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）：这项研究的长期目标是阐明磷团（PLB）抑制心脏肌胞质网（SR）CA泵（SERCA2A同工型）的活性的分子和生物物理机制。 PLB是心脏SR中的一种五聚磷蛋白，由五个相同的单体组成。以前，我们证明了PLB单体负责与SERCA2A结合并抑制它。现在，我们建议在PLB单体和SERCA2A之间定位结合相互作用位点，从而导致酶抑制，并确定分子相互作用如何受到关键的变构调节剂的调节，包括CA浓度，核苷酸和磷酸化。将重点放在识别直接相互作用的氨基酸中，利用我们新开发的化学交联方法。在AIM 1中，我们将执行PLB的Cys扫描诱变，以将不同的位点沿其主要结构进行定位，从而交联SERCA2A的内源性CYS残基。 SERCA2A的交联Cys残基将通过蛋白质纯化/肽测序直接鉴定。在AIM 2中，将定位与PLB不同位点交联的SERCA2A的LYS残基。通过将交联剂用作分子统治者，并结合了目标1和2的结果，我们将开发出PLB单体和SERCA2A之间形成的结合复合物的精确3-D模型。在AIM 3中，将研究Ca浓度，核苷酸和抑制剂Thapsigargin对PLB与SERCA2A的交联的影响。测试的假设是，PLB仅与SERCA2A的无CA形式（E2）结合，但仅结合具有ATP或ADP的E2状态。在AIM 4中，我们将确定如何缓解PLB对SERCA2A的抑制作用。我们假设PLB优先与E2结合，拮抗Ca与SERCA2A的结合，并且Ca优先结合与EL结合，与PLB结合与SERCA2A结合。将确定负责从泵中分离PLB的SERCA2A的CA结合位点，并且将量化PLB对SERCA2A的CA结合亲和力的影响。在AIM 5中，我们将确定蛋白激酶对PLB的磷酸化如何缓解PLB抑制作用。测试的假设是，PLB的磷酸化直接从SERCA2A解离。在这里，我们还将确定PLB，SER 16和THR 17的两个磷酸化残基是否与SERCA2A直接相互作用以帮助酶抑制。 PLB是心肌收缩动力学的关键调节剂。通过定义其对CA泵的分子作用机制，将产生对PLB调节强度的新见解，这最终可能导致设计新药以治疗心力衰竭。