Study of hemagglutinin membrane fusion domain

血凝素膜融合结构域的研究

• 批准号: 8741545
• 负责人: Ad Bax
• 金额: $ 16.92万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8741545
• 关键词: Acute; Adopted; Behavior; Biological; Biological Process; C-terminal; Carbon; Charge; Chemicals; Choline; Complement; Data Analyses; Diffuse; Environment; Equilibrium; Event; Genes; Goals; Hemagglutinin; Hydrogen Bonding; Influenza Hemagglutinin; Influenza Virus Hemagglutinin Glycoproteins; Length; Link; Lipid Bilayers; Liquid substance; Membrane; Membrane Fusion; Membrane Proteins; Micelles; Motion; N-terminal; Nature; Peptides; Phospholipids; Play; Population; Positioning Attribute; Process; Protein Dynamics; Proteins; Relative (related person); Relaxation; Reporting; Role; Sampling; Serotyping; Side; Solvents; Staging; Structure; Surface; System; Time; Titrations; Vesicle; Viral; Water; alpha helix; amino group; base; carbene; carboxylate; dipole moment; mutant; nanosecond; novel; protonation; research study; vector

All but five of the N-terminal 23 residues of the HA2 domain of the influenza virus glycoprotein hemagglutinin (HA) are strictly conserved across all 16 serotypes of HA genes. The structure and function of this HA2 fusion peptide (HAfp) continues to be the focus of extensive biophysical, computational, and functional analysis, but most of these analyses are of peptides that do not include the strictly conserved residues Trp21-Tyr22-Gly23. Our heteronuclear triple resonance NMR study of full length HAfp of sero subtype H1, solubilized in dodecylphosphatidyl choline (DPC), previously revealed a remarkably tight helical hairpin structure, with its N-terminal alpha-helix (Gly1-Glu11) packed tightly against its second alpha-helix (Trp14-Gly23), with six of the seven conserved Gly residues at the interhelical interface. The seventh conserved Gly residue in position 13 adopts a positive phi angle, enabling the hairpin turn that links the two helices. The structure was found to be stabilized by multiple aliphatic interhelical CaH to C=O hydrogen bonds, characterized by strong interhelical HN-Ha; and Ha-Hb NOE contacts. New evidence for an additional stabilizing force of this hairpin structure has now been identified, namely a strong charge dipole interaction between the N-terminal Gly1 amino group and the dipole moment of helix 2. pH titration of the amino-terminal 15N resonance, using a novel methylene-TROSY based 3D NMR experiment, and observation of Gly1 13C' show a strongly elevated pK value of 8.8, considerably higher than expected for an N-terminal amino group in a lipophilic environment. Chemical shifts of three C-terminal carbonyl carbons of helix 2 titrate with the protonation state of Gly1-N, indicative of a close proximity between the N-terminal amino group and the axis of helix 2, thereby providing an optimal charge-dipole stabilization of the antiparallel hairpin fold. pK values of the side chain carboxylate groups of Glu11 and Asp19 are higher by about one and 0.5 unit, respectively, than commonly seen for solvent-exposed side chains in water-soluble proteins, indicative of dielectric constants of epsilon = 30 (Glu11) and epsilon= 60 (Asp19), which places these groups in the headgroup region of the phospholipid micelle. Biological membranes present a highly fluid environment and integration of proteins within such membranes is itself highly dynamic: proteins diffuse laterally within the plane of the membrane, and rotationally about the normal vector of this plane. We have found that whole-body motions of proteins within a lipid bilayer can be determined from NMR 15N relaxation rates collected for different size bicelles. The importance of membrane integration and interaction is particularly acute for proteins and peptides that function on the membrane itself, as is the case for pore-forming and fusion-inducing proteins. For the influenza hemagglutinin fusion peptide, which lies on the surface of membranes and catalyzes the fusion of membranes and vesicles, we find large-amplitude, rigid-body wobbling motions on the nanosecond timescale relative to the lipid bilayer. This behavior complements prior analyses where data were commonly interpreted in terms of a static oblique angle of insertion for the fusion peptide with respect to the membrane. Quantitative disentanglement of the relative motions of two interacting objects by systematically varying the size of one is applicable to a wide range of systems beyond protein-membrane interactions. At low pH (4.0) there is evidence in the NMR spectrum of the transient presence of another lowly populated activated state. By linking relaxation and chemical shift effects, we have found evidence that this activated state is similar to a state that is highly populated in the G8A mutant of the peptide. This latter structure is found to exist as an equilibrium between 20% of the original hairpin structure, and for the remainder of the time samples open, more extended structures without direct interhelical contacts. These extended structures are sufficient in size to span the membrane, and may be involved in formation of the actual fusion pore. The results of our study differ relative to those of earlier studies that focused on a shorter, 20-residue synthetic version of the fusion peptide. This 20-residue peptide was reported to adopt an open boomerang structure that differs significantly from the closed helical-hairpin structure we found for the first 23 residues of the HA2 sequence. By studying shorter versions of the fusion peptide, we found that the absence of several key interactions involving residues 21-23, that stabilize the closed, helical-hairpin structure, causes the 20-residue peptide to be in a dynamic equilibrium between closed and open states, adopting a ca. 11% population of the former when solubilized by DPC micelles. Peptides shorter than 20 residues have even fewer interactions to stabilize a helical hairpin fold, resulting in a vanishing hairpin population. Considering the conserved nature of hairpin-stabilizing interactions across all serotypes, and the minimum of 20 residues needed for membrane fusion, we postulate that the closed state plays an essential role in the fusion process. However, opening of this hairpin structure appears essential to the formation of a membrane pore at the final stage of the fusion process.

除了在HA基因的所有16种血清型中，除了HA2病毒糖蛋白血凝蛋白（HA）的Ha2域的N末端23残基中，所有除5个残基中都严格保存。 该HA2融合肽（HAFP）的结构和功能仍然是广泛的生物物理，计算和功能分析的重点，但是这些分析中的大多数是肽的大多数，这些肽不包括严格保守的TRP21-TYR22-GLY23。 我们在十二烷基磷脂酰胆碱（DPC）中溶解的血清亚型H1的全长HAFP的异质三重共振NMR研究先前揭示了螺旋式发夹的结构，其N末端alpha-Helix（Gly1-GlU11）与Second Alpha-section Alpha the the the theyty（trply3）相对紧密，并与Section Alpha-nix（Gly1-glU11）相对。在螺旋间界面处的保守Gly残基。 位置13中的第七个保守的Gly残留物采用正Phi角，使发夹转弯可以连接两个螺旋。 发现该结构通过多个脂肪族间的CAH稳定在C = O氢键中，其特征在于强螺旋间HN-HA。和HA-HB NOE联系。 现在已经确定了这种发夹结构额外稳定力的新证据，即N末端Gly1氨基组与螺旋螺旋的偶极矩之间的强电荷偶极相互作用。氨基末端15N共振的PH滴定，使用新型基于甲基化的3D NMR实验的新型甲基元素较高的gly iffer a ivery ixter gy a ivery of 8.在亲脂环境中，N末端氨基群预计。 螺旋2滴定的三个C末端羰基碳与Gly1-N的质子化状态的化学位移，表明N末端氨基组与螺旋2的轴紧密接近，从而提供了对抗乳头发pin的最佳电荷 - 二极稳定。 GLU11和ASP19的侧链羧酸盐基团的PK值分别高约1和0.5单位，比在水溶性蛋白中溶剂暴露的侧链通常所见，表明Epsilon = 30（GLU11）和Epsilon = 60（ASP19）的PK = 60（ASP19），这些组合是这些组合，这些组合是这些组合，这些组合是这些组合的组合。 生物膜表现出高度流动的环境，并且在这种膜内蛋白质的整合本身是高度动态的：蛋白质在膜平面内横向扩散，并旋转围绕该平面的正常矢量。我们发现，可以通过针对不同尺寸的双皮细胞收集的NMR 15N弛豫率确定脂质双层中蛋白质的全身运动。膜整合和相互作用的重要性对于在膜本身上起作用的蛋白质和肽尤其是急性，就像孔形成和融合融合蛋白一样。对于位于膜表面并催化膜和囊泡融合的流感血凝素融合肽，我们发现纳米代数时尺度上相对于脂质胆剂的纳米秒时尺度上的大幅度，刚体的摇摆运动。这种行为补充了先前的分析，即通常根据膜相对于膜的融合肽的静态倾斜角度来解释数据。通过系统地变化一个相互作用对象的相对运动的定量分离适用于蛋白质 - 膜相互作用以外的各种系统。 在低pH值（4.0）下，在NMR光谱中有证据表明另一种较低的活化状态的瞬时存在。 通过将松弛和化学位移效应联系起来，我们发现了证据表明，这种激活状态类似于肽的G8A突变体中高度填充的状态。 发现后一种结构是在原始发夹结构的20％之间的平衡而存在的，在其余时间样本中，没有直接的螺旋间接触。 这些扩展的结构的大小足以跨越膜，并且可能参与了实际融合孔的形成。 我们研究的结果相对于较短的20个保留合成版的融合肽的研究结果不同。 据报道，这种20个残留的肽采用了开放的飞旋镖结构，该结构与我们在HA2序列的前23个残基中发现的封闭的螺旋发蛋白结构显着不同。通过研究融合肽的较短版本，我们发现缺乏涉及残基21-23的几种关键相互作用，从而稳定封闭的螺旋发质结构，这会导致20个沉积的肽处于封闭状态和开放状态之间的动态平衡，并采用了CA。通过DPC胶束溶解后，前者的人口为11％。 肽短于20个残留物的相互作用甚至更少以稳定螺旋发夹折叠，从而导致发夹的消失。 考虑到所有血清型中发夹稳定的相互作用的保守性质，以及膜融合所需的20个残基的最小值，我们假设封闭状态在融合过程中起着至关重要的作用。但是，这种发夹结构的打开对于在融合过程的最后阶段的膜孔的形成似乎至关重要。