STRUCTURE/FUNCTION OF S100 PROTEIN

S100 蛋白的结构/功能

• 批准号: 6498756
• 负责人: David Joseph Weber
• 金额: $ 24.29万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6498756
• 关键词: calbindin; calcium binding protein; calcium ion; conformation; dimer; nuclear magnetic resonance spectroscopy; p53 gene /protein; protein binding; protein isoforms; protein kinase C; protein protein interaction; protein purification; protein structure function; site directed mutagenesis; structural biology; thermodynamics; zinc

During the last 3 years we (i) prepared overexpression vectors that produce high yields (greater than 30 mg/liter) of S100B (beta beta), S100A1, and S100L, (ii) determined the solution structures of apo- and Ca2+-bound S100B (beta beta) at high resolution using heteronuclear multidimensional NMR spectroscopy, (iii) collected NMR data in liquid crystalline media necessary to measure dipolar coupling values, (iv) showed that dimeric S100B (beta beta) is the physiologically relevant oligomerization state of this protein, and (v) determined that S100B (beta beta) inhibits p53 phosphorylation by protein kinase C kinase C (PKC) in a Ca2+-dependent manner, and that this inhibition is the result of S100B (beta beta) interacting directly with the C-terminal regulatory domain of p53. We plan to continue characterizing the Ca2+-dependent interaction of S100B (beta beta) with target proteins. First, we will refine our previous NMR structures of apo- and Ca2+-loaded S100B (beta beta) using dipolar coupling constraints. We will also determine the 3D structure of Ca2+-bound S100B (beta beta) complexed with peptide derived from the C-terminal regulatory domain of p53 (residues 367-388). This will represent the first 3D structure of a S100-target protein complex. The binding of Zn2+ to S100B (beta beta) will also be characterized, and we will determine whether the Zn2+ binding site overlaps with the target protein site. Heteronuclear relaxation measurements are planned for all of the structures that we solve in order to clarify how Ca2+ and target protein binding affects dynamic processes in S100B (beta eta). Lastly, the 3D solution structures of S100A and S100L will be determined and compared to S100B (beta beta). This will be done with the goal of identifying the structural properties of these two proteins that lead to their higher affinity for Ca2+ and their specificity in target protein binding. Our effort is directed towards characterizing S100-target protein interactions with the long-range goal of inhibiting them. Therefore, structural studies, together with site-directed mutagenesis, thermodynamic binding, and dynamic measurements will be used to characterize, at atomic resolution, the interaction between specific residues of S100B (beta beta) with those of various protein targets in solution. An inhibitor based on this information could be relevant to treating uncontrolled cell growth found in diseases such as cancer and Alzheimer's disease.

在过去 3 年中，我们 (i) 制备了过表达载体 产生高产量（大于 30 毫克/升）的 S100B（β beta）， S100A1 和 S100L，(ii) 确定了 apo- 和 使用异核进行高分辨率 Ca2+ 结合 S100B (beta beta) 多维核磁共振波谱，(iii) 收集液体中的核磁共振数据 测量偶极耦合值所需的结晶介质，(iv) 表明二聚体 S100B（ββ）与生理相关 该蛋白质的寡聚状态，并且 (v) 确定 S100B (beta beta) 通过蛋白激酶 C 抑制 p53 磷酸化 激酶 C (PKC) 以 Ca2+ 依赖性方式进行，并且这种抑制是结果 S100B (beta beta) 与 C 端调控直接相互作用 p53 的结构域。 我们计划继续表征 Ca2+ 依赖性相互作用 S100B（ββ）与目标蛋白。 首先，我们将完善我们的 之前使用 apo 和 Ca2+ 负载的 S100B (beta beta) 的 NMR 结构 偶极耦合约束。 我们还将确定 3D 结构 Ca2+ 结合的 S100B (beta beta) 与源自 p53 的 C 末端调节域（残基 367-388）。 这将 代表 S100-靶蛋白复合物的第一个 3D 结构。 这 Zn2+ 与 S100B (beta beta) 的结合也将被表征，我们 将确定 Zn2+ 结合位点是否与目标重叠 蛋白质位点。 计划对所有的异核弛豫测量 我们解决的结构是为了阐明 Ca2+ 和靶标如何 蛋白质结合影响 S100B (beta eta) 的动态过程。 最后， S100A和S100L的3D解决方案结构将被确定并 与 S100B（beta beta）相比。 这样做的目的是 确定这两种蛋白质的结构特性 其对 Ca2+ 具有更高的亲和力以及对目标的特异性 蛋白质结合。 我们的努力旨在表征 S100 靶蛋白 相互作用与抑制它们的长期目标。所以， 结构研究，连同定点诱变， 热力学结合和动态测量将用于 以原子分辨率表征特定物质之间的相互作用 S100B（ββ）的残基与各种蛋白质靶标的残基 解决方案。 基于此信息的抑制剂可能与 治疗癌症等疾病中发现的不受控制的细胞生长 阿尔茨海默病。