An Outward-Facing Aromatic Amino Acid Is Crucial for Signaling between the Membrane-Spanning and Nucleotide-Binding Domains of Multidrug Resistance Protein 1 (MRP1; ABCC1)

Abstract

The 190-kDa human MRP1 is an ATP-binding cassette multidrug and multiorganic anion efflux transporter. The 17 transmembrane helices of its three membrane-spanning domains, together with its two nucleotide binding domains (NBDs), form a stabilizing network of domain-domain interactions that ensure substrate binding in the cytoplasm is efficiently coupled to ATP binding and hydrolysis to effect solute efflux into the extracellular milieu. Here we show that Ala substitution of Phe in an outward-facing loop between the two halves of the transporter essentially eliminates the binding of multiple organic anions by MRP1. Conservative substitutions with Trp and Tyr had little or no effect. The F583A mutation also caused a substantial increase in orthovanadate-induced trapping of azidoADP by the cytoplasmic NBDs of MRP1, although the binding of ATP was unaffected. These observations indicate that the loss of the aromatic side chain at position 583 impairs the release of ADP and thus effectively locks the transporter in a low-affinity solute binding state. Phe is the first outward-facing amino acid in MRP1 found to be critical for its transport function. Our data provide evidence for long-range coupling, presumably via allosteric interaction, between this outward-facing region of MRP1 and both the solute binding and nucleotide binding regions of the transporter. Cryoelectron microscopy structural and homology models of MRP1 indicate that the orientation of the Phe side chain is altered by ATP binding but are currently unable to provide insights into the molecular mechanism by which this long-range signaling is propagated.

The extracellular gate shapes the energy profile of an ABC exporter

“…In summary, our results support the notion that ATP hydrolysis is required to drive the transport cycle at the resetting step from the OF to the IF state. We anticipate that our results provide a mechanistic framework to understand the functional role of the extracellular gate of type I ABC exporters and to explain the molecular underpinning of disease-causing mutations found in the extracellular region of medically important ABC exporters as recently investigated for MRP1 and CFTR 32,33 .…”

Structure‐guided probing of the leukotriene C4binding site in human multidrug resistance protein 1 (MRP1; ABCC1)

“…bMrp1 cryo-EM structures to complement previous models based on the bacterial nucleotide-bound homodimeric Sav1866 from Staphylococcus aureus and substrate-free heterodimeric TM287 and TM288 from Thermotoga maritima crystal structures (48,49). The models indicated that replacing the bulky Met 1093 in hMRP1 with a nonconservative, cavity-creating Ala might disrupt the proposed hydrophobic H-pocket around the arachidonic acid moiety of LTC 4 (Fig.…”

The extracellular gate shapes the energy profile of an ABC exporter

“…We hope that our results provide a mechanistic framework to further study the functional role of the extracellular gate of type I ABC exporters and to investigate the molecular underpinning of disease-causing mutations found in the extracellular region of medically important ABC exporters such as MRP1 and CFTR 34,35 .…”