It is known from earlier work that two conserved Glu residues, designated "catalytic carboxylates," are critical for function in P-glycoprotein (Pgp). Here the role of these residues (Glu-552 and Glu-1197 in mouse MDR3 Pgp) was studied further. Mutation E552Q or E1197Q reduced Pgp-ATPase to low but still measurable rates. Two explanations previously offered for effects of these mutations, namely that ADP release is slowed or that a second (drug site-resetting) round of ATP hydrolysis is blocked, were evaluated and appeared unsatisfactory. Thus the study was extended to include E552A, -D, and -K and E1197A, -D, and -K mutants. All reduced ATPase to similar low but measurable rates. Orthovanadate-trapping experiments showed that mutation to Gln, Ala, Asp, or Lys altered characteristics of the transition state but did not eliminate its formation in contrast e.g. with mutation of the analogous catalytic Glu in F 1 -ATPase. Retention of ATP as well as ADP was seen in Ala, Asp, and Lys mutants. Mutation E552A in nucleotide binding domain 1 (NBD1) was combined with mutation S528A or S1173A in the LSGGQ sequence of NBD1 or NBD2, respectively. Synergistic effects were seen. E552A/S1173A had extremely low turnover rate for ATPase, while E552A/S528A showed zero or close to zero ATPase. Both showed orthovanadate-independent retention of ATP and ADP. We propose that mutations of the catalytic Glu residues interfere with formation and characteristics of a closed conformation, involving an interdigitated NBD dimer interface, which normally occurs immediately following ATP binding and progresses to the transition state.

Functional roles of the two ABC signature sequences ("LSGGQ") in the N-and C-terminal nucleotide binding domains of P-glycoprotein were studied by mutating the conserved Ser residues to Ala. The two single mutants (S528A; S1173A) each impaired ATPase activity mildly, and showed generally symmetrical effects on function, consistent with equivalent mechanistic roles of the two nucleotide sites. Synergy between the two mutations when combined was remarkable and resulted in strong catalytic impairment. P-glycoprotein (Pgp)1 is a plasma membrane-located efflux pump which confers multidrug resistance in human cancers by virtue of its ability to exclude chemotherapeutic drugs from cells in an ATP hydrolysis-dependent manner. In recent years it has been recognized as a general mechanism for protecting the body from hydrophobic toxins, playing a particularly important role in specific tissues such as the central nervous system. Its impact on therapy for other diseases, e.g. AIDS, and its potential impact on novel drug therapies in general, have generated significant interest in studies of its structure and mechanism, with the underlying aims of understanding its physiological function and development of new strategies for circumventing or disabling the protein (1-4).Pgp is a member of the ABC transporter superfamily (5), and consists of two six-helix membrane domains and two nucleotide-binding domains (NBDs) contained in one continuous ϳ1280-residue polypeptide. The two NBDs are ϳ60% identical in sequence and each contain Walker A and B consensus sequences, both of which are known to be intimately involved in and required for the ATP hydrolysis reaction (6 -11). Inactivation of the ATP hydrolysis reaction by mutations in these sequences leads, as one would predict, to commensurate loss of ATP-driven drug efflux from cells. Between the Walker A and B sequences is found a third conserved sequence ("LSGGQ") which is named the ABC signature sequence, because it is the hallmark of the ABC transporter superfamily (5, 12). The very strong conservation of this sequence in evolution implies an important role, but what that role is has not yet been determined.New ideas about the role of the ABC signature sequence have come from x-ray crystallography studies and from photocleavage and cross-linking studies. At the time of writing no highresolution structure of Pgp is available, but structures of the dimeric NBD-containing subunits of Rad50, MalK, and thermophilic MJ0796 (13-15) and of the complete ABC transporter BtuCD (16) have been published, and show a most interesting feature, which is that in the dimeric NBD arrangement seen in these structures, the ABC signature sequence of NBD2 lies juxtaposed to the Walker A sequence of NBD1, and vice versa. The two bound nucleotides (where present) are sandwiched between the Walker A and ABC signature sequences, with the hydroxyl side-chain group of the Ser residue of the ABC signature sequence coming close enough to the ␥-phosphate of ATP to possibly form an H-bond with a ␥-phosphate oxy...

Kinetics of inhibition ofP-glycoprotein (Pgp) 1 is a prominent member of the ABCtransporter family of membrane proteins that uses the energy of ATP hydrolysis to exclude hydrophobic compounds from cells. In human, it was first recognized as a major potential obstacle to successful chemotherapy of cancer because of its expression in cancer cells and was later realized to function physiologically in such strategic locations as the blood-brain barrier, intestine, placenta, and elsewhere as an important agent in protection from environmental and dietary toxins. More recent appreciation of its role in impeding the action of anti-AIDS therapy and its potentially general role in reducing the efficacy of many hydrophobic drugs, new and old, have made it a target of intense investigation. Recent reviews of the biochemistry and pharmacology of Pgp may be found in Refs. 1-6.Our research has focused on the catalytic mechanism by which ATP is hydrolyzed and the energy transduced into drug transport. In 1995, we presented schemes for the hydrolysis of ATP at a single catalytic site (7), also for the interaction of the two ATP-binding sites in catalysis and transduction of the energy of hydrolysis to the drug binding site(s) situated in the membrane bilayer (8). Both ATP-binding sites were shown to have catalytic capability. Release of product P i was thought to occur before the release of ADP, which was rate-limiting. Interaction of the two ATP-binding sites was concluded to be an integral and a necessary facet of catalysis with hydrolysis of ATP occurring sequentially and alternately at each site. Formation and collapse of the transition state of catalysis were postulated as critical events in driving the changes in drugbinding site affinity and orientation (inward versus outward facing) necessary for binding and extrusion of drug with a stoichiometry of one ATP hydrolyzed per change in drug-binding site affinity and orientation. Later studies of Pgp and also of ABC transporter homologs such as bacterial LmrA and the maltose transporter have supported and extended the earlier schemes (9 -11). New ideas incorporated into more recent schemes include the concept of reciprocating pairs of drugbinding sites (10) and of the requirement for a second ATP hydrolysis event to "reset" the drug-binding site for drug extrusion (9). Another concept, recently introduced, envisages that the ATP-binding event provides the primary driving force for transport (12). Electron microscopy studies provide evidence for conformational changes in the membrane domains observed upon nucleotide binding (12); however, biochemical evidence has been provided showing that the drug-binding site also changes conformation in response to ATP hydrolysis per se (13,14).Recent work (15) demonstrated that, at concentrations of MgATP and MgADP present in the cytoplasm of mammalian cells, both nucleotide-binding domains (NBD) in "resting" Pgp would be expected to bind MgATP (15). Our original scheme for ATP-driven drug transport (8) envisaged that upon attainment of...