Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
Biochemistry. 2012 May 1;51(17):3651-61. doi: 10.1021/bi300327z. Epub 2012 Apr 16.
Nitric oxide synthase (NOS) plays a major role in a number of key physiological and pathological processes. Knowledge of how this is regulated is important. The small acidic calcium binding protein, calmodulin (CaM), is required to fully activate the enzyme. The exact mechanism of how CaM activates NOS is not fully understood. Studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the transfer of an electron between the reductase and oxygenase domains through a process that is thought to be highly dynamic. To investigate the dynamic properties of CaM-NOS interactions, we determined the solution structure of CaM bound to the inducible NOS (iNOS) and endothelial NOS (eNOS) CaM binding region peptides. In addition, we investigated the effect of CaM phosphorylation. Tyrosine 99 (Y99) of CaM is reported to be phosphorylated in vivo. We have produced a phosphomimetic Y99E CaM to investigate the structural and functional effects that the phosphorylation of this residue may have on nitric oxide production. All three mammalian NOS isoforms were included in the investigation. Our results show that a phosphomimetic Y99E CaM significantly reduces the maximal synthase activity of eNOS by 40% while having little effect on nNOS or iNOS activity. A comparative nuclear magnetic resonance study between phosphomimetic Y99E CaM and wild-type CaM bound to the eNOS CaM binding region peptide was performed. This investigation provides important insights into how the increased electronegativity of a phosphorylated CaM protein affects the binding, dynamics, and activation of the NOS enzymes.
一氧化氮合酶（NOS）在许多关键的生理和病理过程中起着重要作用。了解其如何被调节是很重要的。小的酸性钙结合蛋白，钙调蛋白（CaM），是充分激活酶所必需的。CaM 如何激活 NOS 的具体机制尚未完全理解。研究表明，CaM 像一个开关，导致 NOS 的构象变化，允许电子在还原酶和加氧酶结构域之间通过一个被认为是高度动态的过程转移。为了研究 CaM-NOS 相互作用的动态特性，我们确定了与诱导型 NOS（iNOS）和内皮型 NOS（eNOS）CaM 结合区肽结合的 CaM 的溶液结构。此外，我们还研究了 CaM 磷酸化的影响。CaM 中的酪氨酸 99（Y99）据报道在体内被磷酸化。我们已经产生了一个磷酸模拟 Y99E CaM 来研究该残基磷酸化可能对一氧化氮产生的结构和功能影响。所有三种哺乳动物 NOS 同工酶都包括在研究中。我们的结果表明，磷酸模拟 Y99E CaM 显著降低 eNOS 的最大合酶活性 40%，而对 nNOS 或 iNOS 活性几乎没有影响。对磷酸模拟 Y99E CaM 和与 eNOS CaM 结合区肽结合的野生型 CaM 进行了比较核磁共振研究。这项研究为了解磷酸化的 CaM 蛋白增加的电负性如何影响 NOS 酶的结合、动力学和激活提供了重要的见解。
