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通过交叉连接和质谱测量来绘制calmodulin和神经元NO合成酶的相互作用

Dana Felker¹, Kanghyun Lee², Thomas H Pospiech²

¹Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

The Journal of biological chemistry

|November 18, 2023

概括

这项研究使用交叉连接和质谱学来揭示calmodulin如何与神经元氧化合成酶 (nNOS) 相互作用. 结果表明一个更紧的nNOS结构,澄清酶激活机制.

科学领域:

生物化学生物化学
酶学是一种酶学.
结构生物学结构生物学

背景情况:

神经氧化合成酶 (nNOS) 是NO合成中的关键酶,需要calmodulin (CaM) 来激活.
由于域动态和结构数据有限,激活期间nNOS的精确的CaM结合形状尚不清楚.
之前的研究表明,与nNOS相比,iNOS中的CaM相互作用不同.

研究的目的:

通过共价交联和质谱学阐明CaM和nNOS之间的相互作用点.
为了解决CaM结合的nNOS复合体的结构形状.
研究FMN域在CaM-nNOS相互作用中的作用.

主要方法:

使用MS-cleavable双功能交叉链接器来识别CaM和nNOS之间的交叉链接.
进行质谱 (MS) 分析nNOS内和nNOS与CaM之间的交联.
集成的交叉连接数据与 Cryo-EM 结构和计算对接以建模 nNOS 复合体.

主要成果:

在CaM和nNOS之间确定了13个独特的交叉链接,在nNOS内确定了61个.
提供了证据,证明CaM结合nNOS.的减少酶和氧化酶域.
揭示了FMN域与氧化酶二聚体之间的相互作用.
开发了一个nNOS的交叉连接导向模型,显示了一个紧的形状与heme附近的FMN.

结论:

与MS相结合的共价交联有效地捕捉了nNOS的短暂构造.
当与CaM结合时,nNOS结构比以前认为的更加紧.
这些发现澄清了nNOS的激活机制,并突出了跨链MS在结构生物学中的实用性.

关键词:

卡尔莫杜林是一种卡尔莫杜林.交叉连接 (crosslinking) 是一种交叉连接.质谱测量质谱测量质谱测量质谱测量质量测量质谱测量质量测量质量测量质量测量质量测量质量测量质量测量质量测量质量测量质量测量质量测量质量测量质量测量质量测量质量测量质量测量质量测量质量测量质量测量质量测量氧化合成酶是一种氧化合成酶.蛋白质与蛋白质的相互作用

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