Switching Kinetics in Calmodulin-IQ Domain Complexes

钙调蛋白-IQ 结构域复合物中的转换动力学

• 批准号: 7621040
• 负责人: ANTHONY J PERSECHINI
• 金额: $ 21.84万
• 依托单位: UNIVERSITY OF MISSOURI KANSAS CITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7621040
• 关键词: Affect; Behavior; Binding; Biochemical; Biological Process; Buffers; Calmodulin; Calmodulin-Binding Proteins; Cardiovascular system; Cells; Characteristics; Complex; Cytoskeletal Proteins; Data; Defect; Dissociation; Exhibits; Fluorescence; GTP-Binding Proteins; Glycine; Growth Associated Protein 43; Human; Immune; Impairment; Ion Channel; Kinetics; Knowledge; Length; Lysine; Measurement; Methods; Microcephaly; Molecular; Mutation; Myosin ATPase; Neurologic; Neurons; Process; Protein Binding; Proteins; Relative (related person); Reporter; Research; Research Personnel; Signal Transduction; Tertiary Protein Structure; Testing; Thermodynamics; base; brain size; deafness; heart electrical activity; human disease; insight; interest; mutant; nervous system disorder; programs; response; sensor

DESCRIPTION (provided by applicant): An extensive class of proteins binds the Ca2+-binding protein calmodulin (CaM) through what are termed "IQ domains". The biological functions of many in this class, including Na+, K+, and Ca2+ channels, G protein modulators and unconventional myosins are regulated by Ca2+ via the CaM-IQ domain complex, yet little is known about how these important molecular switches function. We propose to help fill this gap in knowledge by deriving detailed transient and steady-state kinetic mechanisms for Ca2+-dependent switching in the CaM-IQ domain complexes in neuromodulin and PEP-19, which serve as CaM buffers and/or stores in neurons. Both are of fundamental interest because they represent two major classes of IQ domains: A glycine residue found in half of all IQ domains is present in neuromodulin, but has been replaced by a lysine in PEP-19. Structural information indicates that this difference should significantly affect interactions with CaM. These proteins are also of interest because of the control they appear to exert over the distribution of CaM among its many targets in the cell. Knowledge of the mechanisms determining how they interact with CaM will provide insights to this control process, and hence to the way in which the network of CaM targets is orchestrated. The actions of this network are a major determinant of how neurons and other cells respond to Ca2+ signals. SPECIFIC AIM 1: Derive steady-state kinetic mechanisms for Ca2+-dependent switching in the complexes between CaM and the IQ domains in neuromodulin and PEP-19. This will be performed using native and mutant CaMs, and fluorescent protein reporters based on neuromodulin and PEP-19. The final steady state mechanisms will include the kinetic parameters governing formation of the major switching intermediates. SPECIFIC AIM 2: Expand the steady-state mechanisms derived under Aim 1 to encompass the transient kinetic behaviors of the complexes between CaM and the IQ domains in neuromodulin and PEP-19. Mechanisms derived for Ca2+-dependent switching in the two CaM complexes will be rigorously tested and refined by fitting them to the responses exhibited by the fluorescent reporters over a range of transient and steady-state conditions. The health-related benefits of this research program derive from the direct relationship between mutations in proteins containing IQ domains and numerous human diseases: Truncations and other mutations in the ASPM protein, which contains multiple IQ domain repeats, are associated with microcephaly, and the number of repeats appears to be positively correlated with brain size. Mutations in the Na+ channel IQ domain cause irregularities in cardiac electrical activity, and defects in other ion channels regulated via CaM-IQ domain switches are responsible for a host of cardiovascular and neurological disorders. Mutations in unconventional myosins result in immune-deficiencies and neurological impairment, and are the most frequent cause of deafness-blindness in humans.

描述（由申请人提供）：一类广泛的蛋白质通过称为“ IQ域”的蛋白质结合Ca2+结合蛋白钙调蛋白（CAM）。该类别中许多人的生物学功能，包括Na+，K+和Ca2+通道，G蛋白调节剂和非常规的肌球蛋白受CA2+调节的CAM-IQ域复合物调节，但对这些重要分子开关如何功能的知识知之甚少。我们建议通过在神经化蛋白和PEP-19中的CAM-IQ结构域复合物中的Ca2+依赖性切换来得出详细的瞬时和稳态动力学机制，以帮助填补这一空白，这些机制用作神经元中的cam缓冲液和/或储存神经元中的cAM-IQ结构域复合物。两者都具有基本的兴趣，因为它们代表了智商域的两个主要类别：在神经元素中存在的所有智商结构域中发现的甘氨酸残基，但已在PEP-19中被赖氨酸取代。结构信息表明，这种差异应该显着影响与CAM的相互作用。这些蛋白质也引起了人们的关注，因为它们的控制似乎对CAM在细胞中的许多靶标之间的分布似乎作用。了解确定它们如何与CAM相互作用的机制的知识将为此控制过程提供洞察力，从而为CAM目标网络精心策划的方式提供了见解。该网络的作用是神经元和其他细胞如何响应Ca2+信号的主要决定因素。具体目标1：在CAM和iQ域之间的CA2+依赖性切换中，稳态动力学机制在神经调节蛋白和PEP-19中的iQ域之间的切换。这将使用天然和突变体凸轮以及基于神经调节蛋白和PEP-19的荧光蛋白报道器进行。最终的稳态机制将包括有关主要开关中间体形成的动力学参数。特定目标2：扩展在目标1下得出的稳态机制，以涵盖CAM和iQ域之间的复合物的瞬时动力学行为。通过将荧光记者在一系列短暂和稳态条件下拟合的响应中，将对两个CAM复合物中Ca2+依赖性切换的机制进行严格测试和完善。该研究计划与健康相关的益处来自含有智商域和许多人类疾病的蛋白质突变之间的直接关系：ASPM蛋白中的截断和其他突变（其中包含多个智商域重复序列）与小头畸形相关，并且重复次数似乎与大脑大小相关。 Na+通道智商域中的突变会导致心脏电活动中的不规则性，以及通过CAM-IQ域开关调节的其他离子通道中的缺陷负责许多心血管和神经系统疾病。非常规肌球蛋白的突变会导致免疫缺陷和神经系统损害，并且是人类聋哑人的最常见原因。

项目成果

Phosphorylation within an autoinhibitory domain in endothelial nitric oxide synthase reduces the Ca(2+) concentrations required for calmodulin to bind and activate the enzyme.

• DOI: 10.1021/bi8003186
• 发表时间: 2008-07
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Q. Tran;J. Leonard;D. J. Black;A. Persechini