Department of Biochemistry, Ohio State University, Columbus, OH 43210, USA.
Proc Natl Acad Sci U S A. 2010 Aug 31;107(35):15385-90. doi: 10.1073/pnas.1004981107. Epub 2010 Aug 16.
Anti-TRAP (AT) is a small zinc-binding protein that regulates tryptophan biosynthesis in Bacillus subtilis by binding to tryptophan-bound trp RNA-binding attenuation protein (TRAP), thereby preventing it from binding RNA, and allowing transcription and translation of the trpEDCFBA operon. Crystallographic and sedimentation studies have shown that AT can homooligomerize to form a dodecamer, AT(12), composed of a tetramer of trimers, AT(3). Structural and biochemical studies suggest that only trimeric AT is active for binding to TRAP. Our chromatographic and spectroscopic data revealed that a large fraction of recombinantly overexpressed AT retains the N-formyl group (fAT), presumably due to incomplete N-formyl-methionine processing by peptide deformylase. Hydrodynamic parameters from NMR relaxation and diffusion measurements showed that fAT is exclusively trimeric (AT(3)), while (deformylated) AT exhibits slow exchange between both trimeric and dodecameric forms. We examined this equilibrium using NMR spectroscopy and found that oligomerization of active AT(3) to form inactive AT(12) is linked to protonation of the amino terminus. Global analysis of the pH dependence of the trimer-dodecamer equilibrium revealed a near physiological pK(a) for the N-terminal amine of AT and yielded a pH-dependent oligomerization equilibrium constant. Estimates of excluded volume effects due to molecular crowding suggest the oligomerization equilibrium may be physiologically important. Because deprotonation favors "active" trimeric AT and protonation favors "inactive" dodecameric AT, our findings illuminate a possible mechanism for sensing and responding to changes in cellular pH.
抗 TRAP（AT）是一种小的锌结合蛋白，通过与色氨酸结合的 trp RNA 结合衰减蛋白（TRAP）结合，调节枯草芽孢杆菌中的色氨酸生物合成，从而阻止其与 RNA 结合，允许 trpEDCFBA 操纵子的转录和翻译。晶体学和沉降研究表明，AT 可以同源寡聚形成十二聚体，AT（12），由四聚体三聚体组成，AT（3）。结构和生化研究表明，只有三聚体 AT 才能与 TRAP 结合。我们的色谱和光谱数据显示，重组过度表达的 AT 保留了 N-甲酰基（fAT），这可能是由于肽脱甲酰酶不完全加工 N-甲酰基甲硫氨酸。NMR 弛豫和扩散测量的流体力学参数表明，fAT 仅为三聚体（AT（3）），而（去甲酰化）AT 表现出三聚体和十二聚体形式之间的缓慢交换。我们使用 NMR 光谱法研究了这种平衡，发现活性 AT（3）的寡聚化形成无活性的 AT（12）与氨基末端的质子化有关。对三聚体-十二聚体平衡的 pH 依赖性的全局分析揭示了 AT 的 N 末端胺的近生理 pK（a），并产生了 pH 依赖性的寡聚平衡常数。由于分子拥挤而产生的排除体积效应的估计表明，寡聚平衡可能具有生理重要性。由于去质子化有利于“活性”三聚体 AT，而质子化有利于“无活性”十二聚体 AT，因此我们的发现阐明了一种可能的机制，用于感应和响应细胞 pH 的变化。
