McElroy Craig A, Ihms Elihu C, Kumar Yadav Deepak, Holmquist Melody L, Wadhwa Vibhuti, Wysocki Vicki H, Gollnick Paul, Foster Mark P
Ohio State Biochemistry Program, USA.
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
J Struct Biol X. 2024 Jun 11;10:100103. doi: 10.1016/j.yjsbx.2024.100103. eCollection 2024 Dec.
Cellular production of tryptophan is metabolically expensive and tightly regulated. The small zinc binding Anti-TRAP protein (AT), which is the product of the gene, is upregulated in response to accumulating levels of uncharged tRNA through a T-box antitermination mechanism. AT binds to the undecameric axially symmetric ring-shaped protein TRAP ( RNA Binding Attenuation Protein), thereby preventing it from binding to the leader RNA. This reverses the inhibitory effect of TRAP on transcription and translation of the operon. AT principally adopts two symmetric oligomeric states, a trimer (AT) featuring three-fold axial symmetry or a dodecamer (AT) comprising a tetrahedral assembly of trimers, whereas only the trimeric form binds and inhibits TRAP. We apply native mass spectrometry (nMS) and small-angle x-ray scattering (SAXS), together with analytical ultracentrifugation (AUC) to monitor the pH and concentration-dependent equilibrium between the trimeric and dodecameric structural forms of AT. In addition, we use solution nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of AT, while heteronuclear N relaxation measurements on both oligomeric forms of AT provide insights into the dynamic properties of binding-active AT and binding-inactive AT, with implications for TRAP binding and inhibition.
色氨酸的细胞生产在代谢上成本高昂且受到严格调控。小锌结合抗TRAP蛋白（AT）是该基因的产物，它通过T盒抗终止机制响应未负载tRNA积累水平的升高而上调。AT与十一聚体轴向对称环状蛋白TRAP（RNA结合衰减蛋白）结合，从而阻止其与前导RNA结合。这逆转了TRAP对操纵子转录和翻译的抑制作用。AT主要呈现两种对称的寡聚状态，一种是具有三重轴对称性的三聚体（AT），另一种是由三聚体的四面体组装体构成的十二聚体（AT），而只有三聚体形式能结合并抑制TRAP。我们应用原生质谱（nMS）、小角X射线散射（SAXS）以及分析超速离心（AUC）来监测AT三聚体和十二聚体结构形式之间pH和浓度依赖性的平衡。此外，我们使用溶液核磁共振（NMR）光谱来确定AT的溶液结构，同时对AT的两种寡聚形式进行异核N弛豫测量，以深入了解具有结合活性的AT和不具有结合活性的AT的动态特性，这对TRAP的结合和抑制具有重要意义。
