Department of Chemical Engineering, Tennessee Technological University, Cookeville, Tennessee 38505, United States.
J Chem Inf Model. 2021 Sep 27;61(9):4670-4686. doi: 10.1021/acs.jcim.1c00805. Epub 2021 Sep 2.
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--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--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.
人β防御素 3（hBD-3）是一种富含半胱氨酸的小分子抗菌肽。它属于人体先天免疫系统。hBD-3 有 6 个半胱氨酸残基，形成 3 对二硫键，这些键在还原条件下断裂。已知 hBD-3 可以与细菌膜甚至真核细胞膜相互作用，而这些细胞膜中含有低浓度的磷脂酰肌醇 4,5-二磷酸（PIP2）脂质。PIP2 是细胞膜的重要组成部分，研究发现它在抗菌肽（AMP）与膜相互作用过程中发挥着重要作用。为了了解 hBD-3 与含 PIP2 细胞膜相互作用的功能机制，使用两种方法预测了 hBD-3 在 1-棕榈酰基-2-油酰基--甘油-3-磷酸胆碱（POPC）双层膜与 10%PIP2 混合时与膜的结合结构。第一种方法是将 hBD-3 单体以不同的取向放置在 POPC+10%PIP2 膜上方，建立五个不同的初始模拟系统，并对每个系统进行长时间模拟，以预测最稳定的结合结构。结果表明，hBD-3 类似物结合在混合脂质膜的两个环区上。第二种方法是从固态核磁共振（ssNMR）建议的取向开始，对一个或九个 hBD-3 二聚体在与 10%PIP2 脂质双层混合的 POPC 上的结合进行长时间模拟。二聚体解离，野生型 hBD-3 在混合膜上的最稳定结合也是通过两个环区，这与第一种方法和脂质自组装结果的预测一致。PIP2 脂质可以与 hBD-3 上带正电荷的残基（如 Arg 和 Lys）形成持久的氢键，从而与 hBD-3 形成簇。相比之下，还研究了 hBD-3 与上有 1,2-棕榈酰基-油酰基--甘油-3-磷酸丝氨酸（POPS）、下有 POPC 的双层结合以及混合有 10%PIP2 的 POPS+POPC 双层的结合。长时间模拟结果表明，hBD-3 可以与带负电荷的 POPS 和 PIP2 脂质的头部结合并形成氢键。hBD-3 在 PIP2 或 POPS 混合双层上的稳定结合位点仍在两个环区。在与 10%PIP2 混合的 POPS+POPC 双层上，由于与 POPS 脂质的结合竞争，hBD-3 与 PIP2 脂质的结合变得不稳定且减少。此外，结合 hBD-3 可以降低 POPC+PIP2、POPS+POPC 和 POPS+POPC+PIP2 双层的膜厚度，并根据序参数计算使 POPS 和 PIP2 脂质更具柔韧性。我们的结果提供了 AMP 与不同膜结合的分子见解，并有助于理解 hBD-3 破坏含 PIP2 细胞膜的功能机制。
