Solution NMR Studies of Interactions of Ligands With Plasma Proteins

配体与血浆蛋白相互作用的溶液核磁共振研究

• 批准号: RGPIN-2014-04514
• 负责人: Melacini, Giuseppe
• 金额: $ 5.17万
• 依托单位: McMaster University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=659503
• 关键词: Solution; NMR; Studies; Interactions; Ligands

Plasma proteins, although well known as carriers of serum solutes (e.g. fatty acids), have been recently shown to function also as extra-cellular chaperones, which inhibit the aggregation of unfolded proteins and amyloidogenic peptides (e.g. Aß). The long-term goal of our research program is to define fundamental molecular mechanisms governing ligand binding and aggregation inhibition by plasma proteins. My laboratory has made significant contributions to the current understanding of how the prototypical human serum albumin (HSA) acts as the most potent inhibitor of Aß fibrillization in plasma. Using a multidisciplinary combination of nuclear magnetic resonance (NMR) spectroscopy and other biophysical and biochemical approaches (e.g. fluorescence, dynamic light scattering, electron microscopy and site-directed mutagenesis), we have shown that HSA prevents the Aß peptide from forming insoluble amyloid fibrils by selectively binding to soluble Aß oligomers and competing with the further addition of Aß monomers. We have determined the stoichiometry and the affinities of the HSA - Aß oligomer complexes and through comparative mutational analyses we have shown that HSA recognizes the Aß oligomers through sites that are unique and distinct from those interacting with low molecular weight (MW) ligands. Our preliminary data suggest the hypothesis that the critical contacts with the Aß oligomers are mediated by flexible loops of HSA with partial sequence homology to Aß. However, the current knowledge about the location of the Aß oligomer binding sites within HSA is at best scant. Our first short-term objective will therefore be to: (i) Map at single-residue resolution the binding sites within HSA for the Aß oligomers, by combining the multidisciplinary approach of our previous publications with new NMR experiments designed to probe reversible interactions with the Aß oligomers. This will be the first time that binding sites of a protein for oligomers of an amyloidogenic peptide are mapped at single-residue resolution. In the long term, this objective will be extended to a plethora of other amyloidogenic peptides and other extracellular chaperones. We will also elucidate the mechanisms underlying the other primary function of plasma proteins, i.e. ligand transport. The structures of ligand-bound HSA reveal the HSA architecture and the location of the binding sites for low MW ligands. However, structures alone cannot address several outstanding questions about ligand binding. How do ligands access buried binding sites? How does conformational entropy drive ligand binding? How are the apo conformational pre-equilibria allosterically coupled to ligand binding? To address these fundamental questions our second objective will be to: (ii) Comparatively analyze the dynamics of HSA in the absence and presence of fatty acids and other ligands, benefiting from recent NMR advances. These include an approach we pioneered to map otherwise elusive allosteric networks through the covariance analysis of NMR chemical shifts. We will start with the analysis of isolated HSA domains and will expand to progressively longer HSA constructs to reveal how dynamics modulates ligand binding, gating and allostery. The preliminary data available so far indicate that both objectives are feasible in terms of available materials and of experimental methods, capitalizing on the NMR approaches employed in our past publications. The resulting program will have a broad impact that goes well beyond HSA, as it will reveal general molecular mechanisms for the extra-cellular chaperones and for the allosteric coupling of ligand binding to protein dynamics. The impact will be both scientific and educational, as the proposed program provides a unique HQP training opportunity.

血浆蛋白虽然是众所周知的血清溶质(例如脂肪酸)的载体，但最近被证明也具有细胞外伴侣的功能，它抑制未折叠的蛋白质和淀粉样多肽(例如A？)的聚集。我们研究计划的长期目标是确定控制配体结合和血浆蛋白抑制聚集的基本分子机制。我的实验室对目前理解典型的人血清白蛋白(HSA)如何作为血浆中最有效的A？纤化抑制因子做出了重大贡献。利用核磁共振光谱和其他生物物理和生化方法(如荧光、动态光散射、电子显微镜和定点突变)的多学科组合，我们已经证明，HSA通过选择性地与可溶低聚物结合并与进一步添加的A？单体竞争，防止A？肽形成不溶的淀粉样原纤维。我们测定了HSA-A？低聚物复合体的化学计量比和亲和力，并通过比较突变分析表明，HSA通过与低分子量(MW)配体相互作用的独特和不同的位点识别A？低聚物。我们的初步数据表明，与A？寡聚体的关键接触是由部分序列与A？同源的HSA柔性环介导的。然而，目前对低聚物结合位点在HSA中的位置的了解充其量是很少的。因此，我们的第一个短期目标将是：(I)通过将我们以前发表的多学科方法与新的核磁共振实验相结合，在单一残基分辨率下映射HSA中A？低聚物的结合位置，以探索与A？低聚物的可逆相互作用。这将是第一次在单一残基分辨率下绘制蛋白质与淀粉样肽低聚体的结合位点图。从长远来看，这一目标将扩展到过多的其他淀粉样多肽和其他细胞外伴侣蛋白。我们还将阐明血浆蛋白的另一主要功能，即配体运输的潜在机制。配体结合人血清白蛋白的结构揭示了低分子量配体的人血清白蛋白结构和结合位置。然而，单靠结构不能解决有关配体结合的几个悬而未决的问题。配体如何访问埋藏的结合位点？构象熵是如何驱动配体结合的？载脂蛋白构象预平衡是如何以变构方式与配体结合的？为了解决这些基本问题，我们的第二个目标将是：(Ii)比较分析在没有和存在脂肪酸和其他配体的情况下，受益于最近的核磁共振进展的HSA的动力学。其中包括我们首创的一种方法，通过对核磁共振化学位移的协方差分析，绘制出原本难以捉摸的变构网络。我们将从分离的HSA结构域的分析开始，并将扩展到逐渐变长的HSA结构，以揭示动力学如何调节配体结合、门控和变构。到目前为止获得的初步数据表明，这两个目标在现有材料和实验方法方面都是可行的，利用了我们过去出版物中采用的核磁共振方法。由此产生的计划将产生远远超出HSA的广泛影响，因为它将揭示细胞外伴侣以及配体与蛋白质动力学结合的变构耦合的一般分子机制。其影响将是科学的和教育的，因为拟议的计划提供了一个独特的HQP培训机会。