Expression and Characterization of the N- and C-Terminal ATP-Binding Domains of MRP1

Multiple flavonoid-binding sites within multidrug resistance protein MRP1

“…Our studies have therefore focused on nucleotide binding in order to characterize the interaction of flavonoids within NBDs. The high-affinity binding observed, in the micromolar concentration range, for the hydrophobic TNP derivative of ATP was comparable to that found previously with NBDs fused to maltose-binding protein [42], as well as other NBDs from P-glycoproteins [25,36,37] or CFTR [45,46]. The millimolar dissociation constants for ATP, which is able to almost completely displace bound TNP-ATP, are also in the same range as K m values for ATP hydrolysis by either recombinant…”

“…NBDs [41,42,44] or full-length MRP1 [39,47]; this further confirms that TNP-ATP indeed binds to the ATP site.…”

“…In contrast, a similar NBD2 length (234 -243 residues) was used by all groups. The highly critical conditions of solubility might therefore be responsible for the failure to ob- tain soluble NBD2 either by direct overexpression [42] or after thrombin cleavage [41], whereas it was successfully obtained in a cytosolic fraction from insect cells [9,43]. NBD2 indeed appears here to be intrinsically much less soluble than NBD1.…”

“…A slightly higher activity was reported for fusions with either maltose-binding protein or gluthathione S-transferase, but contradictory results were obtained with respect to vanadate and N-ethylmaleimide inhibitions [41,42,44]. In addition, no differential behavior of NBDs could be found [41,42], whereas the two ATP sites have clearly distinct roles within the whole MRP1 transporter; this was certainly due to the lack of cooperativity [12] which requires interactions between the two NBDs. Consequently, the ATPase activity of isolated NBDs is not suited to monitor events occurring within the entire MRP1.…”

“…The length of the NBD1 sequence appears to be critical for solubility of the overexpressed protein, since a shorter domain lacking the 11-residue Nterminal hydrophilic sequence was much less soluble than the 254-residue NBD1 (data not shown). This may explain, at least partly, why Kern et al [42] recovered a quite short 200-residue protein only in inclusion bodies, whereas a longer unfused NBD1, of 346 residues, was soluble after liberation by thrombin cleavage [41]. In contrast, a similar NBD2 length (234 -243 residues) was used by all groups.…”