ATP-binding cassette sub-family G member 2, ABCG2, was initially cloned from drug-resistant cancer cells (Austin Doyle et al., 1998, Miyake et al., 1999) and, around the same time, also from the normal placenta (Allikmets R et al., 1998). It is expressed in the apical membranes of many tissues. Its important physiological function is centered around the extrusion of endogenous compounds and xenobiotics such as uric acid across tissue barriers, including the blood-brain barrier (BBB), intestine, liver, and kidney. In cancer cells, ABCG2 exports many structurally diverse and mechanistically unrelated chemotherapeutic drugs out of the cells using the energy of ATP hydrolysis, resulting in their reduced efficiency. ABCG2 is also a key drug transporter which can alter the pharmacokinetics of its substrate drugs.

ABCG2 mutations are common, with more than 1,000 germline and/or acquired somatic mutations (gnomAD (gnomAD v3.1.1) and COSMIC (Tate et al., 2019) databases), which might affect therapeutic efficacy and survival. ABCG2 was initially cloned from a drug-resistant cancer cell, but this version had different substrate specificities than ABCG2 cloned from normal tissues (Honjo et al., 2001). ABCG2 from the drug-resistant cancer cells harbored a mutation at R482 (R482G/R482T) (Honjo et al., 2001). One of the most common dysfunctional mutants, ABCG2-Q141K, is a germline mutant and a risk factor for hyperuricemia/gout (Dehghan et al., 2008). The functionally impaired Q141K mutant reduces renal and intestinal urate excretion thereby increasing serum levels of uric acid (Hoque et al., 2020). The Q141K mutant also affects statin-based hyperlipidemia treatment (Zhang et al., 2006) and increases the side effects of statins (Mirošević Skvrce et al., 2015). ABCG2-Q141K is reportedly associated with a lower risk of certain cancers including leukemia, suggesting ABCG2 function might affect therapeutic efficacy and/or cancer cell survival as in some cases it seems important for the cancer development (Chen et al., 2012, Fukuda et al., 2017). Numerous ABCG2 mutations have been classified by in vitro cell-based assays. Defective ABCG2 function in some cases was attributed to their lack of expression on the cell surface and in others due to altered drug transport capability (Homolya, 2021). However, this classification scheme does not differentiate between transport defects in ABCG2 due to altered ATPase activity or due to an inability to perform the conformational changes necessary to expel the drug substrate. Most of the mutations are not located at the substrate-binding site and seem unlikely to interact directly with the substrate (Heyes et al., 2018). Therefore, substantial structure-based analysis is required to gain insights into their functional impact.

The ABCG2 cryo-EM structures (apo-closed, nucleotide-, substrate-, and inhibitor-bound states) complemented by molecular dynamics (MD) simulations provide an excellent framework for better understanding of the effects of mutations on substrate recognition but also inferring if mutations affect the catalytic transport cycle. Recent ABCG2 cryo-EM structures in turnover states revealed a structural role of R482, which is located in transmembrane helix 3 (TMH3) and does not directly interact with the bound substrate (Yu et al., 2021). These findings provided insights into the R482 gain-of-function mutation as this residue contacts TMH2 in which the residue F439 interacts with all substrates and inhibitors in the binding cavity (Gose et al., 2020). The results suggested that the altered substrate specificity of the R482 mutation might be due to an allosteric effect (Yu et al., 2021).

Studying the ABCG2 genomic alterations in pediatric cancer using the ProteinPaint data portal (pecan.stjude.org) (Zhou et al., 2015) revealed a novel somatic mutation ABCG2-Q393K (Sample ID; SJMB012) in a sonic-hedgehog-driven medulloblastoma patient (Wijaya et al., 2017). Q393K is located between the elbow helix and TMH1, lining the intracellular region of the transmembrane lumen. Using in vitro studies complemented by MD simulations, we suggest a model for how the mutation in the conserved Q393 affects ABCG2 function. Here, our studies reveal that the Q393K mutant retains cell-surface expression and substrate recognition but the mutation impedes the substrate-driven transition from the inward-facing (IF) state to the outward-facing (OF) state, an effect that we attribute to the formation of a salt bridge between K393 and E446.