Structure of a zosuquidar and UIC2-bound human-mouse chimeric ABCB1

Alam, Amer; Küng, Raphael; Kowal, Julia; McLeod, Robert A.; Tremp, Nina; Broude, Eugenia V.; Roninson, Igor B.; Stahlberg, Henning; Locher, Kaspar P.

doi:10.1073/pnas.1717044115

Cited by 171 publications

(225 citation statements)

References 68 publications

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“…Intriguingly, a cryo-EM structure of the mouse P-gp-ZDR complex was recently reported. 43 Through structural alignment, we observe that the experimentally determined protein structure is very similar to our simulated structure at the local minimum of Z…”

Section: Connection Of Statistically Averaged Microscopic Events Tosupporting

confidence: 52%

Substrate Transport is Mediated not only by P-glycoprotein but also by Lipid Penetration

Wang

Chen

et al. 2019

Preprint

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In association with large-scale conformational changes, the members of the ATP-binding cassette (ABC) transporter superfamily such as P-glycoprotein (P-gp) pump endogenous cytotoxic substances and exogenous drugs out of cells. Here, a series of nonequilibrium-driven molecular dynamics (MD) simulations are sophisticatedly combined to provide a generally effective access to quantitatively investigate such a complex biological process that has been posing a great challenge for experiments and computational simulations. Both common features and unique characteristics of multiple ligands (substrates or inhibitors) that are recognized by P-gps from mouse and human species are quantitatively explored, providing additional insights into experimentally suggested ligand transport pathways and summarizing the important roles of not only different P-gps but also lipids in regulating ligand transport. These findings reveal the molecular mechanism underlying the transport of ligands by P-gps from different species and emphasize the consideration of lipid effects on the future design of effective P-gp inhibitors.

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Section: Connection Of Statistically Averaged Microscopic Events Tosupporting

confidence: 52%

Substrate Transport is Mediated not only by P-glycoprotein but also by Lipid Penetration

Wang

Chen

et al. 2019

Preprint

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“…The strategy of generating state-specific binders against type I ABC exporters has a long history going back to the 90's of the last century, when the state-specific ABCB1 antibody UIC2 was identified 17 . A recent cryo-EM structure of UIC2 in complex with ABCB1 revealed that the antibody clamps the extracellular loops together, thereby preventing extracellular gate opening 18 . Molecular clamping of the closed extracellular gate was also achieved by a cyclic peptide raised against CmABCB1 19 .…”

Section: Discussionmentioning

confidence: 99%

The extracellular gate shapes the energy profile of an ABC exporter

Hutter

Timachi

Hürlimann

et al. 2018

Preprint

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ABC exporters harness the energy of ATP to pump substrates across membranes. Extracellular gate opening and closure are key steps of the transport cycle, but the underlying mechanism is poorly understood. Here, we generated a synthetic single domain antibody (sybody) that recognizes the heterodimeric ABC exporter TM287/288 exclusively in the presence of ATP, which was essential to solve a 3.2 Å crystal structure of the outward-facing transporter. The sybody binds to an extracellular wing and strongly inhibits ATPase activity by shifting the transporter's conformational equilibrium towards the outward-facing state, as shown by double electron-electron resonance (DEER). Mutations that facilitate extracellular gate opening resulted in a comparable equilibrium shift and strongly reduced ATPase activity and drug transport. Using the sybody as conformational probe, we demonstrate that efficient extracellular gate closure is required to dissociate the NBD dimer after ATP hydrolysis to reset the transporter back to its inward-facing state. RESULTS Conformational trapping of TM287/288Having solved two closely related IF structures of TM287/288, our aim was to obtain an atomic structure of this heterodimeric ABC exporter in its OF state. DEER analyses revealed that TM287/288 carrying the TM288 E517Q mutation in the Walker B motif of the consensus site (EtoQ mutation) was almost completely trapped in the OF state in the presence of ATP-Mg and ATPγS-Mg 9 . To further decrease the residual ATPase activity of the EtoQ mutant (turnover of 0.02 min -1 ) by a factor of 6.5, we instead introduced the EtoA mutation. In addition, we generated single domain antibodies (nanobodies) that exclusively recognize the OF state of TM287/288. To this end, alpacas were immunized with outward-facing TM287/288 containing a cross-linked tetrahelix bundle motif 13 (see Materials and Methods). This approach yielded nanobody Nb_TM#1 binding exclusively to TM287/288 in the presence (but not in the absence) of ATP, as shown by surface plasmon resonance (SPR) (Fig. 1d). However, crystals obtained with Nb_TM#1 did not diffract well enough to build a reliable model. Therefore, we selected synthetic nanobodies (sybodies) against TM287/288(EtoA) in the presence of ATP-Mg completely in vitro 14 . Thereby, more than ten OF-specific sybodies were generated and sybody Sb_TM#35 was successfully used to solve the OF structure of TM287/288(EtoA) in the presence of ATPγS-Mg at 3.2 Å resolution ( Fig. 1a, Table S1). Structure of OF TM287/288 with a sybody bound to an extracellular wingSybody Sb_TM#35 binds on top of an extracellular wing of TM287/288 ( Fig. 1a) and was crucially involved in establishing crystal contacts (Fig. S1). Binding is mediated by aromatic residues of all three complementary determining regions (CDRs) of the sybody, which are wedged between transmembrane helices (TMs) 1 and 2 of TM287 and TMs 5' and 6' of TM288 ( Fig. 2a). Since Sb_TM#35 only binds in the presence of ATP (Fig. 1d), we hypothesized that it interferes with the catalytic cycle o...

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“…A Fourier shell correlation value of 0.143 was reached at 7.9Å. The structural data obtained was therefore at lower resolution than was possible by cross-linking and extensive mutagenesis of the protein (6,13); however it was at higher resolution than data previously obtained by cryo-EM for active P-gp in the presence of a stabilising antibody fragment (14). Flexibility may be a prerequisite for active protein, hence may be an intrinsic limitation to structural studies of single particles where there are no constraints due to a crystal lattice.…”

Section: Resultsmentioning

confidence: 91%

“…Compared to the map presented in Figure 3, the cavity for Zosuquidar appears to be slightly smallerperhaps because of the cross-linking of the NBDs or the addition of the UIC2 antibody. These factors may have forced a narrower angle to be subtended between the two TMDs in the Zosuquidar study (13). Similarly, a study of Pglycoprotein with cyclic peptides designed to probe the allocrite-binding pocket showed two molecules bound in the same locations as in Figure 3.…”

Section: Drug Binding Sites (Purple Density)mentioning

confidence: 99%

“…There are now many atomic models and experimental density maps for P-gp deposited in the Protein Databank (PDB) and Electron Micoscopy Databank (EMDB) (6)(7)(8)(9)(10)(11)(12)(13)(14), and data are available at a resolution allowing a direct modeling of the amino acid residue side-chains (6)(7)(8)(9)(10)(11)(12)(13). Murine P-gp has so far been predominant in these studies.…”

Section: Introductionmentioning

confidence: 99%

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Novel features in the structure of P-glycoprotein (ABCB1) in the post-hydrolytic state as determined at 7.9Å resolution

Thongin

Collins

Barbieri

et al. 2018

Preprint

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P-glycoprotein (ABCB1) is a ATP-binding cassette transporter that plays an important role in the removal of drugs and xenobiotic compounds from the cell.It is also associated with multi-drug resistance in cancer. Here we report novel features of the cryo-EM-derived structure of P-glycoprotein in the posthydrolytic state: The cytosolic nucleotide-binding domains (NBDs) are separated despite ADP remaining bound to the NBDs. Gaps in the TMDs that connect to the inner hydrophilic cavity are back-filled by detergent head-groups from the annular detergent micelle and are close to two regions predicted to delineate two pseudo-symmetry-related drug-binding sites. In this conformation, the (newly-resolved) N-terminal extension, NBD-TMD linker region and gap-filling detergents all appear to impede NBD dimerisation. We propose a model for the mechanism of action of the exporter where ATP will be bound to the protein for most of the time, consistent with the high physiological ATP concentrations in vivo. KeywordsP-glycoprotein; ABCB1; ATP-binding cassette; transporter, membrane protein, protein structure, cryo-electron microscopy.

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Structure of a zosuquidar and UIC2-bound human-mouse chimeric ABCB1

Cited by 171 publications

References 68 publications

Substrate Transport is Mediated not only by P-glycoprotein but also by Lipid Penetration

Substrate Transport is Mediated not only by P-glycoprotein but also by Lipid Penetration

The extracellular gate shapes the energy profile of an ABC exporter

Novel features in the structure of P-glycoprotein (ABCB1) in the post-hydrolytic state as determined at 7.9Å resolution

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