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: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=611082
• 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.

血浆蛋白虽然众所周知是血清溶质（例如脂肪酸）的载体，但最近被证明也可以作为细胞外伴侣，抑制未折叠蛋白和淀粉样肽（例如 Aß）的聚集。我们研究计划的长期目标是确定血浆蛋白控制配体结合和聚集抑制的基本分子机制。我的实验室为目前了解原型人血清白蛋白 (HSA) 如何充当血浆中最有效的 Aß 纤维化抑制剂做出了重大贡献。通过核磁共振 (NMR) 光谱和其他生物物理和生化方法（例如荧光、动态光散射、电子显微镜和定点诱变）的多学科组合，我们发现 HSA 通过选择性结合可溶性 Aß 寡聚物并与 进一步添加Aß单体。我们已经确定了 HSA - Aß 寡聚物复合物的化学计量和亲和力，并且通过比较突变分析，我们表明 HSA 通过独特且不同于与低分子量 (MW) 配体相互作用的位点来识别 Aß 寡聚物。我们的初步数据表明，与 Aß 寡聚体的关键接触是由与 Aß 具有部分序列同源性的 HSA 柔性环介导的。然而，目前关于 Aß 寡聚体结合位点在 HSA 内的位置的知识充其量也很少。因此，我们的第一个短期目标是：（i）通过将我们之前出版物的多学科方法与旨在探测与 Aß 寡聚物的可逆相互作用的新 NMR 实验相结合，以单残基分辨率绘制 HSA 内 Aß 寡聚物的结合位点。这将是首次以单残基分辨率绘制蛋白质与淀粉样肽寡聚体的结合位点。从长远来看，这一目标将扩展到大量其他淀粉样蛋白肽和其他细胞外伴侣。我们还将阐明血浆蛋白其他主要功能（即配体转运）的机制。配体结合的 HSA 的结构揭示了 HSA 的结构和低分子量配体的结合位点的位置。然而，结构本身并不能解决有关配体结合的几个突出问题。配体如何进入埋藏的结合位点？构象熵如何驱动配体结合？ apo 构象预平衡如何与配体结合变构耦合？为了解决这些基本问题，我们的第二个目标是：(ii) 受益于最近的 NMR 进展，比较分析在不存在和存在脂肪酸和其他配体的情况下 HSA 的动力学。其中包括我们首创的一种方法，通过 NMR 化学位移的协方差分析来绘制原本难以捉摸的变构网络。我们将从分析分离的 HSA 结构域开始，逐步扩展到更长的 HSA 结构，以揭示动力学如何调节配体结合、门控和变构。迄今为止可用的初步数据表明，利用我们过去出版物中采用的核磁共振方法，这两个目标在可用材料和实验方法方面都是可行的。由此产生的程序将产生远远超出 HSA 范围的广泛影响，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偶联的一般分子机制。其影响将是科学性和教育性的，因为拟议的计划提供了独特的 HQP 培训机会。