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.

流感病毒糖蛋白血凝素（HA）的HA2结构域的N-末端23个残基中除5个之外的所有残基在HA基因的所有16种血清型中是严格保守的。 这种HA2融合肽（HAfp）的结构和功能仍然是广泛的生物物理、计算和功能分析的焦点，但这些分析中的大多数是不包括严格保守的残基Trp21-Tyr22-Gly23的肽。 我们的异源三重共振核磁共振研究全长HAfp血清亚型H1，溶解在十二烷基磷脂酰胆碱（DPC），以前揭示了一个非常紧密的螺旋发夹结构，其N-末端α-螺旋（Gly1-Glu11）紧密包装对第二个α-螺旋（Trp14-Gly23），与六个保守的Gly残基在螺旋间的接口。 第七个保守的Gly残基在位置13采取一个积极的phi角，使发夹的转折，连接两个螺旋。 该结构被认为是稳定的多个脂肪族螺旋间的CaH到C = O氢键，其特征在于强螺旋间HN-Ha;和Ha-Hb NOE接触。 现在已经确定了这种发夹结构的额外稳定力的新证据，即N-末端Gly 1氨基和螺旋2的偶极矩之间的强电荷偶极相互作用。 使用基于新型亚甲基-TROSY的3D NMR实验的氨基末端15 N共振的pH滴定和Gly 113 C '的观察显示出8.8的强烈升高的pK值，显著高于亲脂性环境中N末端氨基的预期。 螺旋2的三个C-末端羰基碳的化学位移与Gly1-N的质子化状态滴定，指示N-末端氨基与螺旋2的轴之间的紧密接近，从而提供反平行发夹折叠的最佳电荷偶极稳定。Glu11和Asp19的侧链羧酸基团的pK值分别比水溶性蛋白质中溶剂暴露侧链的常见值高约1和0.5单位，表明介电常数分别为30（Glu11）和60（Asp19），这将这些基团置于磷脂胶束的头基区域。 生物膜呈现高度流动的环境，并且蛋白质在这种膜内的整合本身是高度动态的：蛋白质在膜平面内横向扩散，并且围绕该平面的法向矢量旋转。我们已经发现，全身运动的脂质双层内的蛋白质可以确定从NMR 15 N弛豫速率收集不同大小的bicelles。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尖锐，如孔形成和融合诱导蛋白的情况。对于流感血凝素融合肽，它位于膜的表面上，催化膜和囊泡的融合，我们发现大振幅，刚体摆动运动的纳秒时间尺度相对于脂质双层。这种行为补充了先前的分析，其中数据通常根据融合肽相对于膜的静态倾斜插入角来解释。通过系统地改变一个物体的大小来定量地解开两个相互作用物体的相对运动，这适用于蛋白质-膜相互作用之外的广泛系统。 在低pH（4.0）下，在NMR谱中存在另一种低填充活化状态的瞬时存在的证据。 通过将弛豫和化学位移效应联系起来，我们发现了这种激活状态与肽的G8A突变体中高度聚集的状态相似的证据。 发现后一种结构作为20%的原始发夹结构与样品开放的剩余时间之间的平衡存在，更延伸的结构没有直接的螺旋间接触。 这些延伸的结构在尺寸上足以跨越膜，并且可能参与实际融合孔的形成。 我们的研究结果与早期研究的结果不同，早期研究的重点是融合肽的较短的20个残基的合成版本。 据报道，这20个残基的肽采用开放的回飞棒结构，这与我们在HA2序列的前23个残基中发现的封闭的螺旋发夹结构显著不同。通过研究较短版本的融合肽，我们发现，涉及残基21 - 23的几个关键相互作用的缺乏，稳定了封闭的螺旋发夹结构，导致20个残基的肽处于封闭和开放状态之间的动态平衡，采用了一种近似平衡。当被DPC胶束增溶时，前者的群体为11%。 短于20个残基的肽具有甚至更少的相互作用以稳定螺旋发夹折叠，导致发夹群体消失。 考虑到所有血清型中发夹稳定相互作用的保守性，以及膜融合所需的最少20个残基，我们假设闭合状态在融合过程中起着至关重要的作用。然而，这种发夹结构的开放似乎是在融合过程的最后阶段形成膜孔所必需的。