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: 7226021
• 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 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7226021
• 关键词: Affect; Behavior; Binding; Biochemical; Biological Process; Buffers; Calmodulin; Calmodulin-Binding Proteins; Cardiovascular system; Cells; Characteristics; Class; Complex; Condition; 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; Numbers; Process; Protein Binding; Proteins; Range; Rate; Relative (related person); Reporter; Research; Research Personnel; Signal Transduction; Tertiary Protein Structure; Testing; Thermodynamics; Thinking; 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two major classes of IQ domains: A glycine residue found in half of all IQ domains is present in neuromodulin, but has been replaced by a lysine in PEP-19.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所谓的“IQ结构域”结合Ca2+结合蛋白钙调蛋白（CaM）。此类中许多分子的生物学功能，包括 Na+、K+ 和 Ca2+ 通道、G 蛋白调节剂和非常规肌球蛋白，均由 Ca2+ 通过 CaM-IQ 结构域复合体进行调节，但人们对这些重要分子开关的功能知之甚少。我们建议通过推导神经调节蛋白和 PEP-19 中 CaM-IQ 结构域复合物中 Ca2+ 依赖性开关的详细瞬态和稳态动力学机制来帮助填补这一知识空白，这些复合物充当神经元中的 CaM 缓冲区和/或存储。两者都具有根本意义，因为它们代表了 IQ 结构域的两大类：一半 IQ 结构域中发现的甘氨酸残基存在于神经调节蛋白中，但在 PEP-19 中已被赖氨酸取代。结构信息表明这种差异应显着影响与 CaM 的相互作用。这些蛋白质也引起人们的兴趣，因为它们似乎可以控制 CaM 在细胞内许多靶标中的分布。了解决定它们如何与 CaM 交互的机制将为了解该控制过程提供见解，从而了解 CaM 目标网络的编排方式。该网络的活动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：导出神经调节蛋白和 PEP-19 中 CaM 与 IQ 结构域之间复合物中 Ca2+ 依赖性转换的稳态动力学机制。这将使用天然和突变 CaM 以及基于神经调节蛋白和 PEP-19 的荧光蛋白报告基因来进行。最终的稳态机制将包括控制主要转换中间体形成的动力学参数。具体目标 2：扩展目标 1 下导出的稳态机制，以涵盖 CaM 与神经调节蛋白和 PEP-19 中 IQ 域之间复合物的瞬态动力学行为。两个 CaM 复合物中 Ca2+ 依赖性开关的衍生机制将通过将其拟合到荧光报告基因在一系列瞬态和稳态条件下表现出的响应来进行严格测试和完善。该研究项目与健康相关的益处源自包含 IQ 结构域的蛋白质突变与多种人类疾病之间的直接关系：包含多个 IQ 结构域重复的 ASPM 蛋白中的截短和其他突变与小头畸形有关，并且数量重复次数似乎与大脑大小呈正相关。 Na+ 通道 IQ 结构域的突变会导致心电活动不规则，而通过 CaM-IQ 结构域开关调节的其他离子通道的缺陷则导致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损伤，也是人类耳聋失明的最常见原因。