Structure /Function of Phospholamban in Heart

磷脂班在心脏中的结构/功能

• 批准号: 6761959
• 负责人: LARRY R. JONES
• 金额: $ 45.32万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6761959
• 关键词: active sites; biological signal transduction; calcium transporting ATPase; chemical models; crosslink; crystallization; dogs; enzyme complex; enzyme inhibitors; genetic manipulation; heart contraction; intermolecular interaction; molecular biology information system; monomer; nucleotides; phospholamban; phosphorylation; protein binding; protein protein interaction; protein sequence; protein structure function; sarcoplasmic reticulum; thapsigargin; tissue /cell culture; western blottings

DESCRIPTION (provided by applicant): The long term goal of this research is to elucidate the molecular and biophysical mechanism by which phospholamban (PLB) inhibits the activity of the Ca pump (SERCA2a isoform) in cardiac sarcoplasmic reticulum (SR). PLB is a pentameric phosphoprotein in cardiac SR, which is composed of five identical monomers. Previously, we demonstrated the PLB monomer is responsible for binding to SERCA2a and inhibiting it. Now, we propose to localize the binding-interaction sites between the PLB monomer and SERCA2a that lead to enzyme inhibition, and determine how the molecular interaction is regulated by key allosteric modulators including Ca concentration, nucleotides, and phosphorylation. Emphasis will be placed upon identifying amino acids in the inhibitory complex that interact directly, taking advantage of our newly developed chemical cross-linking method. In Aim 1, we will perform Cys-scanning mutagenesis of PLB to localize distinct sites along its primary structure that cross-link to endogenous Cys residues of SERCA2a. The cross-linked Cys residues of SERCA2a will be directly identified by protein purification/peptide sequencing. In Aim 2, Lys residues of SERCA2a that cross-link to distinct sites of PLB will be localized. By use of crosslinking agents as molecular rulers and combining results from Aims 1 and 2, we will develop an accurate 3-D model of the binding-complex formed between the PLB monomer and SERCA2a. In Aim 3, the effects of Ca concentration, nucleotides, and the inhibitor thapsigargin on cross-linking of PLB to SERCA2a will be investigated. The hypothesis tested is that PLB binds exclusively to the Ca-free form (E2) of SERCA2a, but only that E2 state that has bound ATP or ADP. In Aim 4, we will determine how Ca relieves PLB inhibition of SERCA2a. We hypothesize that PLB binds preferentially to E2, antagonizing Ca binding to SERCA2a, and that Ca binds preferentially to El, antagonizing PLB binding to SERCA2a. The Ca-binding site of SERCA2a responsible for dissociating PLB from the pump will be identified, and the effect of PLB on the Ca-binding affinity of SERCA2a will be quantified. In Aim 5, we will determine how phosphorylation of PLB by protein kinases relieves PLB inhibition. The hypothesis tested is that phosphorylation of PLB directly dissociates it from SERCA2a. Here we will also determine if the two phosphorylated residues of PLB, Ser 16 and Thr 17, interact directly with SERCA2a to aid in enzyme inhibition. PLB is a key regulator of myocardial contractile dynamics. By defining its molecular mechanism of action on the Ca pump, new insights on PLB regulation of the strength of the heartbeat will result, that may ultimately lead to the design of new drugs to treat heart failure.

描述（由申请人提供）：本研究的长期目标是阐明受磷蛋白（PLB）抑制心脏肌浆网（SR）中钙泵（SERCA2a亚型）活性的分子和生物物理机制。 PLB是心脏SR中的五聚体磷蛋白，由五个相同的单体组成。之前，我们证明了 PLB 单体负责与 SERCA2a 结合并抑制它。现在，我们建议定位 PLB 单体和 SERCA2a 之间导致酶抑制的结合相互作用位点，并确定关键变构调节剂（包括 Ca2+ 浓度、核苷酸和磷酸化）如何调节分子相互作用。重点将放在利用我们新开发的化学交联方法识别抑制复合物中直接相互作用的氨基酸。在目标 1 中，我们将对 PLB 进行 Cys 扫描诱变，以沿着其一级结构定位与 SERCA2a 的内源 Cys 残基交联的不同位点。 SERCA2a 的交联 Cys 残基将通过蛋白质纯化/肽测序直接鉴定。在目标 2 中，SERCA2a 与 PLB 不同位点交联的赖氨酸残基将被定位。通过使用交联剂作为分子标尺并结合目标 1 和 2 的结果，我们将开发 PLB 单体和 SERCA2a 之间形成的结合复合物的精确 3D 模型。在目标 3 中，将研究 Ca 浓度、核苷酸和抑制剂毒胡萝卜素对 PLB 与 SERCA2a 交联的影响。测试的假设是 PLB 仅与 SERCA2a 的无 Ca 形式 (E2) 结合，但仅与结合 ATP 或 ADP 的 E2 状态结合。在目标 4 中，我们将确定 Ca 如何减轻 PLB 对 SERCA2a 的抑制。我们假设PLB优先结合E2，拮抗Ca结合SERCA2a，并且Ca优先结合E1，拮抗PLB结合SERCA2a。将鉴定负责将 PLB 从泵解离的 SERCA2a 的 Ca 结合位点，并量化 PLB 对 SERCA2a 的 Ca 结合亲和力的影响。在目标 5 中，我们将确定蛋白激酶对 PLB 的磷酸化如何缓解 PLB 抑制。测试的假设是 PLB 的磷酸化直接将其与 SERCA2a 解离。在这里，我们还将确定 PLB 的两个磷酸化残基（Ser 16 和 Thr 17）是否直接与 SERCA2a 相互作用以帮助酶抑制。 PLB 是心肌收缩动力学的关键调节因子。通过定义其对 Ca 泵作用的分子机制，将产生对 PLB 调节心跳强度的新见解，这可能最终导致治疗心力衰竭的新药物的设计。