Replacement of the positively charged Walker A lysine residue with a hydrophobic leucine residue and conformational alterations caused by this mutation in MRP1 impair ATP binding and hydrolysis

Frederic Buyse, Yue Xian Hou, Catherine Vigano, Qing Zhao, Jean Marie Ruysschaert, Xiu Bao Chang

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

MRP1 (multidrug resistance protein 1) couples ATP binding/ hydrolysis at its two non-equivalent NBDs (nucleotide-binding domains) with solute transport. Some of the NBD1 mutants, such as W653C, decreased affinity for ATP at the mutated site, but increased the rate of ATP-dependent solute transport. In contrast, other NBD1 mutants, such as K684L, had decreased ATP binding and rate of solute transport. We now report that mutations of the Walker A lysine residue, K684L and K1333L, significantly alter the tertiary structure of the protein. Due to elimination of the positively charged group and conformational alterations, the K684L mutation greatly decreases the affinity for ATP at the mutated NBD1 and affects ATP binding at the unmutated NBD2. Although K684L-mutated NBD1 can bind ATP at higher concentrations, the bound nucleotide at that site is not efficiently hydrolysed. All these alterations result in decreased ATP-dependent solute transport to approx. 40% of the wild-type. In contrast, the K1333L mutation affects ATP binding and hydrolysis at the mutated NBD2 only, leading to decreased ATP-dependent solute transport to approx. 11% of the wild-type. Consistent with their relative transport activities, the amount of vincristine accumulated in cells is in the order of K1333L ≥ CFTR (cystic fibrosis transmembrane conductance regulator) > K684L ≫ wild-type MRP1. Although these mutants retain partial solute transport activities, the cells expressing them are not multidrug-resistant owing to inefficient export of the anticancer drugs by these mutants. This indicates that even partial inhibition of transport activity of MRP1 can reverse the multidrug resistance caused by this drug transporter.

Original languageEnglish (US)
Pages (from-to)121-130
Number of pages10
JournalBiochemical Journal
Volume397
Issue number1
DOIs
StatePublished - Jul 1 2006

Keywords

  • ATP binding/hydrolysis
  • ATP-dependent transport
  • Conformation
  • Multidrug resistance
  • Multidrug resistance protein 1 (MRP1)
  • Nucleotide-binding domain

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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