ATP Binding to the First Nucleotide Binding Domain of Multidrug Resistance-associated Protein Plays a Regulatory Role at Low Nucleotide Concentration, whereas ATP Hydrolysis at the Second Plays a Dominant Role in ATP-dependent Leukotriene C4 Transport

• Published in: The Journal of biological chemistry 2003-08, Vol.278 (33), p.30764-30771
• Main Authors: Yang, Runying, Cui, Liying, Hou, Yue-xian, Riordan, John R., Chang, Xiu-bao
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Amino Acid Sequence; Amino Acid Substitution; Animals; Binding Sites; Biological Transport - physiology; Cell Line; Cricetinae; Cysteine - genetics; Enzyme Inhibitors - pharmacology; Ethylmaleimide - pharmacology; Humans; Hydrolysis; Kidney - cytology; Leukotriene C4 - metabolism; Mutagenesis; Protein Structure, Tertiary
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M304118200

Multidrug resistance-associated protein (MRP1) transports solutes in an ATP dependent manner by utilizing its two nonequivalent nucleotide binding domains (NBDs) to bind and hydrolyze ATP. The two NBDs possess different properties (Gao, M., Cui, H. R., Loe, D. W., Grant, C. E., Almquist, K. C., Cole, S. P., and Deeley, R. G. (2000) J. Biol. Chem. 275, 13098–13108; Hou, Y., Cui, L., Riordan, J. R., and Chang, X. (2000) J. Biol. Chem. 275, 20280–20287) and may play different roles during solute transport. We now report that NBD1 has moderately higher affinity for ATP than NBD2. The consequence of this difference is that the overall Kd value for wild-type MRP1 is mainly determined by ATP binding at NBD1. This conclusion is supported by the following: 1) mutation of the cysteine residue at 682 to alanine (C682A) in Walker A motif in NBD1 decreases the Kd value, indicating increased affinity for ATP; 2) mutation of the alanine residue at 1331 to cysteine (A1331C) in the Walker A motif of NBD2 does not have an effect on the Kd value; and 3) photolabeling of the protein with a cysteine residue in the Walker A motif of NBD1 is much more sensitive to N-ethylmaleimide modification than the protein with a cysteine residue in the Walker A motif of NBD2. In contrast, the Km for ATP in support of LTC4 transport is mainly determined by ATP hydrolysis at NBD2. This conclusion is supported by the following: 1) although mutation of A1331C does not have an effect on the Kd value, the Km values measured from LTC4 transport by proteins with this mutation in NBD2 are much higher than the proteins with wild-type NBD2, implying that the A1331C mutation affects ATP binding/hydrolysis at NBD2; and 2) ATP-dependent LTC4 transport by the protein with a cysteine residue in the Walker A motif of NBD2 is much more sensitive to N-ethylmaleimide modification than the protein with a cysteine residue in the Walker A motif of NBD1. Our previous results indicated that ATP binding at NBD1 at low concentration enhanced ATP binding/hydrolysis at NBD2. All of these results support the notion that ATP binding at NBD1 at low concentration plays a more important regulatory role than the binding at high ATP concentration and that ATP hydrolysis at NBD2 plays a dominant role in the ATP-dependent LTC4 transport.