Investigation of the Role of Glutamine-471 and Glutamine-1114 in the Two Catalytic Sites of P-Glycoprotein

Abstract

P-glycoprotein, also known as multidrug resistance protein, pumps drugs out of cells using ATP hydrolysis as the energy source. Glutamine-471 and the corresponding glutamine-1114 in the two catalytic sites of P-glycoprotein are conserved in ABC transporters. X-ray structures show that they lie close to the bound nucleotide. Proposed functional roles are (1) activation of the attacking water for ATP hydrolysis, (2) coordination of the essential Mg(2+) cofactor in Mg nucleotide, and (3) signal communication between catalytic site reaction chemistry and drug-binding sites. We made mutations Q471A, Q471E, Q1114A, and Q1114E in mouse MDR3 P-glycoprotein. Pure mutant and wild-type proteins were prepared and subjected to enzymatic and biochemical characterization. We conclude from the results that the primary role of this glutamine residue is in interdomain signal communication. Coordination of the Mg(2+) cofactor is not a critical functional role, neither is activation of the attacking water molecule, although an auxiliary role in positioning the water cannot be ruled out. We found that equivalent mutations (Ala or Glu) in either of the two P-glycoprotein catalytic sites produced the same effects, implying functional symmetry of the two sites.