Boyd Stefanie D, Liu Li, Bulla Lee, Winkler Duane D
Department of Biological Sciences, The University of Texas at Dallas, 800 W. Campbell Road, Richardson, TX 75080, USA.
J Proteomics Bioinform. 2018;11(4). doi: 10.4172/jpb.1000473. Epub 2018 May 14.
Immature copper-zinc superoxide dismutase (Sod1) is activated by its copper chaperone (Ccs1). Ccs1 delivers a single copper ion and catalyzes oxidation of an intra-subunit disulfide bond within each Sod1 monomer through a mechanistically ambiguous process. Here, we use residue specific fluorescent labeling of immature Sod1 to quantitate the thermodynamics of the Sod1•Ccs1 interaction while determining a more complete view of Ccs1 function. Ccs1 preferentially binds a completely immature form of Sod1 that is metal deficient and disulfide reduced (E, E-Sod1). However, binding induces structural changes that promote high-affinity zinc binding by the Ccs1-bound Sod1 molecule. This adds further support to the notion that Ccs1 likely plays dual chaperoning roles during the Sod1 maturation process. Further analysis reveals that in addition to the copper-dependent roles during Sod1 activation, the N- and C-terminal domains of Ccs1 also have synergistic roles in securing both Sod1 recognition and its own active conformation. These results provide new and measurable analyses of the molecular determinants guiding Ccs1-mediated Sod1 activation.
未成熟的铜锌超氧化物歧化酶（Sod1）由其铜伴侣蛋白（Ccs1）激活。Ccs1传递单个铜离子，并通过一个机制尚不明确的过程催化每个Sod1单体亚基内二硫键的氧化。在此，我们使用未成熟Sod1的残基特异性荧光标记来定量Sod1•Ccs1相互作用的热力学，同时确定Ccs1功能的更完整视图。Ccs1优先结合金属缺乏且二硫键还原的完全未成熟形式的Sod1（E，E-Sod1）。然而，结合会诱导结构变化，促进与Ccs1结合的Sod1分子对锌的高亲和力结合。这进一步支持了Ccs1可能在Sod1成熟过程中发挥双重伴侣作用的观点。进一步分析表明，除了在Sod1激活过程中依赖铜的作用外，Ccs1的N端和C端结构域在确保Sod1识别及其自身活性构象方面也具有协同作用。这些结果为指导Ccs1介导的Sod1激活的分子决定因素提供了新的可测量分析。
