STRUCTURAL FLEXIBILITY OF THE LINKER REGION OF HUMAN P-GLYCOPROTEIN PERMITS ATP HYDROLYSIS AND DRUG TRANSPORT

P-Glycoprotein (Pgp), an energy-dependent drug efflux pump responsible for multidrug resistance of many cancer cells, is comprised of two ho mologous halves connected by a peptide segment approximately 75 amino acids (aa) in length. The effects of length and composition of this co nnecting region on Pgp cell surface expression and the ability of the two halves to interact were explored using both stable transfections o f Pgp mutants in mammalian cell lines and a vaccinia virus transient e xpression system. A 17 aa insertion of predicted flexible structure be tween amino acids 681 and 682 resulted in a functional Pgp molecule th at was capable of conferring drug resistance. In contrast, an 18 aa pe ptide insertion with a predicted alpha-helical structure was unstable when expressed transiently. A 34 aa deletion from the central core of the Linker region (Delta 653-686) resulted in a protein expressed at t he cell surface in amounts comparable to that of wild-type Pgp but una ble to confer drug resistance. No apparent differences in drug or [alp ha-P-32]-8-azido-ATP photoaffinity labeling were observed. However, bo th ATP hydrolysis and drug transport activities of the deletion mutant were completely abrogated, indicating that the linker deletion discon nected substrate binding from ATP hydrolysis and transport. This mutan t also failed to exhibit an ATP hydrolysis-dependent enhancement of bi nding of a conformation-sensitive monoclonal antibody, UIC2. Upon repl acement with a 17 aa linker peptide having a predicted flexible second ary structure, but bearing no homology to the deleted 34 aa segment, n ormal Pgp transport and basal and drug-stimulated ATPase activities we re restored along with increased UIC2 binding in the presence of subst rate, suggesting a dramatic conformational change between the nonfunct ional and functional molecules. Taken together, these data suggest a f lexible secondary structure of the connector region is sufficient for the coordinate functioning of the two halves of Pgp, likely specifical ly required for the proper interaction of the two ATP binding sites.