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

<p>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.</p

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

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

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

Comparison of the Interaction and Structure of Lignin in Pure Systems and in Asphalt Media by Molecular Dynamics Simulations

Lignin is a class of organic aromatic polymers contributing to the rigidity and strength of plants and has been proposed as a modifier to improve asphalt performance on road pavement. However, contradicting experimental results on the lignin miscibility in asphalt were found from different studies, and lignin has been found to self-assemble in different solutions. Thus, investigating the interaction and microstructure of lignin in asphalt media in molecular detail is necessary. Molecular dynamics (MD) simulations using both the LAMMPS program with the OPLS-aa force field and the NAMD program with the CHARMM force field have been conducted on pure lignin (including lignin monomer, dimer, and polymer with 17 and 31 units) and their mixtures with model asphalt molecules at different temperatures. Consistent results were observed from both programs and force fields in terms of density, hydrogen bonds, diffusion coefficient, radius of gyration, and radial distribution function. Glass transition was observed in the pure lignin systems based on density and diffusion coefficient calculations at different temperatures. Lignin can form intramolecular hydrogen bonds and intermolecular hydrogen bonds with other lignin and 1,7-dimethylnapthalene in the asphalt mixture, which has dependence on temperature and lignin chain length. Correlating the lignin size and chain length using the power-law relationship showed that lignin polymers in pure systems are in quasi-relaxed structures at different temperatures; lignin molecules stay in quasi-relaxed structures in asphalt mixtures at high temperatures but in collapsed structures at low temperatures. Implementing lignin monomer, dimer, and polymer into the model asphalt mixture can improve its density. Although lignin in different chain lengths aggregates in asphalt, lignin can modify the packing between different components in asphalt media at different temperatures. The work suggests that temperature can significantly influence the miscibility of lignin polymer in asphalt and that lignin can function as both a modifier and a resin in asphalt

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

Molecular Dynamics Simulations Reveal Isoform Specific Contact Dynamics between the Plexin Rho GTPase Binding Domain (RBD) and Small Rho GTPases Rac1 and Rnd1

The Plexin family of transmembrane receptors are unique in that their intracellular region interacts directly with small GTPases of the Rho family. The Rho GTPase binding domain of plexin (RBD)which is responsible for these interactionscan bind with Rac1 as well as Rnd1 GTPases. GTPase complexes have been crystallized with the RBDs of plexinA1, -A2, and -B1. The protein association is thought to elicit different functional responses in a GTPase and plexin isoform specific manner, but the origin of this is unknown. In this project, we investigated complexes between several RBD and Rac1/Rnd1 GTPases using multimicrosecond length all atom molecular dynamics simulations, also with reference to the free forms of the RBDs and GTPases. In accord with the crystallographic data, the RBDs experience more structural changes than Rho-GTPases upon complex formation. Changes in protein dynamics and networks of correlated motions are revealed by analyzing dihedral angle fluctuations in the proteins. The extent of these changes differs between the different RBDs and also between the Rac1 and Rnd1 GTPases. While the RBDs in the free and bound states have similarif not decreasedcorrelations, correlations within the GTPases are increased upon binding. Mapping highly correlated residues to the structures, it is found that the plexinA1, -B1, and -A2 RBDs all have similar communication pathways within the ubiquitin fold, but that different residues are involved. Dynamic network analyses indicate that plexinA1 and -B1 RBDs interact with small GTPases in a similar manner, whereas complexes with the plexinA2 RBD display different features. Importantly complexes with Rnd1 have a considerable number of dynamic correlations and network connections between the proteins, whereas such features are missing in the RBD–Rac1 complexes. Overall, the simulations suggest mechanisms that are consistent with the experimental data on plexinB1 and indicate RBD and GTPase isoform specific changes in protein dynamics upon complex formation

Analyzing Properties of Model Asphalts Using Molecular Simulation

Molecular simulations have been used to estimate the properties of three-component mixtures whose constituents were chosen to represent the chemical families found in paving asphalts. Naphthene aromatics and saturates were represented by 1,7-dimethylnaphthalene and n-C22, respectively. Two different asphaltene model structures were considered. The first has a large aromatic core with a few short side chains; the second contains a moderate size aromatic core with larger branches. Both types have been proposed in the recent literature based on experimental characterizations of asphaltene fractions. Properties calculated from atomistic molecular simulations of the mixtures include density and isothermal compressibility (inverse of bulk modulus). The thermodynamic properties suggest a high-frequency glass transition above 25 °C for both model mixtures. The mixture based on the more aromatic asphaltene shows a more pronounced transition and has a higher bulk modulus. For a polymer-modified model asphalt, the calculations are consistent with increases in the bulk modulus

ΔΔG predicted from FEP in comparison with available literature data.

ΔΔG predicted from FEP in comparison with available literature data.</p