Does the ATP‐bound EQ mutant reflect the pre‐ or post‐ATP hydrolysis state in the catalytic cycle of human P‐glycoprotein (ABCB1)?

Lusvarghi, Sabrina; Durell, Stewart R.; Ambudkar, Suresh V.

doi:10.1002/1873-3468.14054

Cited by 9 publications

(5 citation statements)

References 55 publications

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“…In the presence of ATP, these structures are outward-facing with no evidence of bound substrates. Based on spectroscopic 14,33 and biochemical data 34 , the underlying mutations conspire to reshape the energy landscape further confounding the mechanistic interpretation of the structural record.…”

Section: Discussionmentioning

confidence: 99%

Cryo-EM structure of an elusive pre-transport intermediate of the multidrug ABC transporter BmrCD reveals modes of asymmetric drug binding

Mishra

Zhou

et al. 2021

Preprint

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Vectorial substrate efflux by ATP binding cassette (ABC) transporters, which play a major role in multidrug resistance, entails the ATP-powered interconversion of the transporter between stable intermediates. Despite recent progress in structure elucidation of ABC transporters, a number of such intermediates have yet to be visualized and mechanistically interpreted. Here, we combine single particle cryo-EM, Double Electron Electron Resonance (DEER) spectroscopy with Molecular Dynamics simulations to profile and mechanistically frame the conformation of a hitherto unobserved intermediate in the context of BmrCD, a heterodimeric multidrug ABC exporter from Bacillus subtilis. In our cryo-EM structure, BmrCD adopts an inward facing architecture bound to both ATP and the substrate Hoechst-33342 and is capped by an extracellular domain which undergoes ATP-dependent conformational changes. A striking feature of the structure is a symmetric arrangement of the nucleotide-binding domain (NBD) in the presence of ATP whereas binding of Hoechst at two distinct sites in an acidic pocket stabilizes an asymmetric arrangement of the transmembrane domain architecture (TMD). Mutation of residues coordinating Hoechst in the structure abrogates the cooperative stimulation of ATP hydrolysis. In conjunction with previous studies, our findings suggest a mechanistic role for symmetry mismatch between NBDs and TMDs in the conformational cycle of ABC transporters. Moreover, the resolved structures of bimodally-bound drugs are of notable importance for future rational design and optimization of molecules for targeted transport inhibition of ABC transporters.

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Section: Discussionmentioning

confidence: 99%

Cryo-EM structure of an elusive pre-transport intermediate of the multidrug ABC transporter BmrCD reveals modes of asymmetric drug binding

Mishra

Zhou

et al. 2021

Preprint

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“…To date, each study of the effect of Aβ42 upon the ATPase activity of P-gp has used membrane vesicles derived from different cellular systems [12,20,27]. Combined with our findings, these data support the growing consensus that the lipid environment strongly affects the behavior and transport activity of P-gp [51,[58][59][60][61][62]. In our ATPase activity assays, we found that monomeric Aβ42 does not stimulate the ATPase activity of P-gp in micelles, but does stimulate the ATPase activity of P-gp in nanodiscs (Fig 3).…”

Section: Discussionsupporting

confidence: 87%

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“…Indeed, occlusion of one site during the transition state (in the presence of vanadate) has been observed for several ABC transporters (P-glycoprotein, LmrA, or the maltose transporter). This NBD asymmetry in the functioning mechanism also led to alternative models where ATP hydrolysis could be directly coupled to drug efflux …”

Section: Introductionmentioning

confidence: 99%

Solid-State NMR Reveals Asymmetric ATP Hydrolysis in the Multidrug ABC Transporter BmrA

et al. 2022

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The detailed mechanism of ATP hydrolysis in ATPbinding cassette (ABC) transporters is still not fully understood. Here, we employed 31 P solid-state NMR to probe the conformational changes and dynamics during the catalytic cycle by locking the multidrug ABC transporter BmrA in prehydrolytic, transition, and posthydrolytic states, using a combination of mutants and ATP analogues. The 31 P spectra reveal that ATP binds strongly in the prehydrolytic state to both ATP-binding sites as inferred from the analysis of the nonhydrolytic E504A mutant. In the transition state of wild-type BmrA, the symmetry of the dimer is broken and only a single site is tightly bound to ADP:Mg 2+ :vanadate, while the second site is more 'open' allowing exchange with the nucleotides in the solvent. In the posthydrolytic state, weak binding, as characterized by chemical exchange with free ADP and by asymmetric 31 P− 31 P two-dimensional (2D) correlation spectra, is observed for both sites. Revisiting the 13 C spectra in light of these findings confirms the conformational nonequivalence of the two nucleotide-binding sites in the transition state. Our results show that following ATP binding, the symmetry of the ATP-binding sites of BmrA is lost in the ATP-hydrolysis step, but is then recovered in the posthydrolytic ADP-bound state.

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Does the ATP‐bound EQ mutant reflect the pre‐ or post‐ATP hydrolysis state in the catalytic cycle of human P‐glycoprotein (ABCB1)?

Cited by 9 publications

References 55 publications

Cryo-EM structure of an elusive pre-transport intermediate of the multidrug ABC transporter BmrCD reveals modes of asymmetric drug binding

Cryo-EM structure of an elusive pre-transport intermediate of the multidrug ABC transporter BmrCD reveals modes of asymmetric drug binding

Untitled

Solid-State NMR Reveals Asymmetric ATP Hydrolysis in the Multidrug ABC Transporter BmrA

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