Studying Factors Affecting Human β Defensin Type 3 Crossing Model Bacterial Lipid Membranes

Human β defensin type 3 (hBD-3) is a cysteine-rich positively charged antimicrobial peptide (AMP), which is originally secreted from the human innate immune system. It has 45 residues, with one short alpha helix and three β sheets connected by 2 major loops. It can disrupt bacterial cell membranes, break the membrane and kill the bacteria. Because the chemical compositions of bacterial membranes are stable, the bacteria-killing by hBD-3 will not cause drug resistance. In order to design novel drugs based on hBD-based natural antimicrobial peptides, understanding their functional mechanism in molecular detail is important. In this study, factors affecting the capabilities of hBD-3 breaking different model bacterial membranes were studied using molecular dynamics simulations. hBD-3 monomer and dimer in both wildtype and linear analog forms were embedded in model Gram-positive and Gram-negative bacterial lipid membranes represented by a mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) and 1-Palmitoyl-2-oleoylsn-glycero-3-phosphoglycerol (POPG) lipids at a molar ratio of 1:3 and 3:1 respectively, plus a model lipid bilayer having 1-palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine (POPC) mixed with 10% of Phosphatidylinositol 4,5-bisphosphate (PIP2). Based on microsecond-long all-atom molecular dynamics simulation trajectories, the distance map between protein and lipids was analyzed, which shows that hBD3 can bind with both kinds of model bacterial membranes on similar binding sites stably. During the long-term simulations, aggregation of negatively charged lipids towards hBD-3 dimer was observed based on the distance histograms comparison, and the reorientation of lipid heads towards hBD-3 monomer and dimer was detected based on the distance maps between hBD-3 and lipid heads, tails individually. Also, there are water molecules translocating through the lipid bilayers induced by hBD-3, and more water molecules translocated in the hBD-3 dimer systems than in the monomer systems. hBD-3 monomer can move to the surface of the lipid bilayer during microsecond-long simulations especially in the Gram-negative lipid bilayer and at high salt concentrations, although hBD-3 dimer prefers to span through the membrane in a back-to-back structure. Based on the order parameter calculation on lipids, hBD-3 disrupted the surrounding POPE and POPG lipids with similar significance. In order to find out the effect of different factors, such as oligomerization, salt concentration, disulfide bonding status, and membrane chemical composition, on the structure and dynamics of hBD-3 embedding inside model bacterial membranes, principal component analysis (PCA) was conducted based on the contact degree of freedom calculation. It was found that the major conformational variance corresponds to the expansion of hBD-3 monomer inside lipid membrane, while the major conformational variance in hBD-3 dimers corresponds to the dissociation of the hBD-3 dimer inside the membrane. Adding salt can break the contacts inside hBD-3 monomer, while forming contacts between hBD-3 and lipids. Adding salt can contribute to the dissociation of the hBD-3 dimer and the formation of local contacts. With the breaking of disulfide bonds, the contacts inside hBD-3 monomer break. With the breaking of disulfide bonds, the contacts inside hBD-3 dimer also break. As the chemical composition of lipid membrane changes from POPC+10%PIP2 to Gram-positive or Gram-negative, the head of hBD-3 monomer moves from the β2 sheet to the loop region. Our results provide unique insights into the structural and dynamic characteristics of hBD-3 translocating through model bacterial membranes and factors contributing to the disruption and translocation process. These findings can help in understanding the bacteria-killing process of hBD-3 with molecular details, offering valuable clues for the design of novel AMP-based antibiotics.