Won H S, Yamazaki T, Lee T W, Yoon M K, Park S H, Kyogoku Y, Lee B J
College of Pharmacy, Seoul National University, San 56-1, Shinlim-Dong, Kwanak-Gu, Seoul 151-742, Korea.
Biochemistry. 2000 Nov 14;39(45):13953-62. doi: 10.1021/bi000012x.
Cyclic AMP receptor protein (CRP) plays a key role in the regulation of more than 150 genes. CRP is allosterically activated by cyclic AMP and binds to specific DNA sites. A structural understanding of this allosteric conformational change, which is essential for its function, is still lacking because the structure of apo-CRP has not been solved. Therefore, we performed various NMR experiments to obtain apo-CRP structural data. The secondary structure of apo-CRP was determined by analyses of the NOE connectivities, the amide proton exchange rates, and the (1)H-(15)N steady-state NOE values. A combination of the CSI-method and TALOS prediction was also used to supplement the determination of the secondary structure of apo-CRP. This secondary structure of apo-CRP was compared with the known structure of cyclic AMP-bound CRP. The results suggest that the allosteric conformational change of CRP caused by cyclic AMP binding involves subunit realignment and domain rearrangement, resulting in the exposure of helix F onto the surface of the protein. Additionally, the results of the one-dimensional [(13)C]carbonyl NMR experiments show that the conformational change of CRP caused by the binding of cyclic GMP, an analogue of cyclic AMP, is different from that caused by cyclic AMP binding.
环腺苷酸受体蛋白（CRP）在150多个基因的调控中起关键作用。CRP被环腺苷酸别构激活并与特定的DNA位点结合。由于无配体CRP的结构尚未解析，因此仍缺乏对这种对其功能至关重要的别构构象变化的结构理解。因此，我们进行了各种核磁共振实验以获得无配体CRP的结构数据。通过对核Overhauser效应（NOE）连接性、酰胺质子交换率和（1）H-（15）N稳态NOE值的分析来确定无配体CRP的二级结构。CSI方法和TALOS预测的组合也用于补充无配体CRP二级结构的测定。将无配体CRP的这种二级结构与环腺苷酸结合型CRP的已知结构进行比较。结果表明，环腺苷酸结合引起的CRP别构构象变化涉及亚基重排和结构域重排，导致螺旋F暴露于蛋白质表面。此外，一维[（13）C]羰基核磁共振实验结果表明，环腺苷酸类似物环鸟苷酸结合引起的CRP构象变化与环腺苷酸结合引起的不同。
