Expression, purification, and characterization of cysteine-free mouse P-glycoprotein.
Cysteine-free mouse MDR3 P-glycoprotein (Pgp) was constructed by mutagenesis of the nine natural Cys to Ala. The Cys-free protein was expressed in Pichia pastoris and purified. Yield, purity, ATPase activity, K(m)(MgATP), and stimulation of ATPase by verapamil, were similar to wild-type mouse Ppg. Mouse Cys-free Pgp was superior in yield and stability to Cys-free human MDR1 Pgp. Mutants Y1040A and Y1040C were constructed in mouse Cys-free Pgp background. Both showed extremely low ATPase activity, strongly-impaired vanadate-trapping of ADP, and reduced photolabeling by 8-azido-ATP. The results are consistent with the conclusion that Tyr-1040 is located in the MgATP-binding site in NBD2 and is required for correct binding and/or orientation of bound MgATP substrate in Pgp as previously suggested by X-ray structures of other ABC transporters and by sequencing of photolabeled Pgp. The results also support our previous conclusion that both catalytic sites must be intact for normal function in Pgp.
Tombline
Gregory
G
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY 14642, USA.
Urbatsch
Ina L
IL
Virk
Navneet
N
Muharemagic
Alma
A
White
Lori Bartholomew
LB
Senior
Alan E
AE
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics; D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D019943:Amino Acid Substitution; D000818:Animals; D001386:Azides / Q000737:chemistry; D020134:Catalytic Domain; D003545:Cysteine / Q000235:genetics*; D006801:Humans; D051379:Mice; D009154:Mutation; D019920:Photoaffinity Labels / Q000737:chemistry; D010843:Pichia / Q000378:metabolism
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D001386:Azides; D019920:Photoaffinity Labels; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; C095122:multidrug resistance protein 3; D003545:Cysteine