Allosteric Interactions between the Two Non-equivalent Nucleotide Binding Domains of Multidrug Resistance Protein MRP1

Abstract

Membrane transporters of the adenine nucleotide binding cassette (ABC) superfamily utilize two either identical or homologous nucleotide binding domains (NBDs). Although the hydrolysis of ATP by these domains is believed to drive transport of solute, it is unknown why two rather than a single NBD is required. In the well studied P-glycoprotein multidrug transporter, the two appear to be functionally equivalent, and a strongly supported model proposes that ATP hydrolysis occurs alternately at each NBD (Senior, A. E., al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett 377, 285-289). To assess how applicable this model may be to other ABC transporters, we have examined adenine nucleotide interactions with the multidrug resistance protein, MRP1, a member of a different ABC family that transports conjugated organic anions and in which sequences of the two NBDs are much less similar than in P-glycoprotein. Photoaffinity labeling experiments with 8-azido-ATP, which strongly supports transport revealed ATP binding exclusively at NBD1 and ADP trapping predominantly at NBD2. Despite this apparent asymmetry in the two domains, they are entirely interdependent as substitution of key lysine residues in the Walker A motif of either impaired both ATP binding and ADP trapping. Furthermore, the interaction of ADP at NBD2 appears to allosterically enhance the binding of ATP at NBD1. Glutathione, which supports drug transport by the protein, does not enhance ATP binding but stimulates the trapping of ADP. Thus MRP1 may employ a more complex mechanism of coupling ATP utilization to the export of agents from cells than P-glycoprotein.The multidrug resistance protein, MRP1, 1 is believed to function as an active exporter of many conjugated organic anions from cells (1-7). Among agents that are transported are also some unconjugated compounds, including certain cancer drugs, provided that glutathione is also present (8, 9). In this case, the hydrophilic conjugating compound such as glutathione may be co-transported along with the hydrophobic drug (8). As a member of the ABC super family of membrane transporters (10), MRP1 is an ATPase that is stimulated by agents that it transports (11, 12). However, as yet there is little further information about how the two nucleotide binding domains of the protein act to bring about the transport event. In the case of the better studied P-glycoprotein, which belongs to a different family of this super family (13), the two NBDs have been shown to be functionally equivalent with identical ATP hydrolysis steps occurring alternatively at each domain (14, 15). According to an insightful model based largely on this information, binding and translocation of the hydrophobic compound exported is controlled in an ordered fashion by the two hydrolysis steps (15). The objective of the present study was to determine whether MRP1 performs a similar symmetrical cycle of partial reactions of ATP hydrolysis to accomplish the export of conjugated organic anions. To follow the interaction of the nucleotide substr...

The First Nucleotide Binding Domain of Cystic Fibrosis Transmembrane Conductance Regulator Is a Site of Stable Nucleotide Interaction, whereas the Second Is a Site of Rapid Turnover

“…both domains are labeled in the absence of added vanadate; this confirms earlier findings (15,16) and hence is not dwelt upon here. However, it is significant to note that this is different from the situation for P-glycoprotein and MRP1, where vanadate is necessary for NDP trapping (6,12). The third major point made by the results in Fig.…”

“…The retention of activity by one domain when the other is compromised parallels the channel gating that persists when either one of the domains is similarly mutagenized (30), which, along with other observations, has led to the suggestion that either of the NBDs may gate the channel (23). Such individual action of the two domains is different from the situation with one other ABCC subfamily relative of CFTR, the MRP1-conjugated anion transporter in which nucleotide interactions at one NBD strongly influence those at the other (11)(12)(13). It is possible that this type of allosteric coupling between NBDs in CFTR could require phosphorylation by cAMP-dependent protein kinase, which is essential for channel activation under normal circumstances (42).…”

“…Indeed, in the P-glycoprotein multidrug transporter in which studies supporting each of these models have been performed, the amino acid sequences of the two domains are very similar and only minor functional asymmetry has been observed (8). However, in some other members of the large family, including the ABCC subfamily, this similarity is far less and distinctive properties have been observed in the case of SUR1 (9, 10), MRP1 (11)(12)(13)(14), and CFTR (15,16). Utilizing [␣- 32 P]8-N 3 ATP, both NBDs of CFTR could be photolabeled (15,16).…”

Distinct functions and cooperative interaction of the subunits of the transporter associated with antigen processing (TAP)

“…This biochemical asymmetry suggests a functional dichotomy between the two NBDs of CFTR, SUR1, and MRP1. Functional studies have confirmed that the NBDs of these proteins are non-equivalent, each involved in a different stage of the transport or conductance cycle (29,(40)(41)(42).…”

“…Vanadate may trap the hydrolysis product of the photolabel, [␣-32 P]8N 3 ADP, in the NBD, or, if a cooperative interaction exists between two NBDs, the effect may be indirect. For many ABC transporters, binding of ADP to one NBD leads to an increase in ATP binding by the second (27)(28)(29). By trapping an ADP at one NBD, vanadate can enhance photolabeling of the second by this increase in affinity.…”