Molecular modeling correctly predicts the functional importance of Phe594 in transmembrane helix 11 of the multidrug resistance protein, MRP1 (ABCC1).
The human ATP-binding cassette (ABC) transporter, multidrug resistance protein 1 (MRP1/ABCC1), confers resistance to a broad range of anti-cancer agents and transports a variety of organic anions. At present, essentially no structural data exists for MRP1 that might be used to elucidate its mechanism of transport. Consequently, we have applied a modeling strategy incorporating crystal and indirect structural data from other ABC transporters to construct a model of the transmembrane domains of the core region of MRP1 that includes the amino acid side chains. Three conserved Trp residues and one non-conserved Tyr residue, shown previously to be of functional importance (Koike, K., Oleschuk, C. J., Haimeur, A., Olsen, S. L., Deeley, R. G., and Cole, S. P. C. (2002) J. Biol. Chem. 277, 49495-49503), were found to line the "pore" in our model proximal to the membrane cytosol interface. A fifth aromatic residue (Phe594) was identified that, with the Trp and Tyr residues, completed a ring or "basket" of aromatic amino acids and, accordingly, we postulated that it would also be of functional importance. To test this idea, MRP1-Phe594 mutants were expressed in human embryonic kidney cells, and their properties were examined using membrane vesicles. Substitution of Phe594 with Ala substantially reduced or eliminated the transport of five organic anion substrates by MRP1 and abrogated the binding of leukotriene C4. On the other hand, the conservatively substituted F594W and F594Y mutants remained transport competent, although significant substrate- and substitution-specific changes were observed. These studies provide some structural insight into a possible substrate binding/transport site of MRP1 at the beginning of a putative substrate translocation pathway and demonstrate the usefulness of modeling for directing structure-function analyses of this transporter.
Campbell
Jeff D
JD
Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.
Koike
Koji
K
Moreau
Christophe
C
Sansom
Michael S P
MS
Deeley
Roger G
RG
Cole
Susan P C
SP
D000345:Affinity Labels; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D017124:Conserved Sequence; D017931:DNA Primers; D006801:Humans; D017997:Leukotriene C4; D008958:Models, Molecular; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry
D000345:Affinity Labels; D017931:DNA Primers; D017997:Leukotriene C4; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010649:Phenylalanine; D011994:Recombinant Proteins; C421109:multidrug resistance-associated protein 1