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.

血浆蛋白虽然被认为是血清溶质（如脂肪酸）的载体，但最近也被证明具有细胞外伴侣的功能，可以抑制未折叠蛋白和淀粉样蛋白肽（如β）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。我的实验室对目前对典型的人血清白蛋白（HSA）如何作为血浆中最有效的α -纤维化抑制剂的理解做出了重大贡献。通过多学科结合核磁共振（NMR）波谱和其他生物物理和生化方法（如荧光、动态光散射、电子显微镜和定点诱变），我们已经证明，HSA通过选择性地结合可溶性asß寡聚物并与进一步添加的asß单体竞争，阻止了asß肽形成不溶性淀粉样蛋白原纤维。我们已经确定了HSA -亚ß寡聚物复合物的化学计量学和亲和性，并通过比较突变分析表明，HSA通过独特的位点识别亚ß寡聚物，这些位点与与低分子量配体相互作用的位点不同。我们的初步数据表明，与asas低聚物的关键接触是由部分序列与asas同源的HSA柔性环介导的。然而，目前关于HSA内亚ß寡聚物结合位点的位置的知识最多是很少的。因此，我们的第一个短期目标将是：(i)通过将我们之前发表的多学科方法与旨在探测与asas低聚物可逆相互作用的新NMR实验相结合，以单残基分辨率绘制HSA内asas低聚物的结合位点。这将是第一次在单残基分辨率下绘制淀粉样蛋白肽低聚物蛋白的结合位点。从长远来看，这一目标将扩展到过多的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的机制，即配体运输。配体结合的HSA结构揭示了低分子量配体的HSA结构和结合位点的位置。然而，结构本身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？构象熵是如何驱动配体结合的？载脂蛋白构象预平衡是如何与配体结合变构耦合的？为了解决这些基本问题，我们的第二个目标将是：（ii）受益于最近的核磁共振进展，比较分析在脂肪酸和其他配体存在和不存在的情况下HSA的动力学。其中包括我们首创的一种方法，通过核磁共振化学位移的协方差分析来绘制其他难以捉摸的变构网络。我们将从分析分离的HSA结构域开始，并逐步扩展到更长的HSA结构，以揭示动力学如何调节配体结合，门控和变构。到目前为止，现有的初步数据表明，就可用材料和实验方法而言，利用我们过去出版物中采用的核磁共振方法，这两个目标都是可行的。由此产生的程序将产生广泛的影响，远远超出HSA，因为它将揭示细胞外伴侣和配体与蛋白质动力学结合的变构偶联的一般分子机制。影响将是科学和教育，因为拟议的计划提供了一个独特的HQP培训机会。