LIBERATION OF AN INTERACTION DOMAIN FROM THE PHOSPHOTRANSFER REGION OF CHEA, A SIGNALING KINASE OF ESCHERICHIA-COLI

The CheA protein of Escherichia coli is a histidine autokinase that do nates its phosphate groups to two target proteins, CheY and CheB, to r egulate flagellar rotation and sensory adaptation during chemotactic r esponses. The amino-terminal third of CheA contains the autophosphoryl ation site, determinants needed to interact with the catalytic center of the molecule, and determinants needed for specific recognition of i ts phosphorylation targets. To understand the structural basis for the se activities, we examined the domain organization of the CheA phospho transfer region by using DNA sequence analysis, limited proteolytic di gestion, and a genetic technique called domain liberation. Comparison of the functionally interchangeable CheA proteins of E. coli and Salmo nella typhimurium revealed two extensively mismatched segments within the phosphotransfer region, 22 and 25 aa long, with sequences characte ristic of domain linkers. Both segments were readily susceptible to pr oteases, implying that they have an extended, flexible structure. In c ontrast, the intervening segments of the phosphotransfer region, desig nated P1 and P2 (roughly 140 and 65 aa, respectively), were relatively insensitive, suggesting they correspond to more compactly folded stru ctural domains. Their functional properties were explored by identifyi ng portions of the cheA coding region capable of interfering with chem otactic behavior when ''liberated'' and expressed as polypeptides. P1 fragments were not inhibitory, but P2 fragments blocked the interactio n of CheY with the rotational switch at the flagellar motor, leading t o incessant forward swimming. These results suggest that P2 contains C heY-binding determinants which are normally responsible for phosphotra nsfer specificity. Domain-liberation approaches should prove generally useful for analyzing multidomain proteins and their interaction targe ts.