Di Marino Daniele, Bruno Agostino, Grimaldi Manuela, Scrima Mario, Stillitano Ilaria, Amodio Giuseppina, Della Sala Grazia, Romagnoli Alice, De Santis Augusta, Moltedo Ornella, Remondelli Paolo, Boccia Giovanni, D'Errico Gerardino, D'Ursi Anna Maria, Limongelli Vittorio
Department of Life and Environmental Sciences, New York-Marche Structural Biology Center (NY-MaSBiC), Polytechnic University of Marche, Ancona, Italy.
Department of Pharmacy, University of Naples "Federico II", Naples, Italy.
Front Chem. 2020 Jun 26;8:493. doi: 10.3389/fchem.2020.00493. eCollection 2020.
Gp36 is the virus envelope glycoproteins catalyzing the fusion of the feline immunodeficiency virus with the host cells. The peptide C8 is a tryptophan-rich peptide corresponding to the fragment W-I of gp36 exerting antiviral activity by binding the membrane cell and inhibiting the virus entry. Several factors, including the membrane surface charge, regulate the binding of C8 to the lipid membrane. Based on the evidence that imperceptible variation of membrane charge may induce a dramatic effect in several critical biological events, in the present work we investigate the effect induced by systematic variation of charge in phospholipid bilayers on the aptitude of C8 to interact with lipid membranes, the tendency of C8 to assume specific conformational states and the re-organization of the lipid bilayer upon the interaction with C8. Accordingly, employing a bottom-up multiscale protocol, including CD, NMR, ESR spectroscopy, atomistic molecular dynamics simulations, and confocal microscopy, we studied C8 in six membrane models composed of different ratios of zwitterionic/negatively charged phospholipids. Our data show that charge content modulates C8-membrane binding with significant effects on the peptide conformations. C8 in micelle solution or in SUV formed by DPC or DOPC zwitterionic phospholipids assumes regular β-turn structures that are progressively destabilized as the concentration of negatively charged SDS or DOPG phospholipids exceed 40%. Interaction of C8 with zwitterionic membrane surface is mediated by Trp1 and Trp4 that are deepened in the membrane, forming H-bonds and cation-π interactions with the DOPC polar heads. Additional stabilizing salt bridge interactions involve Glu2 and Asp3. MD and ESR data show that the C8-membrane affinity increases as the concentration of zwitterionic phospholipid increases. In the lipid membrane characterized by an excess of zwitterionic phospholipids, C8 is adsorbed at the membrane interface, inducing a stiffening of the outer region of the DOPC bilayer. However, the bound of C8 significantly perturbs the whole organization of lipid bilayer resulting in membrane remodeling. These events, measurable as a variation of the bilayer thickness, are the onset mechanism of the membrane fusion and vesicle tubulation observed in confocal microscopy by imaging zwitterionic MLVs in the presence of C8 peptide.
Gp36是催化猫免疫缺陷病毒与宿主细胞融合的病毒包膜糖蛋白。肽C8是一种富含色氨酸的肽，对应于gp36的W-I片段，通过结合细胞膜并抑制病毒进入发挥抗病毒活性。包括膜表面电荷在内的几个因素调节C8与脂质膜的结合。基于膜电荷的细微变化可能在几个关键生物学事件中产生显著影响的证据，在本研究中，我们研究了磷脂双层中电荷的系统变化对C8与脂质膜相互作用能力、C8呈现特定构象状态的趋势以及与C8相互作用后脂质双层的重组所诱导的影响。因此，我们采用自下而上的多尺度方案，包括圆二色光谱（CD）、核磁共振（NMR）、电子自旋共振光谱（ESR）、原子分子动力学模拟和共聚焦显微镜，研究了由不同比例的两性离子/带负电荷的磷脂组成的六种膜模型中的C8。我们的数据表明，电荷含量调节C8与膜的结合，对肽构象有显著影响。在由DPC或DOPC两性离子磷脂形成的胶束溶液或小单层囊泡（SUV）中，C8呈现规则的β-转角结构，随着带负电荷的SDS或DOPG磷脂浓度超过40%，这种结构会逐渐不稳定。C8与两性离子膜表面的相互作用由Trp1和Trp4介导，它们深入膜内，与DOPC极性头部形成氢键和阳离子-π相互作用。额外的稳定盐桥相互作用涉及Glu2和Asp3。分子动力学（MD）和ESR数据表明，C8与膜的亲和力随着两性离子磷脂浓度的增加而增加。在以过量两性离子磷脂为特征的脂质膜中，C8吸附在膜界面，导致DOPC双层外层区域变硬。然而，C8的结合显著扰乱了脂质双层的整体组织，导致膜重塑。这些事件可通过双层厚度的变化来衡量，是在共聚焦显微镜下通过对存在C8肽的两性离子多层囊泡（MLV）成像观察到的膜融合和囊泡微管形成的起始机制。
