Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain of mouse P-glycoprotein: structure-activity relationships for flavonoid binding.
Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain (NBD2) of mouse P-glycoprotein were investigated by using two recombinantly expressed soluble proteins of different lengths and photoactive ATP analogues, 8-azidoadenosine triphosphate (8N(3)-ATP) and 2',3',4'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP). The two proteins, Thr(1044)-Thr(1224) (NBD2(short)) and Lys(1025)-Ser(1276) (NBD2(long)), both incorporated the four consensus sequences of ABC (ATP-binding cassette) transporters, Walker A and B motifs, the Q-loop, and the ABC signature, while differing in N-terminal and C-terminal extensions. Radioactive photolabeling of both proteins was characterized by hyperbolic dependence on nucleotide concentration and high-affinity binding with K(0.5)(8N(3)-ATP) = 36-37 microM and K(0.5)(TNP-8N(3)-ATP) = 0.8-2.6 microM and was maximal at acidic pH. Photolabeling was strongly inhibited by TNP-ATP (K(D) = 0.1-5 microM) and ATP (K(D) = 0.5-2.7 mM). Since flavonoids display bifunctional interactions at the ATP-binding site and a vicinal steroid-interacting hydrophobic sequence [Conseil, G., Baubichon-Cortay, H., Dayan, G., Jault, J.-M., Barron, D., and Di Pietro, A. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9831-9836], a series of 30 flavonoids from different classes were investigated for structure-activity relationships toward binding to the ATP site, monitored by protection against photolabeling. The 3-OH and aromaticity of conjugated rings A and C appeared important, whereas opening of ring C abolished the binding in all but one case. It can be concluded that the benzopyrone portion of the flavonoids binds at the adenyl site and the phenyl ring B at the ribosyl site. The Walker A and B motifs, intervening sequences, and small segments on both sides are sufficient to constitute the ATP site.
de Wet
H
H
Department of Chemical Pathology, University of Cape Town Medical School, Observatory 7925, Cape Town, South Africa.
McIntosh
D B
DB
Conseil
G
G
Baubichon-Cortay
H
H
Krell
T
T
Jault
J M
JM
Daskiewicz
J B
JB
Barron
D
D
Di Pietro
A
A
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000378:metabolism*; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000595:Amino Acid Sequence; D026923:Amino Acid Transport Systems, Basic; D000818:Animals; D001426:Bacterial Proteins; D001665:Binding Sites; D002599:Chalcone / Q000378:metabolism; D005419:Flavonoids / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry; D051379:Mice; D008956:Models, Chemical; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D019920:Photoaffinity Labels / Q000493:pharmacokinetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012486:Salmonella typhimurium / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D026923:Amino Acid Transport Systems, Basic; D001426:Bacterial Proteins; D005419:Flavonoids; D026901:Membrane Transport Proteins; D019920:Photoaffinity Labels; D011994:Recombinant Proteins; D002599:Chalcone; C043828:histidine permease, Bacteria; D000255:Adenosine Triphosphate