Attempts to characterize the NBD heterodimer of MRP1: transient complex formation involves Gly⁷⁷¹ of the ABC signature sequence but does not enhance the intrinsic ATPase activity

MRP1 (multidrug-resistance-associated protein 1; also known as ABCC1) is a member of the human ABC (ATP-binding cassette) transporter superfamily that confers cell resistance to chemotherapeutic agents. Considering the structural and functional similarities to the other ABC proteins, the interaction between its two NBDs (nucleotide-binding domains), NBD1 (N-terminal NBD) and NBD2 (C-terminal NBD), is proposed to be essential for the regulation of the ATP-binding/ATP-hydrolysis cycle of MRP1. We were interested in the ability of recombinant NBD1 and NBD2 to interact with each other and to influence ATPase activity. We purified NBD1 (Asn⁶⁴²–Ser⁸⁷¹) and NBD2 (Ser¹²⁸⁶–Val¹⁵³¹) as soluble monomers under native conditions. We measured extremely low intrinsic ATPase activity of NBD1 (10⁻⁵ s⁻¹) and NBD2 (6×10⁻⁶ s⁻¹) and no increase in the ATP-hydrolysis rate could be detected in an NBD1+NBD2 mixture, with concentrations up to 200 μM. Despite the fact that both monomers bind ATP, no stable NBD1·NBD2 heterodimer could be isolated by gel-filtration chromatography or native-PAGE, but we observed some significant modifications of the heteronuclear single-quantum correlation NMR spectrum of ¹⁵N-NBD1 in the presence of NBD2. This apparent NBD1·NBD2 interaction only occurred in the presence of Mg²⁺ and ATP. Partial sequential assignment of the NBD1 backbone resonances shows that residue Gly⁷⁷¹ of the LSGGQ sequence is involved in NBD1·NBD2 complex formation. This is the first NMR observation of a direct interaction between the ABC signature and the opposite NBD. Our study also reveals that the NBD1·NBD2 heterodimer of MRP1 is a transient complex. This labile interaction is not sufficient to induce an ATPase co-operativity of the NBDs and suggests that other structures are required for the ATPase activation mechanism.