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Nucleotide binding, ATP hydrolysis, and mutation of the catalytic carboxylates of human P-glycoprotein cause distinct conformational changes in the transmembrane segments.
- Source :
-
Biochemistry [Biochemistry] 2007 Aug 14; Vol. 46 (32), pp. 9328-36. Date of Electronic Publication: 2007 Jul 18. - Publication Year :
- 2007
-
Abstract
- P-Glycoprotein (P-gp, ABCB1) transports a variety of structurally unrelated cytotoxic compounds out of the cell. Each homologous half of P-gp has a transmembrane (TM) domain containing six TM segments and a nucleotide-binding domain (NBD) and is joined by a linker region. It has been postulated that binding of two ATP molecules at the NBD interface to form a "nucleotide sandwich" induces drug efflux by altering packing of the TM segments that make up the drug-binding pocket. To test if ATP binding alone could alter packing of the TM segments, we introduced catalytic carboxylate mutations (E556Q in NBD1 and E1201Q in NBD2) into double-cysteine mutants that exhibited ATP-dependent cross-linking so that the mutants could bind but not hydrolyze ATP. It was found that ATP binding alone could alter disulfide cross-linking between the TM segments. For example, ATP inhibited cross-linking of mutant L339C(TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2) but promoted cross-linking of mutant F343C(TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2). Cross-linking of some mutants, however, appeared to require ATP hydrolysis as introduction of the catalytic carboxylate mutations into mutant L332C(TM6)/L975C(TM12) inhibited ATP-dependent cross-linking. Cross-linking between cysteines in the TM segments also could be altered via introduction of a single catalytic carboxylate mutation into mutant L332C(TM6)/L975C(TM12) or by using the nonhydrolyzable ATP analogue, AMP.PNP. The results show that the TM segments are quite sensitive to changes within the ATP-binding sites because different conformations could be detected in the presence of ATP, AMP.PNP, during ATP hydrolysis or through mutation of the catalytic carboxylates.
- Subjects :
- ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism
Amino Acid Substitution drug effects
Amino Acid Substitution genetics
Catalysis
Cell Line
Cross-Linking Reagents metabolism
Cysteine chemistry
Cysteine genetics
Cysteine metabolism
Disulfides metabolism
Energy Metabolism genetics
Extracellular Space chemistry
Extracellular Space genetics
Extracellular Space metabolism
Humans
Hydrolysis
Membrane Proteins genetics
Membrane Proteins metabolism
Peptide Fragments genetics
Peptide Fragments metabolism
Protein Binding drug effects
Protein Binding genetics
Protein Conformation drug effects
Protein Structure, Tertiary drug effects
Protein Structure, Tertiary genetics
Vinblastine metabolism
ATP Binding Cassette Transporter, Subfamily B, Member 1 chemistry
ATP Binding Cassette Transporter, Subfamily B, Member 1 genetics
Adenosine Triphosphate metabolism
Carboxylic Acids metabolism
Membrane Proteins chemistry
Mutation
Nucleotides metabolism
Peptide Fragments chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2960
- Volume :
- 46
- Issue :
- 32
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 17636884
- Full Text :
- https://doi.org/10.1021/bi700837y