Supertertiary structure of the synaptic MAGuK scaffold proteins is conserved
Supertertiary structure of the synaptic MAGuK scaffold proteins is conserved
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DOI：
10.1073/pnas.1200254109
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发表时间：
2012-09
期刊：
影响因子：
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通讯作者：
James J McCann;Liqiang Zheng;D. Rohrbeck;S. Felekyan;R. Kühnemuth;R B Sutton;C. Seidel;M. Bowen
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文献类型：
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作者：
James J McCann;Liqiang Zheng;D. Rohrbeck;S. Felekyan;R. Kühnemuth;R B Sutton;C. Seidel;M. Bowen
支架蛋白通过结合信号通路内的多个伴侣形成组织信号转导的框架。这塑造了信号响应的输出，并提供了特异性和定位。膜相关鸟苷酸激酶（MAGuKs）是位于细胞连接处的支架蛋白，其定位细胞表面受体并将它们连接到下游信号传导酶。支架蛋白通常含有通过无序接头串联连接的蛋白质结合结构域。折叠结构域的三级结构是很好的理解，但描述动态域间的相互作用（superteritary结构），这样的多结构域蛋白质仍然是一个挑战，结构生物学。我们使用了65个来自单分子荧光共振能量转移（smFRET）的距离限制来描述经典MAGuK支架蛋白PSD-95的超空间结构。通过结合多种荧光技术，PSD-95的构象动力学可以在蛋白质结构域运动的生物学相关时间尺度上表征。仅依赖于FRET数据的定性解释，我们能够区分稳定的域间相互作用自由取向的域。这表明PSD-95的5个结构域分为两个独立的超模块：PDZ 1-PDZ 2和PDZ 3-SH 3-GuK。我们使用我们的smFRET数据的混合结构细化模型的PDZ 3-SH 3-GuK超模块，并包括明确的染料模拟，以提供完整的表征固有的FRET作为距离的定量解释的潜在不确定性。突触MAGuK同源物的比较结构分析表明，这种超三级结构的保护。我们的方法是一个通用的解决方案来描述超三级结构的多结构域蛋白质。
Scaffold proteins form a framework to organize signal transduction by binding multiple partners within a signaling pathway. This shapes the output of signal responses as well as providing specificity and localization. The Membrane Associated Guanylate Kinases (MAGuKs) are scaffold proteins at cellular junctions that localize cell surface receptors and link them to downstream signaling enzymes. Scaffold proteins often contain protein-binding domains that are connected in series by disordered linkers. The tertiary structure of the folded domains is well understood, but describing the dynamic inter-domain interactions (the superteritary structure) of such multidomain proteins remains a challenge to structural biology. We used 65 distance restraints from single-molecule fluorescence resonance energy transfer (smFRET) to describe the superteritary structure of the canonical MAGuK scaffold protein PSD-95. By combining multiple fluorescence techniques, the conformational dynamics of PSD-95 could be characterized across the biologically relevant timescales for protein domain motions. Relying only on a qualitative interpretation of FRET data, we were able to distinguish stable interdomain interactions from freely orienting domains. This revealed that the five domains in PSD-95 partitioned into two independent supramodules: PDZ1-PDZ2 and PDZ3-SH3-GuK. We used our smFRET data for hybrid structural refinement to model the PDZ3-SH3-GuK supramodule and include explicit dye simulations to provide complete characterization of potential uncertainties inherent to quantitative interpretation of FRET as distance. Comparative structural analysis of synaptic MAGuK homologues showed a conservation of this supertertiary structure. Our approach represents a general solution to describing the supertertiary structure of multidomain proteins.