Park Sang-Youn, Quezada Cindy M, Bilwes Alexandrine M, Crane Brian R
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, USA.
Biochemistry. 2004 Mar 2;43(8):2228-40. doi: 10.1021/bi0352419.
Dimerization of the chemotaxis histidine kinase CheA is required for intersubunit autophosphorylation [Swanson, R. V., Bourret, R. B., and Simon, M. I. (1993) Mol. Microbiol. 8, 435-441]. Here we show that CheA dimers exchange subunits by the rate-limiting dissociation of a central four-helix bundle association domain (P3), despite the high stability of P3 versus unfolding. P3 alone determines the stability and exchange properties of the CheA dimer. For CheA proteins from the mesophile Escherichia coli and the thermophile Thermotoga maritima, subunit dissociation activates at temperatures where the respective organisms live (37 and 80 degrees C). Under destabilizing conditions, P3 dimer dissociation is cooperative with unfolding. Chemical denaturation is reversible for both EP3 and TP3. Aggregation accompanies thermal unfolding for both proteins under most conditions, but thermal unfolding is reversible and two-state for EP3 at low protein concentrations. Residue differences within interhelical loops may account for the contrasted thermodynamic properties of structurally similar EP3 and TP3 (41% sequence identity). Under stabilizing conditions, greater correlation between activation energy for dimer dissociation and P3 stability suggests more unfolding in the dissociation of EP3 than TP3. Furthermore, destabilization of extended conformations by glycerol slows relative dissociation rates more for EP3 than for TP3. Nevertheless, at physiological temperatures, neither protein likely unfolds completely during subunit exchange. EP3 and TP3 will not exchange subunits with each other. The receptor coupling protein CheW reduces the subunit dissociation rate of the T. maritima CheA dimer by interacting with the regulatory domain P5.
趋化组氨酸激酶CheA的二聚化是亚基间自磷酸化所必需的[斯旺森，R. V.，布雷特，R. B.，和西蒙，M. I.（1993年）《分子微生物学》8，435 - 441]。在此我们表明，尽管中央四螺旋束结合结构域（P3）相对于解折叠具有高稳定性，但CheA二聚体通过P3的限速解离来交换亚基。单独的P3决定了CheA二聚体的稳定性和交换特性。对于嗜温菌大肠杆菌和嗜热菌海栖热袍菌的CheA蛋白，亚基解离在各自生物体生存的温度（37和80摄氏度）下被激活。在不稳定条件下，P3二聚体解离与解折叠协同进行。化学变性对EP3和TP3两者都是可逆的。在大多数条件下，两种蛋白质的热解折叠都伴随聚集，但在低蛋白浓度下，EP3的热解折叠是可逆的且呈两态。螺旋间环内的残基差异可能解释了结构相似的EP3和TP3（序列同一性为41%）的对比热力学性质。在稳定条件下，二聚体解离的活化能与P3稳定性之间的更大相关性表明，EP3解离过程中的解折叠比TP3更多。此外，甘油对伸展构象的去稳定作用使EP3的相对解离速率比TP3减慢得更多。然而，在生理温度下，两种蛋白质在亚基交换过程中都不太可能完全解折叠。EP3和TP3不会相互交换亚基。受体偶联蛋白CheW通过与调节结构域P5相互作用降低了海栖热袍菌CheA二聚体的亚基解离速率。
