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

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dc.authoridStockner, Thomas/0000-0002-7071-8283en_US
dc.authoridSzöllösi, Dániel/0000-0003-4641-3065en_US
dc.authoridÇakıl, Yaprak Dönmez/0000-0002-4605-1167en_US
dc.authoridTurk, Dora/0000-0003-1215-3453en_US
dc.authoridTarapcsak, Szabolcs/0000-0001-7182-0135en_US
dc.authoridGoda, Katalin/0000-0003-2001-7400en_US
dc.contributor.authorGoda, Katalin
dc.contributor.authorDoenmez-Çakıl, Yaprak
dc.contributor.authorTarapcsak, Szabolcs
dc.contributor.authorSzaloki, Gabor
dc.contributor.authorSzoellosi, Daniel
dc.contributor.authorParveen, Zahida
dc.contributor.authorTuerk, Dora
dc.date.accessioned2024-07-12T21:37:50Z
dc.date.available2024-07-12T21:37:50Z
dc.date.issued2020en_US
dc.department[Belirlenecek]en_US
dc.description.abstractSeveral 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. Author summary ABC transporters are one of the largest membrane protein superfamilies, present in all organisms from archaea to humans. They transport a wide range of molecules including amino acids, sugars, vitamins, nucleotides, peptides, lipids, metabolites, antibiotics, and xenobiotics. ABC transporters energize substrate transport by hydrolyzing ATP in two symmetrically arranged nucleotide binding sites (NBSs). The human multidrug resistance transporter ABCB1 has two active NBSs, and it is generally believed that integrity and cooperation of both sites are needed for transport. Several human ABC transporters, such as the bile salt transporter ABCB11, have one degenerate NBS, which has significantly reduced ATPase activity. Interestingly, unilateral mutations affecting one of the two NBSs completely abolish the function of symmetrical ABC transporters. Here we engineered an ABCB1 variant with a degenerate, ABCB11-like NBS1, which can nevertheless transport substrates. Our results indicate that ABCB1 can mediate active transport with a single active site, questioning the validity of models assuming strictly alternating catalysis.en_US
dc.description.sponsorshipAustrian Science Fund (FWF) [F3509, F3524, F3525]; Hungarian National Research, Development and Innovations Office (NKFIH) [K124815]en_US
dc.description.sponsorshipWe are grateful for financial support by the Austrian Science Fund (FWF; https://www.fwf.ac.at/), grant number F3509 to PC, F3524 to TS and F3525 to GS and by the Hungarian National Research, Development and Innovations Office (NKFIH; https://nkfih.gov.hu/english), grant number K124815 to KG. We are also grateful for computing time allocated to TS by the Vienna Scientific Cluster (VSC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.en_US
dc.identifier.doi10.1371/journal.pgen.1009016
dc.identifier.issn1553-7404
dc.identifier.issue10en_US
dc.identifier.pmid33031417en_US
dc.identifier.scopus2-s2.0-85092684596en_US
dc.identifier.scopusqualityQ1en_US
dc.identifier.urihttps://doi.org/10.1371/journal.pgen.1009016
dc.identifier.urihttps://hdl.handle.net/20.500.12415/6955
dc.identifier.volume16en_US
dc.identifier.wosWOS:000581778600004en_US
dc.identifier.wosqualityQ1en_US
dc.indekslendigikaynakWeb of Science
dc.indekslendigikaynakScopus
dc.indekslendigikaynakPubMed
dc.language.isoenen_US
dc.publisherPublic Library Scienceen_US
dc.relation.ispartofPlos Geneticsen_US
dc.relation.publicationcategoryMakale - Uluslararası Hakemli Dergi - Kurum Öğretim Elemanıen_US
dc.rightsinfo:eu-repo/semantics/openAccessen_US
dc.snmzKY04297
dc.titleHuman ABCB1 with an ABCB11-like degenerate nucleotide binding site maintains transport activity by avoiding nucleotide occlusionen_US
dc.typeArticle
dspace.entity.typePublication

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