An Evolutionarily Conserved Residue in ABCG2 Regulates the Transport Cycle and Inhibitor Activity.

ABCG2 is a medically significant ATP-binding cassette (ABC) transporter restricting the cellular penetration of more than 200 toxins and drugs, as well as promoting physiological detoxification and multidrug resistance in cancers. Although ABCG2 cryo-EM structural studies reveal an overlap between substrate and inhibitor binding sites in the ABCG2 binding pocket, it is unknown how much these substrate and inhibitor interactions will translate into functional knowledge that is exploitable in live cells. We generated multiple single point-mutations in the residues of the ABCG2 binding pocket to determine if there is a relationship between binding (defined by thermal shift stabilization) and transport. Unexpectedly, the relationship between substrate-induced thermal stabilization and transport was poor and an alanine substitution in a highly conserved asparagine residue in ABCG2 markedly impaired substrate-induced thermal stabilization, but paradoxically enhanced substrate transport. To determine if the substitution altered ABCG2 conformational response to substrate binding, we used live cells and performed copper phenanthroline induced disulfide cross-linking analysis on cells expressing the appropriate cysteine substituted ABCG2 (1). Consistent with the enhanced transport, the substitution promoted a substrate-induced conformational change from an inward- to an outward-facing ATP-bound state. Molecular dynamics (MD) simulations using an ABCG2 structure (PDB: 6VXH) suggested a mechanism for this enhanced transport by the alanine mutant: disruption of a water-bridge interaction with the asparagine residue coupled with an alteration in contacts with certain residues in the binding pocket. We next investigated if this alanine substitution affected inhibitor binding and transport inhibition. The substitution strongly reduced inhibitor-induced thermal stabilization and transport inhibition. The disulfide cross-linking analysis revealed that the substitution abolished the ability of many inhibitors to block the transition to an outward-facing state. For these inhibitors, MD simulations revealed that the substitution disrupted a hydrogen bond interaction with the asparagine residue and impacted inhibitor interactions with multiple residues. Overall these changes indicate that inhibitors have mostly lost their ability to act like "wedges" to lock ABCG2 into an inward-facing conformation. These findings reveal that an evolutionarily conserved asparagine residue, in the binding pocket, appears to have a key role in regulating the conformational dynamics of ABCG2. Reference: (1) Orlando, B. J. and Liao, M., ABCG2 transports anticancer drugs via a closed-to-open switch. Nat. Commun., 11, 2264 (2020).