Human ABCB1 with an ABCB11-like degenerate nucleotide binding site maintains transport activity by avoiding nucleotide occlusion

Abstract

Several ABC exporters carry a degenerate nucleotide binding site (NBS) that is unable to hydrolyze ATP at a rate sufficient for sustaining transport activity. A hallmark of a degenerate NBS is the lack of the catalytic glutamate in the Walker B motif in the nucleotide binding domain (NBD). The multidrug resistance transporter ABCB1 (P-glycoprotein) has two canonical NBSs, and mutation of the catalytic glutamate E556 in NBS1 renders ABCB1 transport-incompetent. In contrast, the closely related bile salt export pump ABCB11 (BSEP), which shares 49% sequence identity with ABCB1, naturally contains a methionine in place of the catalytic glutamate. The NBD-NBD interfaces of ABCB1 and ABCB11 differ only in four residues, all within NBS1. Mutation of the catalytic glutamate in ABCB1 results in the occlusion of ATP in NBS1, leading to the arrest of the transport cycle. Here we show that despite the catalytic glutamate mutation (E556M), ABCB1 regains its ATP-dependent transport activity, when three additional diverging residues are also replaced. Molecular dynamics simulations revealed that the rescue of ATPase activity is due to the modified geometry of NBS1, resulting in a weaker interaction with ATP, which allows the quadruple mutant to evade the conformationally locked pre-hydrolytic state to proceed to ATP-driven transport. In summary, we show that ABCB1 can be transformed into an active transporter with only one functional catalytic site by preventing the formation of the ATP-locked pre-hydrolytic state in the non-canonical site.

The role of the degenerate nucleotide binding site in type I ABC exporters

“…Insertion of methionine (as present in ABCB11) in place of the Walker B glutamate of NBS1 abolished ATPase activity and substrate transport [82]. Inserting all four residues by which NBS1 of ABCB11 differs from NBS1 of ABCB1 resulted in an ABCB1 variant that can hydrolyze ATP and is substrate transport competent [82], showing that ABCB1 can in principle be active with a degenerate NBS1.…”

“…The single Walker B glutamate mutants revealed that NBS1 or NBS2 is not completely identical [41]: The verapamil stimulated ATP hydrolysis of single glutamate to glutamine mutants in NBS1 or NBS2 differed 2‐ to 3‐fold, the K m(MgATP) differed 5‐fold, while ADP is more likely retained in the Walker B mutant of NBS1 compared to NBS2. Insertion of methionine (as present in ABCB11) in place of the Walker B glutamate of NBS1 abolished ATPase activity and substrate transport [82]. Inserting all four residues by which NBS1 of ABCB11 differs from NBS1 of ABCB1 resulted in an ABCB1 variant that can hydrolyze ATP and is substrate transport competent [82], showing that ABCB1 can in principle be active with a degenerate NBS1.…”

Nucleotide binding is the critical regulator of ABCG2 conformational transitions

“…Apparent affinity of nucleotide binding (K A ) was determined as described previously (Barsony et al, 2016, Goda et al, 2020. Permeabilized cells (1 × 10 6 ml − 1 ) were pre-treated with nucleotides added at different concentrations in the presence of equimolar concentrations of Mg 2+ at 37°C for 10 min and then further incubated with 5 μg/ml 5D3-A647 at 37 °C for 20 min.…”

The Bile Salt Export Pump: Molecular Structure, Study Models and Small-Molecule Drugs for the Treatment of Inherited BSEP Deficiencies

“…When compared to ABCB1, only four amino acids differ in NBS1. These are E502, M584, R1221 and E1223 in BSEP corresponding to S474, E556, G1178 and Q1180 in the ABCB1 protein sequence [ 71 ]. The amino acid changes result in a catalytically inactive ATP binding site (NBS1), which is also variably called “degenerate” NBS.…”