Zhou H, Dahlquist F W
Institute of Molecular Biology, University of Oregon, Eugene 97403, USA.
Biochemistry. 1997 Jan 28;36(4):699-710. doi: 10.1021/bi961663p.
Bacterial chemotaxis involves autophosphorylation of a histidine kinase and transfer of the phosphoryl group to response regulators to control flagellar rotation and receptor adaptation. The phosphotransfer domain, CheA1-134, of the chemotaxis-specific histidine autokinase CheA from Escherichia coli contains the site of phosphorylation, His48, and two other histidine residues, His26 and His67. Two-dimensional 1H-15N NMR techniques were applied to characterize the protonation states of these histidine residues and to evaluate the structural changes in the domain that occur upon phosphorylation of His48. The pKa of His48 was determined to be 7.8 (in 50 mM NaPO4 buffer at 30 degrees C). At high pH, its imidazole ring exists primarily as the normally unfavored N delta 1H tautomer, suggesting hydrogen bond formation to the ring nitrogen atom(s) to stabilize this state. The pKa values and predominant tautomeric states of the imidazole rings of His26 (pKa approximately 7.1, N epsilon 2H tautomer) and His67 (pKa approximately 6.5, N delta 1H tautomer) were also determined. His48 of CheA1-134 and CheA1-233 was phosphorylated by full-length CheA. The phosphorylation site was confirmed to be the N epsilon 2 position in the imidazole ring. Phosphorylation of His48 only results in small changes in the amide 1H and 15N chemical shifts of a few residues from helices B and C, suggesting that only very small changes in structure are associated with phosphorylation of the phosphotransfer domain of CheA. These residues occupy a small surface area of the helix bundle and form the active site of the protein. At the active site, in addition to His48, residues Gly52, His67, and Glu70 are conserved in the CheA homologous phosphotransfer domains from 10 different organisms. Sequence comparison of these CheA homologs suggest that the phosphotransfer domains likely fold in a similar helix-bundle structure and the structural features at the active site are well-conserved.
细菌趋化性涉及组氨酸激酶的自磷酸化以及磷酰基转移至响应调节因子，以控制鞭毛旋转和受体适应性。来自大肠杆菌的趋化性特异性组氨酸自激酶CheA的磷转移结构域CheA1-134包含磷酸化位点His48以及另外两个组氨酸残基His26和His67。应用二维1H-15N NMR技术来表征这些组氨酸残基的质子化状态，并评估His48磷酸化后结构域中发生的结构变化。His48的pKa值被确定为7.8（在30℃的50 mM NaPO4缓冲液中）。在高pH值下，其咪唑环主要以通常不太有利的Nδ1H互变异构体形式存在，这表明与环氮原子形成氢键以稳定该状态。还确定了His26（pKa约为7.1，Nε2H互变异构体）和His67（pKa约为6.5，Nδ1H互变异构体）咪唑环的pKa值和主要互变异构状态。CheA1-134和CheA1-233的His48被全长CheA磷酸化。磷酸化位点被确认为咪唑环中的Nε2位置。His48的磷酸化仅导致来自螺旋B和C的少数残基的酰胺1H和15N化学位移发生微小变化，这表明与CheA磷转移结构域的磷酸化相关的结构变化非常小。这些残基占据螺旋束的小表面积并形成蛋白质的活性位点。在活性位点，除了His48之外，来自10种不同生物体的CheA同源磷转移结构域中，残基Gly52、His67和Glu70是保守的。这些CheA同源物的序列比较表明，磷转移结构域可能折叠成类似的螺旋束结构，并且活性位点的结构特征得到了很好的保守。
