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 (CheA1-233) 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 CheA1-233 remain nearly the same as in the two individual domain fragments, CheA1-134 and CheA124-257. Relaxation properties of the backbone 15N 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 T1 and T2 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 CheA1-233 were also compared with those of CheA1-134. 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 controlling 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.