Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, United States.
Biochemistry. 2012 Sep 18;51(37):7263-77. doi: 10.1021/bi300750w. Epub 2012 Sep 5.
Structural cysteine-rich Zn(2+) sites that stabilize protein folds are considered to be unreactive. In this article, we identified a reactive cysteine residue, Cys151, in a treble-clef zinc finger with a Cys(3)His coordination sphere. The protein in question is the C1B domain of Protein Kinase Cα (PKCα). Mass-tagging cysteine assays of several C1B variants were employed to ascertain the site specificity of the covalent modification. The reactivity of Cys151 in C1B also manifests itself in the structural dynamics of the Zn(2+) coordination sphere where the Sγ of Cys151 alternates between the Zn(2+)-bound thiolate and free thiol states. We used NMR-detected pH titrations, ZZ-exchange spectroscopy, and residual dipolar coupling (RDC)-driven structure refinement to characterize the two exchanging conformations of C1B that differ in zinc coordination. Our data suggest that Cys151 serves as an entry point for the reactive oxygen species that activate PKCα in a process involving Zn(2+) release.
结构上富含半胱氨酸的锌（2+）结合位点被认为是无反应性的。在本文中，我们在具有 Cys（3）His 配位球的三重锌指中鉴定出一个反应性半胱氨酸残基 Cys151。所研究的蛋白质是蛋白激酶 Cα（PKCα）的 C1B 结构域。我们使用标记半胱氨酸的质谱分析了几种 C1B 变体，以确定共价修饰的位点特异性。C1B 中 Cys151 的反应性也表现在 Zn（2+）配位球的结构动力学中，其中 Cys151 的 Sγ 在 Zn（2+）结合的硫醇盐和游离硫醇状态之间交替。我们使用 NMR 检测的 pH 滴定、ZZ 交换光谱和残余偶极耦合（RDC）驱动的结构精修来表征锌配位不同的 C1B 的两种交换构象。我们的数据表明，Cys151 充当活性氧物质的进入点，该物质在涉及 Zn（2+）释放的过程中激活 PKCα。
