Defensin Type Research Articles

Molecular Dynamics Simulations on Spike Protein Mutants Binding with Human β Defensin Type 2.

Human β defensin type 2 (hBD-2), a cationic cysteine-rich peptide secreted from the human innate immune system, can bind Spike-RBD at the same site as receptor ACE2, thus blocking viral entry into ACE2-expressing cells. In order to find out the impact of CoV-2 mutations on hBD-2's antiviral activity, it is important to investigate the binding and interaction of hBD-2 with RBD mutants. All-atom molecular dynamics simulations were conducted on typical RBD mutants, including N501Y, E484K, P479S, T478I, S477N, N439K, K417N, and N501Y-E484K-K417N, binding with hBD-2. Starting from the stable binding structure of hBD-2 and wt-RBD and ClusPro and HADDOCK docking-predicted initial structures, the RBD variants bound with hBD-2 simulations were set up, and NAMD simulations were conducted. Based on the structure and dynamics analysis, it was found that most RBD variants can still form a similar number of hydrogen bonds with hBD-2, in addition to having a similar-sized buried surface area (BSA) and a similar binding interface to the RBD wildtype. However, the RBD triple mutant formed a less stable binding structure with hBD-2 than other variants. Additionally, the free energy perturbation (FEP) method was applied to calculate the contribution of key mutant residues to the binding and the free energy change caused by the mutations. The result shows that N439K, K417N, and the trimutation increase the binding free energy of RBD with hBD-2; thus, RBD should bind less stably with hBD-2. E484K decreases the binding free energy, thus it should bind more stably with hBD-2, while N501Y, S477N, T478I, and P479S almost do not change the binding free energy with hBD-2. The MM-GBSA method predicted the binding interaction energy which shows that the trimutant should be able to escape the binding with hBD-2 but N501Y should not. The result can provide insight into understanding the functional mechanism of hBD-2 combating SARS-CoV-2 mutants.

Differential activity on trypanosomatid parasites of a novel recombinant defensin type 1 from the insect Triatoma (Meccus) pallidipennis

Defensins are one of the major families of antimicrobial peptides (AMPs) that are widely distributed in insects. In Triatomines (Hemiptera: Reduviidae) vectors of Trypanosoma cruzi the causative agent of Chagas disease, two large groups of defensin isoforms have been described: type 1 and type 4. The aim of this study was to analyze the trypanocidal activity of a type 1 recombinant defensin (rDef1.3) identified in Triatoma (Meccus) pallidipennis, an endemic specie from México. The trypanocidal activity of this defensin was evaluated in vitro, against the parasites T. cruzi, T. rangeli, and two species of Leishmania (L. mexicana and L. major) both causative agents of cutaneous leishmaniasis. Our data demonstrated that the defensin was active against all the parasites although in different degrees. The defensin altered the morphology, reduced the viability and inhibited the growth of T.cruzi. When tested against T. rangeli (a parasite that infects a variety of mammalian species), stronger morphological effects where observed. Surprisingly the greatest effects were observed against the two Leishmania species, of which L. major was the parasite most affected with 50% of dead cells or with damaged membranes, in addition of a reduction in its proliferative capacity in culture. These results suggest that rDef1.3 has an important antimicrobial effect against trypanosomatids which cause some of the more important neglected tropical diseases transmitted by insect vectors.

Interaction of Human β Defensin Type 3 (hBD-3) with Different PIP2-Containing Membranes, a Molecular Dynamics Simulation Study.

Human β defensin type 3 (hBD-3) is a cysteine-rich small antibacterial peptide. It belongs to the human innate immune system. hBD-3 has six cysteine residues, which form three pairs of disulfide bonds, and those bonds break in the reducing condition. It is known that hBD-3 can interact with bacterial membrane, and even eukaryotic cell membrane, which has a low concentration of phosphatidylinositol 4,5-bisphosphate (PIP2) lipids. PIP2 is a vital component in cell membranes and has been found to play important roles during antimicrobial peptide (AMP) interaction with membranes. To understand the functional mechanism of hBD-3 interacting with PIP2-containing membranes, the binding structures of hBD-3 on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers mixed with 10% of PIP2 were predicted using two kinds of methods. The first one is by placing the hBD-3 monomer in different orientations above the POPC + 10%PIP2 membrane to set up five different initial simulation systems and performing long-term simulations on each to predict the most stable binding structure. It was found that hBD-3 analogue binds on the mixed lipid membrane on the two loop regions. The second method is by running long-term simulations on one or nine hBD-3 dimers binding on POPC mixed with 10%PIP2 lipid bilayer starting from the solid-state NMR (ssNMR)-suggested orientation. The dimer dissociated, and the most stable binding of hBD-3 in wild-type on the mixed membrane is also through the two loop regions, which agrees with the prediction from both the first method and the lipid self-assembly result. The PIP2 lipids can form long-lasting hydrogen bonds with positively charged residues such as Arg and Lys on hBD-3, thus forming clusters with hBD-3. As a comparison, hBD-3 dimers binding with a combined bilayer having 1,2-palmitoyl-oleoyl-sn-glycero-3-phosphoserine (POPS) on the upper and POPC on the lower leaflets and the combined POPS + POPC bilayer mixing with 10%PIP2 were also studied. The long-term simulation result shows that hBD-3 can bind with the heads of negatively charged POPS and PIP2 lipids and form hydrogen bonds. The stable binding sites of hBD-3 on PIP2 or POPS mixed bilayers are still on the two loop regions. On the combined POPS + POPC mixed with 10%PIP2 bilayer, the binding of hBD-3 with PIP2 lipids became less stable and fewer because of the competition of binding with the POPS lipids. Besides that, binding with hBD-3 can decrease the membrane thickness of the POPC + PIP2, POPS + POPC, and POPS + POPC + PIP2 bilayers and make POPS and PIP2 lipids more flexible based on the order parameter calculations. Our results supply molecular insight on AMP binding with different membranes and can help understand the functional mechanism of hBD-3 disrupting PIP2-containing membranes.

Binding free energy calculation of human beta defensin 3 with negatively charged lipid bilayer using free energy perturbation method

Human β defensin type 3 (hBD-3) is a cationic peptide having strong antimicrobial activities even at high salt concentrations. The conserved sequence is believed to contribute to its unique antibacterial activities. To design novel drugs based on hBD-3, predicting the binding free energy contribution of each residue on hBD-3 with bacterial membrane is important. Firstly, the stable binding structure of hBD-3 dimer in analog form bound on POPG lipid bilayer was predicted using NAMD simulations, which was confirmed by RMSD, buried surface area, hydrogen bonds, distance map, and insertion depth map calculations. Then, free energy perturbation (FEP) method was applied to calculate the binding free energy of each residue by mutating it into Alanine. It was found that the positively charged residues on the tail region of hBD-3 contribute significantly to its binding with membrane. The result emphasized the importance of electrostatic interactions to hBD-3's binding with bacterial membrane.

Molecular Dynamics Simulations of Human Beta-Defensin Type 3 Crossing Different Lipid Bilayers.

Human β defensin type 3 (hBD-3) is a small cationic cysteine-rich peptide. It has a broad spectrum of antimicrobial activities. However, at high concentrations, it also shows hemolytic activity by interrupting red blood cells. To understand the selectivity of hBD-3 disrupting cell membranes, investigating the capability of hBD-3 translocating through different membranes is important. Since hBD-3 in the analogue form in which all three pairs of disulfide bonds are broken has similar antibacterial activities to the wild-type, this project investigates the structure and dynamics of an hBD-3 analogue in monomer, dimer, and tetramer forms through both zwitterionic and negatively charged lipid bilayers using molecular dynamics (MD) simulations. One tetramer structure of hBD-3 was predicted by running all-atom MD simulations on hBD-3 in water at a high concentration, which was found to be stable in water during 400 ns all-atom simulations based on root-mean-squared deviation, root-mean-squared fluctuation, buried surface area, and binding interaction energy calculations. After that, hBD-3 in different forms was placed inside different membranes, and then steered MD simulation was conducted to pull the hBD-3 out of the membrane along the z-direction to generate different configurational windows to set up umbrella-sampling (US) simulations. Because extensive sampling is important to obtain accurate free energy barriers, coarse-grained US MD simulations were performed in each window. Based on the long-term simulation result, membrane thinning was found near hBD-3 in different lipid bilayers and in different hBD-3 oligomer systems. By calculating the root-mean-squared deviation of the z-coordinate of hBD-3 molecules, rotation of the oligomer inside the bilayer and stretching of the oligomer structure along the z-direction were observed. Although reorientation of lipid heads toward the hBD-3 tetramer was observed based on the density profile calculation, the order parameter calculation shows that hBD-3 disrupts 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS) lipids more significantly and makes it less ordered than on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids. Calculating the free energy of hBD-3 through different lipid bilayers, it was found that generally hBD-3 encounters a lower energy barrier through negatively charged lipid membranes than the zwitterionic membrane. hBD-3 in different forms needs to overcome a lower energy barrier crossing the combined POPC+POPS bilayer through the POPS leaflet than through the POPC leaflet. Besides that, the potential of mean force result suggests that hBD-3 forms an oligomer translocating negatively charged lipid membranes at a low concentration. This study supplied new insight into the antibacterial mechanism of hBD-3 through different membranes.

Disulfide Bonds Affect the Binding Sites of Human β Defensin Type 3 on Negatively Charged Lipid Membranes.

Human β defensin type 3 (hBD-3) is a small natural antimicrobiotic. It is strongly cationic and has six cysteine residues which can form three pairs of intramolecular disulfide bonds under oxidized condition. Those disulfide bonds can break under reducing condition. However, the antibacterial activities of hBD-3 in its wild-type and analog forms are similar. In this project, the structure and dynamics of hBD-3 were investigated by running simulations on hBD-3 in its wild-type and analog forms in solvent, binding to negatively charged lipid bilayers, and self-assembly with POPG lipids. It was found that the RMSFs of hBD-3 in both its wild-type and analog forms are similar in solvent, while they are very diverse depending on the binding sites of hBD-3 with negatively charged bilayers. Calculating both the distance map and insertion depths for 18 hBD-3 molecules binding on the POPG bilayer, hBD-3 in its analog form binds stably with the POPG bilayer through the head and loop regions, while hBD-3 wild-type binds with the POPG bilayer on the two loop regions stably. hBD-3 analog caused membrane thinning and disrupted the POPG lipids more significantly than the wildtype. Based on the self-assembly simulations, hBD-3 monomer can bind with and embed inside the negatively charged POPG lipid membrane and have more contacts with the POPG lipid heads than with tails. The current work emphasized the structural diversity of hBD-3 interacting with negatively charged lipid membrane affected by the disulfide bonding states.

Binding and Interaction of Human Beta Defensin Type 3 with Mixed PIP2 Lipid Membranes

Defensins are cationic cysteine-rich small molecules with molecular masses ranging from 3 to 5 kDa. Human β defensin type 3 (hBD-3) belongs to the human innate immune system, and is mainly secreted from the epithelial cells. It has a broad-spectrum of antimicrobial activities on virus, fungi, and both Gram-positive and Gram-negative bacteria. hBD-3 can bind to the negatively charged and even zwitterionic (but charge-neutral) lipid membrane, disrupt and then translocate through the lipid membrane. In order to understand its dynamics and functional mechanism in molecular level, all-atom molecular dynamics simulations on hBD-3 binding with and crossing mixed lipid membranes were investigated. Since PIP2 is the key minor component in cell membranes, mixed PIP2 lipid membrane represented by neutrally charged POPC lipids mixed with 10% of PIP2 lipids were studied. Simulations on hBD-3 placed inside the mixed lipid bilayer, and on the surface of the lipid bilayer were conducted. In order to consider the influence of the initial binding orientation on the hBD-3 binding structure, in total five different initial binding orientations of hBD-3 on the lipid bilayer were studied. Since hBD-3 can form a dimer, the binding of hBD-3 in both monomer and dimer forms with lipid membranes were explored. The structure of hBD-3 on lipid membranes and the key residues on the binding interface were predicted and compared. During the micro-second long simulations, the assembly of PIP2 lipids towards hBD-3 was observed in both hBD-3 on the lipid membrane surface systems and inside the lipid membrane systems. hBD-3 staying inside the mixed lipid bilayer can behave as the channel to facilitate water molecules to cross the mixed lipid bilayer. The result emphasized the importance of PIP2 in hBD-3's disruption capability with the mixed cell membrane.

Binding of Human Beta Defensin Type 3 with Negatively Charged Lipid Membranes

Binding of Human Beta Defensin Type 3 with Negatively Charged Lipid Membranes

Translocation of Human β Defensin Type 3 through a Neutrally Charged Lipid Membrane: A Free Energy Study.

Human β defensin type 3 (hBD-3) is a cationic (+11 charged) antimicrobial peptide. It has three pairs of intramolecular disulfide bonds which can break under reducing conditions to convert hBD-3 into the linear analog form. hBD-3 can disrupt both Gram-positive and Gram-negative cell membranes, and even the mammalian cell membrane at high concentrations. However, the structural basis for the membrane-disrupting function of hBD-3 is still unknown. In order to understand the interaction mechanism of hBD-3 with a neutrally charged lipid membrane, explicit solvent and lipid umbrella-sampling simulations were performed using the NAMD program on the hBD-3 wild-type and the linear analog, in both the monomer and dimer forms. During the insertion and translocation process, most of the protein structure changes take place near the membrane-solvent interface, while the membrane interior appears to stabilize and rigidify the native-like hBD-3 structure. An energy barrier of 20 kcal/mol (domain unit) should be overcome by hBD-3 dimer in wild-type to cross the POPC bilayer but only 13 kcal/mol (domain unit) to insert into the bilayer center, and 20 kcal/mol for hBD-3 monomers to insert into the membrane center. Significant reorientation of lipids around hBD-3 inside the membranes was observed, which suggests a toroidal model for the membrane disruption process.

Investigation on two human defensin dimers: structure prediction and refinement using a combined simulation strategy

Defensins are cationic cysteine-rich small molecules belonging to the innate immune system. Most defensins form dimers that can enhance their function. Thus, predicting their dimer structures, if unavailable, is important. In this project, a combined simulation strategy is applied to predict dimer structures of two defensins, human defensin type 2 (hBD-2) and human defensin type 5 (HD5), which includes predicting the initial dimer structure running implicit solvent replica-exchange (REX) simulations with a GBSW module, then running microsecond-long all-atom simulations with the CHARMM36 forcefield to refine the prediction. The combined simulations predicted the dimer structures in good agreement with crystal structures within simulation uncertainty. Microsecond-long refinement on the crystal structures of hBD-2 and HD5 dimers shows that CHARMM36 forcefield could contribute a structural deviation of 1.0–3.0 Å from their crystal structures. Comparing the RMSD, RMSF, radius of gyration, accessible surface area (ASA), number of hydrogen bonds (H-bonds) and residue distance map for simulations starting from the REX initiated structure and the crystal structure, consistent agreements were reached for both dimers. However, hBD-2 dimer has a larger hydrophobic ASA, while HD5 has a larger hydrophilic ASA; HD5 forms 45 H-bonds on the binding interface while 12 for hBD-2 dimer.

Free Energy Study on HBD-3 Translocation on Lipid Membrane

Human defensins are a class of antimicrobial peptides that are crucial components of the innate immune system. Human β defensin type 3 (hBD-3) has been discovered recently, and has a charge density of +11. It includes 3 pairs of intramolecular disulfide bonds, which can break and converts hBD-3 into the linear analog form under reducing conditions. hBD-3 can selectively disrupt bacterial lipid membrane, cause cell leakage thus kill the bacterial cell. But the structural basis for the membrane-disrupting function of hBD is still unknown. In order to understand the interaction mechanism of hBD-3 with zwitterionic (but charge-neutral) lipid membrane, umbrella-sampling simulations were performed on the hBD-3 wildtype as well as the linear analog, both in the monomer and dimer forms. Based on the free energy calculation, both hBD-3 wildtype and linear analog need to overcome a very high energy barrier in order to translocate through the neutrally charged lipid bilayer. hBD-3 without disulfides is much more flexible than the wildtype, and thus the hBD-3 analog can pass through the lipid bilayer relatively easier energetically. Furthermore, forming a dimer can also help to decrease the energy barrier significantly. Based on this simple model, forming a higher order oligomer at high concentration is necessary for hBD-3 to transpass the normal cell lipid membrane.

The Arabidopsis defensin gene, AtPDF1.1, mediates defence against Pectobacterium carotovorum subsp. carotovorum via an iron-withholding defence system

Plant defensins (PDFs) are cysteine-rich peptides that have a range of biological functions, including defence against fungal pathogens. However, little is known about their role in defence against bacteria. In this study, we showed that the protein encoded by ARABIDOPSIS THALIANA PLANT DEFENSIN TYPE 1.1 (AtPDF1.1) is a secreted protein that can chelate apoplastic iron. Transcripts of AtPDF1.1 were induced in both systemic non-infected leaves of Arabidopsis thaliana plants and those infected with the necrotrophic bacterium Pectobacterium carotovorum subsp. carotovorum (Pcc). The expression levels of AtPDF1.1 with correct subcellular localization in transgenic A. thaliana plants were positively correlated with tolerance to Pcc, suggesting its involvement in the defence against this bacterium. Expression analysis of genes associated with iron homeostasis/deficiency and hormone signalling indicated that the increased sequestration of iron by apoplastic AtPDF1.1 overexpression perturbs iron homeostasis in leaves and consequently activates an iron-deficiency-mediated response in roots via the ethylene signalling pathway. This in turn triggers ethylene-mediated signalling in systemic leaves, which is involved in suppressing the infection of necrotrophic pathogens. These findings provide new insight into the key functions of plant defensins in limiting the infection by the necrotrophic bacterium Pcc via an iron-deficiency-mediated defence response.

Different dynamics and pathway of disulfide bonds reduction of two human defensins, a molecular dynamics simulation study.

Human defensins are a class of antimicrobial peptides that are crucial components of the innate immune system. Both human α defensin type 5 (HD5) and human β defensin type 3 (hBD-3) have 6 cysteine residues which form 3 pairs of disulfide bonds in oxidizing condition. Disulfide bond linking is important to the protein structure stabilization, and the disulfide bond linking and breaking order have been shown to influence protein function. In this project, microsecond long molecular dynamics simulations were performed to study the structure and dynamics of HD5 and hBD-3 wildtype and analogs which have all 3 disulfide bonds released in reducing condition. The structure of hBD-3 was found to be more dynamic and flexible than HD5, based on RMSD, RMSF, and radius of gyration calculations. The disulfide bridge breaking order of HD5 and hBD-3 in reducing condition was predicted by two kinds of methods, which gave consistent results. It was found that the disulfide bonds breaking pathways for HD5 and hBD-3 are very different. The breaking of disulfide bonds can influence the dimer interface by making the dimer structure less stable for both kinds of defensin. In order to understand the difference in dynamics and disulfide bond breaking pathway, hydrophilic and hydrophobic accessible surface areas (ASA), buried surface area between cysteine pairs, entropy of cysteine pairs, and internal energy were calculated. Comparing to the wildtype, hBD-3 analog is more hydrophobic, while HD5 is more hydrophilic. For hBD-3, the disulfide breaking is mainly entropy driven, while other factors such as the solvation effects may take the major role in controlling HD5 disulfide breaking pathway. Proteins 2017; 85:665-681. © 2016 Wiley Periodicals, Inc.

Abstract LB-288: Investigation on the interaction between human beta defensin type 3 (hBD-3) and ectodomain of EphA2 receptor using molecular dynamics simulation method

Abstract Defensins are a family of cationic cysteine-rich small molecules. In general, human beta defensins (hBD) are produced by epithelial cells, and they are important to the human innate immune system. Experimental results showed that defensins can directly bind with receptors such as chemokine receptors and act through several mechanisms that may trigger antitumor immunity or facilitate the host's immune response (or evasion) and promote tumor progression. hBD type 3 (hBD-3) is newly discovered and is unique that it has a net charge density much higher than other family members, and has the ability to undergo oligomerization. On the other hand, EPH receptors and their corresponding ligands (ephrins) are membrane-bound proteins that require direct cell-cell interactions for the activation of the receptors. Eph-ephrin system plays a critical role in tumor and vascular functions during carcinogenesis. Once EPH receptor A2 (EphA2) is ligand-independent, it can over-express itself in many types of tumors, and behave like an oncogene. Besides, many studies suggested that mutations that affect EPH receptor function play a role in cancer initiation or progression. Therefore, the EphA2-ephrin5 system can be a novel target for the development of anti-tumorigenic and anti-angiogenic therapies, and understanding its interaction with other molecules is important to demystify the cancer mechanism. Both EphA2 ectodomain (ECD) and hBD-3 exist outside of the cell membrane. In order to find out the interaction mechanism of hBD-3 with EphA2 receptor, initially blind docking using Cluspro software was performed to predict all the possible binding sites between hBD-3 oligomer and EphA2 ECD in the free and bound forms. After that, all atom molecular dynamics CHARMM simulations were performed to find out the most probable binding interfaces between hBD-3 oligomer and EphA2 ECD. It was found that hBD-3 has multiple stable binding sites on the EphA2 ECD, and its binding on the ligand-binding domain (LBD) of EphA2 could influence the binding of ephrin5 with EphA2. Those suggest the hBD-3 could play a role in cancer initiation. Citation Format: Liqun Zhang. Investigation on the interaction between human beta defensin type 3 (hBD-3) and ectodomain of EphA2 receptor using molecular dynamics simulation method. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-288.

Evolutionary tinkering of the expression of PDF1s suggests their joint effect on zinc tolerance and the response to pathogen attack

Multigenic families of Plant Defensin type 1 (PDF1) have been described in several species, including the model plant Arabidopsis thaliana as well as zinc tolerant and hyperaccumulator A. halleri. In A. thaliana, PDF1 transcripts (AtPDF1) accumulate in response to pathogen attack following synergic activation of ethylene/jasmonate pathways. However, in A. halleri, PDF1 transcripts (AhPDF1) are constitutively highly accumulated. Through an evolutionary approach, we investigated the possibility of A. halleri or A. thaliana species specialization in different PDF1s in conveying zinc tolerance and/or the response to pathogen attack via activation of the jasmonate (JA) signaling pathway. The accumulation of each PDF1 from both A. halleri and A. thaliana was thus compared in response to zinc excess and MeJA application. In both species, PDF1 paralogues were barely or not at all responsive to zinc. However, regarding the PDF1 response to JA signaling activation, A. thaliana had a higher number of PDF1s responding to JA signaling activation. Remarkably, in A. thaliana, a slight but significant increase in zinc tolerance was correlated with activation of the JA signaling pathway. In addition, A. halleri was found to be more tolerant to the necrotrophic pathogen Botrytis cinerea than A. thaliana. Since PDF1s are known to be promiscuous antifungal proteins able to convey zinc tolerance, we propose, on the basis of the findings of this study, that high constitutive PDF1 transcript accumulation in A. halleri is a potential way to skip the JA signaling activation step required to increase the PDF1 transcript level in the A. thaliana model species. This could ultimately represent an adaptive evolutionary process that would promote a PDF1 joint effect on both zinc tolerance and the response to pathogens in the A. halleri extremophile species.

Antibacterial peptides: basic facts and emerging concepts.

Antibacterial peptides are the effector molecules of innate immunity. Generally they contain 15-45 amino acid residues and the net charge is positive. The cecropin type of linear peptides without cysteine were found first in insects, whilst the defensin type with three disulphide bridges were found in rabbit granulocytes. Now a database stores more than 800 sequences of antibacterial peptides and proteins from the animal and plant kingdoms. Generally, each species has 15-40 peptides made from genes, which code for only one precursor. The dominating targets are bacterial membranes and the killing reaction must be faster than the growth rate of the bacteria. Some antibacterial peptides are clearly multifunctional and an attempt to predict this property from the hydrophobicity of all amino acid side chains are given. Gene structures and biosynthesis are known both in the fruit fly Drosophila and several mammals. Humans need two classes of defensins and the cathelicidin-derived linear peptide LL-37. Clinical cases show that deficiencies in these peptides give severe symptoms. Examples given are morbus Kostmann and atopic allergy. Several antibacterial peptides are being developed as drugs.