Interaction between the bound Mg.ATP and the Walker A serine residue in NBD2 of multidrug resistance-associated protein MRP1 plays a crucial role for the ATP-dependent leukotriene C4 transport.
Structural analysis of human MRP1-NBD1 revealed that the Walker A S685 forms a hydrogen bond with the Walker B D792 and interacts with the Mg (2+) cofactor and the beta-phosphate of the bound Mg.ATP. We have found that substitution of the S685 with an amino acid that potentially prevents the formation of the hydrogen bond resulted in misfolding of the protein and significantly affect the ATP-dependent leukotriene C4 (LTC4) transport. In this report we tested whether the corresponding substitution in NBD2 would also result in misfolding of the protein. In contrast to the NBD1 mutations, none of the mutations in NBD2, including S1334A, S1334C, S1334D, S1334H, S1334N, and S1334T, caused misfolding of the protein. However, elimination of the hydroxyl group at S1334 in mutations including S1334A, S1334C, S1334D, S1334H, and S1334N drastically reduced the ATP binding and the ATP-enhanced ADP trapping at the mutated NBD2. Due to this low efficient ATP binding at the mutated NBD2, the inhibitory effect of ATP on the LTC4 binding is significantly decreased. Furthermore, ATP bound to the mutated NBD2 cannot be efficiently hydrolyzed, leading to almost completely abolishing the ATP-dependent LTC4 transport. In contrast, S1334T mutation, which retained the hydroxyl group at this position, exerts higher LTC4 transport activity than the wild-type MRP1, indicating that the hydroxyl group at this position plays a crucial role for ATP binding/hydrolysis and ATP-dependent solute transport.
Yang
Runying
R
Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 13400 East Shea Boulevard, Scottsdale, Arizona 85259, USA.
Scavetta
Robert
R
Chang
Xiu-bao
XB
D000255:Adenosine Triphosphate / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D006224:Cricetinae; D006801:Humans; D017997:Leukotriene C4 / Q000235:genetics / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D012694:Serine / Q000235:genetics / Q000378:metabolism*
D000255:Adenosine Triphosphate; D017997:Leukotriene C4; D008274:Magnesium; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D012694:Serine; C421109:multidrug resistance-associated protein 1