possessed 5 hydrogen bonds in the interfacial region, between Trp24-Gly1, Gly31-Trp24, Gly31-Ser33, Gly31-Gly31, and Hsd30-Gly16. W39A dimer was further stabilized by pi-stacking between the Trp39 of both monomers.
It has been reported that highly hydrophobic AMPs can easily interact with zwitterionic mammalian membrane and induce hemolysis59, 62. Even though hydrophobicity of AMP had the least role in the binding and permeabilization of Phosphatidyl-glycerol (PG) membrane, in zwitterionic membrane consisting of PC only, there was an approximately 300 fold difference in the permeabilization capacity between highest and least hydrophobic AMPs 54. The majority of the AMPs are not recommended as the lead for therapeutic development as they induce hemolysis or exhibit toxicity against mammalian cells.
FIG. 5: RADIUS OF GYRATION (RG) OF CORVUS AVBD7 DIMERS. A) RG OF WILD AVBD7 DIMER AND ITS MUTANTS OVER 10NS OF MD SIMULATION. B) RG OF CLAW REGION (RESIDUE 36-41) OF WILD AND ITS MUTANTS
Dimeric forms (both wild and mutants) were only selected for MD analysis as beta-defensins are stable and efficient in this form. We performed a 10 ns MD simulation of both wild and mutant analogs of modeled AvBD7 in the presence of a POPC membrane. Several initial orientations of the dimer (both wild and mutants) with respect to membrane head regions were studied. Analysis of the MD results of wild and mutant dimers showed that the interfacial interactions between the dimers after the MD run were seen reduced in Table 1. After MD, W39A was found to have more interacting residues in the interface that may impart more stability to alanine mutant than others. Different parameters, such as gyration of peptides (Rg), hydrogen bonding, and lipid peptide distance in both monomers and dimers, were considered during the analysis of the MD run.
Wild type AvBD7 was found to have the least average Rg compared to its mutant analogs Fig. 5A. The decreased Rg value of wild indicates that it was more compact than its analogs. Analysis of Rg of the claw (36-41 residues) separately showed that W39A claw exhibited greater variation in its radius of gyration which may be due to its flexible claw, whereas Rg for wild showed the least variation Fig. 5B. Hence, we predict that the claw region of the wild dimer remains more intact during the simulation study compared to its mutant analogs.
Even though the mammalian membrane is neutral in charge, cationic AMPs can interact with the anionic PO4 head group of the lipid layer through hydrogen bond formed by basic amino acids 63, 64. Wild type dimer formed a greater number of hydrogen bonds followed by W39F. From the graph, Fig. 6 it is understood that wild type showed a greater tendency to bind to the mammalian membrane, thus exhibiting a greater propensity for mammalian membrane. Trp39 present in the wild type may be the contributing factor for its higher propensity towards the neutral membrane. By comparing peptide-membrane distance and number of hydrogen bonds, the alanine mutant showed the least tendency to bind to the POPC membrane.
FIG. 6: PEPTIDE LIPID DISTANCE OF DIMERS OVER 10NS OF MD SIMULATION
Aliste et al., observed from their studies that trp residues in the pentapeptides interact with the choline groups of lipid bilayer through cation-π interaction 65. The presence of two Trp at both claw region in the wild dimer might be the reason for its greater propensity to bind to the mammalian membrane.
It has been reported that hemolytic peptides interact strongly with the neutrally charged POPC membrane, while the non-hemolytic peptides exhibit the least interaction 62. Chen et al., reported in his study using α helical antimicrobial peptide, V13KL that increased hydrophobicity induced by the replacement of alanine residues by leucine residues enhances the hemolytic activity of the peptide 66. From our MD simulation study, we also found that alanine mutant showed the least tendency to bind with POPC membrane. Hence, conclude that W39A mutant due to its reduced hydrophobicity may be considered as least hemolytic among these.
CONCLUSION: Most of the AMPs are not recommended for therapeutic use as they induce hemolysis or exhibit toxicity against mammalian cells. In this study, we identified and sequenced the AvBD7 gene of Corvus splendens and attempted to develop the least hemolytic analog of this peptide using MD simulation. The translated sequence showed the presence of beta-defensin core motif and disulphide pairing, a characteristic of the beta-defensin family of peptides.
The results indicate that the residues at the N-terminus possess a significant role in determining the stability of the dimer and maintaining the C-terminal claw.
The C-terminal claw region maintained its shape in both wild AvBD7 and its mutants, but the claw region of W39A dimer remained more flexible when compared to others. Studies of Corvus AvBD7 with POPC membrane revealed that a single amino acid substitution in the crucial location of AMPs could alter its interaction with the mammalian membrane, thereby enhance or reduce the hemolytic activity. We found the alanine mutant as the least hemolytic.
ACKNOWLEDGEMENT: The first author, Kannoth Shalini acknowledges the Council of Scientific and Industrial Research (CSIR) for providing fellowship.
CONFLICTS OF INTEREST: The authors have no conflicts of interest.
- Kumar P, Kizhakkedathu JN and Straus SK: Antimicrobial peptides: Diversity, mechanism of action and strategies to improve the activity and biocompatibility in-vivo. Biomolecules 2018; 8(1): 4.
- Zharkova S, Orlov DS, Golubeva OY, Chakchir OB, Eliseev IE, Grinchuk TM and Shamova OV: Application of antimicrobial peptides of the innate immune system in combination with conventional antibiotics–a novel way to combat antibiotic resistance? Frontiers in Cellular and Infection Microbiology 2019; 9: 128.
- Pfalzgraff A, Brandenburg K and Weindl G: Antimicrobial peptides and their therapeutic potential for bacterial skin infections and wounds. Frontiers in Pharm 2018; 9: 281.
- Yasir M, Dutta D and Willcox MD: Comparative mode of action of the antimicrobial peptide melimine and its derivative Mel4 against Pseudomonas aeruginosa. Scientific Reports 2019; 9(1): 1-2.
- Lei J, Sun L, Huang S, Zhu C, Li P, He J, Mackey V, Coy DH and He Q: The antimicrobial peptides and their potential clinical applications. American Journal of Translational Research 2019; 11(7): 3919.
- Taheri B, Mohammadi M, Nabipour I, Momenzadeh N and Roozbehani M: Identification of novel antimicrobial peptide from Asian sea bass (Lates calcarifer) by in-silico and activity characterization. PloS One 2018; 13(10).
- Migoń D, Neubauer D and Kamysz W: Hydrocarbon stapled antimicrobial peptides. The Protein Journal 2018; 37(1): 2-12.
- Kang X, Dong F, Shi C, Liu S, Sun J, Chen J, Li H, Xu H, Lao X and Zheng H: DRAMP 2.0, an updated data repository of antimicrobial peptides. Scientific Data 2019; 6(1): 1-0.
- Ageitos JM, Sánchez-Pérez A, Calo-Mata P and Villa TG: Antimicrobial peptides (AMPs): Ancient compounds that represent novel weapons in the fight against bacteria. Biochemical Pharmacology 2017; 133: 117-38.
- Liu C, Jiang L, Liu L, Sun L, Zhao W, Chen Y, Qi T, Han Z, Shao Y, Liu S and Ma D: Induction of avian β-defensin 2 is possibly mediated by the p38 MAPK signal pathway in chicken embryo fibroblasts after Newcastle disease virus infection. Frontiers in Microbiology 2018; 9: 751.
- Bailleul G, Guabiraba R, Virlogeux-Payant I, Lantier I, Trotereau J, Gilbert FB, Wiedemann A, Trotereau A, Velge P, Schouler C and Lalmanach AC: Systemic Administration of Avian Defensin 7: Distribution, Cellular Target, and Antibacterial Potential in Mice. Frontiers in Microbiology 2019; 10.
- Edilia Avila E: Functions of antimicrobial peptides in vertebrates. Current Protein and Peptide Science 2017; 18(11): 1098-19.
- Wu Q, Patočka J and Kuča K: Insect antimicrobial peptides, a mini review. Toxins 2018; 10(11): 461.
- Le CF, Fang CM and Sekaran SD: Intracellular targeting mechanisms by antimicrobial peptides. Antimicrobial Agents and Chemotherapy 2017; 61(4): e02340-16.
- Soman SS, Sivakumar KC and Sreekumar E: Molecular dynamics simulation studies and in-vitro site directed mutagenesis of avian beta-defensin Apl_AvBD2. BMC Bioinformatics 2010; 11(1): S7.
- Omardien S, Brul S and Zaat SA: Antimicrobial activity of cationic antimicrobial peptides against gram-positives: current progress made in understanding the mode of action and the response of bacteria. Frontiers in Cell and Developmental Biology 2016; 4: 111.
- Latendorf T, Gerstel U, Wu Z, Bartels J, Becker A, Tholey A and Schröder JM: Cationic intrinsically disordered antimicrobial peptides (CIDAMPs) represent a new paradigm of innate defense with a potential for novel anti-infectives. Scientific Reports 2019; 9(1): 1-25.
- Gomes B, Augusto MT, Felício MR, Hollmann A, Franco OL, Gonçalves S and Santos NC: Designing improved active peptides for therapeutic approaches against infectious diseases. Biotechnology Advances 2018; 36(2): 415-29.
- Ciumac D, Gong H, Hu X and Lu JR: Membrane targeting cationic antimicrobial peptides. Journal of Colloid and Interface Science 2019; 537: 163-85.
- Shafee TM, Lay FT, Phan TK, Anderson MA and Hulett MD: Convergent evolution of defensin sequence, structure and function. Cellular and Molecular Life Sciences 2017; 74(4): 663-82.
- Falanga A, Nigro E, De Biasi MG, Daniele A, Morelli G, Galdiero S and Scudiero O: Cyclic peptides as novel therapeutic microbicides: Engineering of Human Defensin Mimetics. Molecules 2017; 22(7): 1217.
- Rai DK and Qian S: Interaction of the antimicrobial peptide aurein 1.2 and charged lipid bilayer. Scientific Reports 2017; 7(1): 1-0.
- Marggraf MB, Panteleev PV, Emelianova AA, Sorokin MI, Bolosov IA, Buzdin AA, Kuzmin DV and Ovchinnikova TV: Cytotoxic potential of the novel horseshoe crab peptide polyphemusin III. Marine Drugs 2018; 16(12): 466.
- Alencar-Silva T, Braga MC, Santana GO, Saldanha-Araujo F, Pogue R, Dias SC, Franco OL and Carvalho JL: Breaking the frontiers of cosmetology with antimicrobial peptides. Biotechnology Advances 2018; 36(8): 2019-31.
- Ahmed TA and Hammami R: Recent insights into structure–function relationships of antimicrobial peptides. Journal of Food Biochemistry 2019; 43(1): e12546.
- Deslouches B and Di YP: Antimicrobial peptides with selective antitumor mechanisms: prospect for anticancer applications. Oncotarget 2017; 8(28): 46635.
- Almaaytah A, Mohammed GK, Abualhaijaa A and Al-Balas Q: Development of novel ultrashort antimicrobial peptide nanoparticles with potent antimicrobial and antibiofilm activities against multidrug-resistant bacteria. Drug Design, Development and Therapy 2017; 11: 3159.
- Suresh A and Verma C: Modelling study of dimerization in mammalian defensins. In BMC Bioinformatics 2006; 7(S5): S17.
- Zhu B, Li Q, Liu R, Zheng M, Wen J and Zhao G: Genome-Wide Association Study of H/L Traits in Chicken. Animals 2019; 9(5): 260.
- Milona P, Townes CL, Bevan RM and Hall J: The chicken host peptides, gallinacins 4, 7, and 9 have antimicrobial activity against Salmonella serovars. Biochemical and Biophysical Research Communications 2007; 356(1): 169-74.
- van Hoek ML, Prickett MD, Settlage RE, Kang L, Michalak P, Vliet KA and Bishop BM: The Komodo dragon (Varanus komodoensis) genome and identification of innate immunity genes and clusters. BMC Genomics 2019; 20(1): 684.
- Lan H, Chen H, Chen LC, Wang BB, Sun L, Ma MY, Fang SG and Wan QH: The first report of a Pelecaniformes defensin cluster: Characterization of β-defensin genes in the crested ibis based on BAC libraries. Scientific Reports 2014; 4: 6923.
- Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD and Bairoch A: ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Research 2003; 31(13): 3784-8.
- Thompson JD, Higgins DG and Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 1994; 22(22): 4673-80.
- Kumar S, Stecher G and Tamura K: MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 2016; 33(7): 1870-4.
- Armenteros JJ, Tsirigos KD, Sønderby CK, Petersen TN, Winther O, Brunak S, von Heijne G and Nielsen H: Signal P 5.0 improves signal peptide predictions using deep neural networks. Nature Biotechnology 2019; 37(4): 420-3.
- Ceroni A, Passerini A, Vullo A and Frasconi P: DISULFIND: a disulfide bonding state and cysteine connectivity prediction server. Nucleic Acids Research 2006; 34(S2): W177-81.
- Kelley LA, Mezulis S, Yates CM, Wass MN and Sternberg MJ: The Phyre2 web portal for protein modeling, prediction and analysis. Nature Protocols 2015; 10(6): 845.
- Pierce BG, Wiehe K, Hwang H, Kim BH, Vreven T and Weng Z: ZDOCK server: interactive docking prediction of protein–protein complexes and symmetric multimers. Bioinformatics 2014; 30(12): 1771-3.
- Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E, Villa E, Chipot C, Skeel RD, Kale L and Schulten K: Scalable molecular dynamics with NAMD. Journal of Computational Chemistry 2005; 26(16): 1781-02.
- Best RB, Zhu X, Shim J, Lopes PE, Mittal J, Feig M and MacKerell Jr AD: Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone ϕ, ψ and side-chain χ1 and χ2 dihedral angles. Journal of chemical theory and computation 2012; 8(9): 3257-73.
- Humphrey W, Dalke A and Schulten K: VMD: visual molecular dynamics. Journal of Molecular Graphics 1996; 14(1): 33-8.
- Ribeiro JV, Bernardi RC, Rudack T, Stone JE, Phillips JC, Freddolino PL and Schulten K: QwikMD-integrative molecular dynamics toolkit for novices and experts. Scientific Reports 2016; 6(1): 1-4.
- Darden T, York D and Pedersen L: Particle mesh Ewald: An N⋅ log (N) method for Ewald sums in large systems. The Journal of Chemical Physics 1993; 98(12): 10089-92.
- Martyna GJ, Tobias DJ and Klein ML: Constant pressure molecular dynamics algorithms. The Journal of Chemical Physics 1994; 101(5): 4177-89.
- Feller SE, Zhang Y, Pastor RW and Brooks BR: Constant pressure molecular dynamics simulation: the Langevin piston method. The Journal of Chemical Physics 1995; 103(11): 4613-21.
- Wang G: Antimicrobial peptides: discovery, design and novel therapeutic strategies. Cabi 2017.
- Tu J, Li D, Li Q, Zhang L, Zhu Q, Gaur U, Fan X, Xu H, Yao Y, Zhao X and Yang M: Molecular evolutionary analysis of β-defensin peptides in vertebrates. Evolutionary Bioinformatics 2015; 11: EBO-S25580.
- Batra V, Maheshwarappa A, Dagar K, Kumar S, Soni A, Kumaresan A, Kumar R and Datta TK: Unusual interplay of contrasting selective pressures on β-defensin genes implicated in male fertility of the Buffalo (Bubalus bubalis). BMC Evolutionary Biology 2019; 19(1): 1-9.
- Schmitt P, Rosa RD and Destoumieux-Garzón D: An intimate link between antimicrobial peptide sequence diversity and binding to essential components of bacterial membranes. Biochimica et biophysica acta (BBA)-biomembranes 2016; 1858(5): 958-70.
- Wang G: Identification and Characterization of Antimicrobial Peptides with Therapeutic Potential. MDPI 2018.
- Bruggeman M, Ijakipour H and Stamboulis A: Defensin-Like Peptides and Their Antimicrobial Activity in Free-Form and Immobilized on Material Surfaces. InPeptide Synthesis 2019; IntechOpen.
- Koehbach J: Structure-activity relationships of insect defensins. Frontiers in Chemistry 2017; 5: 45.
- Ebenhan T, Gheysens O, Kruger HG, Zeevaart JR and Sathekge MM: Antimicrobial peptides: their role as infection-selective tracers for molecular imaging. BioMed research international 2014; 2014.
- Mahlapuu M, Håkansson J, Ringstad L and Björn C: Antimicrobial peptides: an emerging category of therapeutic agents. Frontiers in Cellular and Infection Microbiology 2016; 6: 194.
- Lombardi L, Falanga A, Del Genio V and Galdiero S: A new hope: self-assembling peptides with antimicrobial activity. Pharmaceutics 2019; 11(4): 166.
- Hoover DM, Rajashankar KR, Blumenthal R, Puri A, Oppenheim JJ, Chertov O and Lubkowski J: The structure of human β-defensin-2 shows evidence of higher order oligomerization. J of Biol Chem 2000; 275(42): 32911-8.
- Hill CP, Yee J, Selsted ME and Eisenberg D: Crystal structure of defensin HNP-3, an amphiphilic dimer: mechanisms of membrane permeabilization. Science 1991; 251(5000): 1481-5.
- Yin LM, Edwards MA, Li J, Yip CM and Deber CM: Roles of hydrophobicity and charge distribution of cationic antimicrobial peptides in peptide-membrane interactions. Journal of Biological Chemistry 2012; 287(10): 7738-45.
- Chang TW, Wei SY, Wang SH, Wei HM, Wang YJ, Wang CF, Chen C and Liao YD: Hydrophobic residues are critical for the helix-forming, hemolytic and bactericidal activities of amphipathic antimicrobial peptide TP4. PloS one 2017; 12(10).
- Hollmann A, Martínez M, Noguera ME, Augusto MT, Disalvo A, Santos NC, Semorile L and Maffía PC: Role of amphipathicity and hydrophobicity in the balance between hemolysis and peptide-membrane interactions of three related antimicrobial peptides. Colloids and Surfaces B: Biointerfaces 2016; 141: 528-36.
- Edwards IA, Elliott AG, Kavanagh AM, Zuegg J, Blaskovich MA and Cooper MA: Contribution of amphipathicity and hydrophobicity to the antimicrobial activity and cytotoxicity of β-hairpin peptides. ACS Infectious Diseases 2016; 2(6): 442-50.
- Li J, Liu S, Lakshminarayanan R, Bai Y, Pervushin K, Verma C and Beuerman RW: Molecular simulations suggest how a branched antimicrobial peptide perturbs a bacterial membrane and enhances permeability. Biochimica et Biophysica Acta (BBA)-Biomembranes 2013; 1828(3): 1112-21.
- Li J, Koh JJ, Liu S, Lakshminarayanan R, Verma CS and Beuerman RW: Membrane active antimicrobial peptides: translating mechanistic insights to design. Frontiers in Neuroscience 2017; 11: 73.
- Aliste MP, MacCallum JL and Tieleman DP: Molecular dynamics simulations of pentapeptides at interfaces: salt bridge and cation- π interactions. Biochemistry 2003; 42(30): 8976-87.
- Chen Y, Guarnieri MT, Vasil AI, Vasil ML, Mant CT and Hodges RS: Role of peptide hydrophobicity in the mechanism of action of α-helical antimicrobial peptides. Antimicrobial Agents and Chemotherapy 2007; 51(4): 1398-06.
How to cite this article:
Shalini K, Prasanth GK, Arvind K and Grace T: C-terminal claw stabilisation in corvus avian beta defensin7 and its mutation studies. Int J Pharm Sci & Res 2020; 11(12): 6086-96. doi: 10.13040/IJPSR.0975-8232.11(12).6086-96.
All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
K. Shalini, G. K. Prasanth, K. Arvind and T. Grace *
Department of Genomic Science, School of Biological Sciences, Central University of Kerala, Kasaragod, Kerala, India.
01 December 2019
27 February 2020
12 March 2020
01 December 2020