IN-VITRO INTERACTION BETWEEN COMPONENTS OF THE INNER MEMBRANE COMPLEXOF THE MALTOSE ABC TRANSPORTER OF ESCHERICHIA-COLI - MODULATION BY ATP

Interactions between domains of ATP-binding cassette (ABC) transporter s are of great functional importance and yet are poorly understood. To gain further knowledge of these protein-protein interactions, we stud ied the inner membrane complex of the maltose transporter of Escherich ia coli. We focused on interactions between the nucleotide-binding pro tein, MalK, and the transmembrane proteins, MalF and MalG. We incubate d purified MalK with inverted membrane vesicles containing MalF and Ma lG. MalK bound specifically to MalF and MalG and reconstituted a funct ional complex. We used this approach and limited proteolysis with tryp sin to show that binding and hydrolysis of ATP, inducing conformationa l changes in MalK, modulate its interaction with MalF and MalG. MalK i n the reconstituted complex was less sensitive to protease added from the cytoplasmic side of the membrane, and one proteolytic cleavage sit e located in the middle of a putative helical domain of MalK was prote cted. These results suggest that the putative helical domain of the nu cleotide-binding domains is involved, through its conformational chang es, in the coupling between the transmembrane domains and ATP binding/ hydrolysis at the nucleotide-binding domains.