Phosphotransfer and CheY-binding domains of the histidine autokinase CheA are joined by a flexible linker

Multidimensional heteronuclear NMR techniques were applied to study a protein fragment of the histidine autokinase CheA from Escherichia coli. This fragment (CheA(1-233)) contains the phosphotransfer domain and the CheY-binding domain joined by a linker region. Comparison of chemical shift and NOE cross-peak patterns indicates that the structures of the two domains in CheA(1-233) remain nearly the same as in the two individual domain fragments, CheA(1-134) and CheA(124-257). Relaxation properties of the backbone N-15 nuclei were measured to study the rotational correlations of the two domains and properties of the linker region. Dynamics data were analyzed both by an isotropic motional model and an anisotropic motional model. The experimental T-1 and T-2 values, the derived rotational correlation times, and motional anisotropy are significantly different for the two domains, indicating the two domains reorient independently and the linker region is highly flexible. Dynamics data of CheA(1-233) were also compared with those of CheA(1-134). Our studies show that flexible domain linkers and extended and flexible terminal polypeptide chains can have significant effects on the motional properties of the adjacent structured regions. These observations suggest a model for the graded regulation of CheA autophosphorylation activity. In this model, the various activity states of the receptor are generated by controling the access of the mean position of the kinase domain to the phosphotransfer domain. This would then modulate the diffusional encounter rate of the domains and hence activity over a wide and graded range of values.