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Quantitative comparison of ABC membrane protein type I exporter structures in a standardized way.

ABSTRACT: An increasing number of ABC membrane protein structures are determined by cryo-electron microscopy and X-ray crystallography, consequently identifying differences between their conformations has become an arising issue. Therefore, we propose to define standardized measures for ABC Type I exporter structure characterization. We set conformational vectors, conftors, which describe the relative orientation of domains and can highlight structural differences. In addition, continuum electrostatics calculations were performed to characterize the energetics of membrane insertion illuminating functionally crucial regions. In summary, the proposed metrics contribute to deeper understanding of ABC membrane proteins' structural features, structure validation, and analysis of movements observed in a molecular dynamics trajectory. Moreover, the concept of standardized metrics can be applied not only to ABC membrane protein structures (http://conftors.hegelab.org).

SUBMITTER: Csizmadia G

PROVIDER: S-EPMC6222291 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Quantitative comparison of ABC membrane protein type I exporter structures in a standardized way.

Csizmadia Georgina G Farkas Bianka B Spagina Zoltán Z Tordai Hedvig H Hegedűs Tamás T

Computational and structural biotechnology journal 20181018

An increasing number of ABC membrane protein structures are determined by cryo-electron microscopy and X-ray crystallography, consequently identifying differences between their conformations has become an arising issue. Therefore, we propose to define standardized measures for ABC Type I exporter structure characterization. We set conformational vectors, conftors, which describe the relative orientation of domains and can highlight structural differences. In addition, continuum electrostatics ca ...[more]

PMID: 30425800

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Project description:Cryo-electron microscopy (cryo-EM) has the capacity to capture molecular machines in action1-3. ATP-binding cassette (ABC) exporters are highly dynamic membrane proteins that extrude a wide range of substances from the cytosol4-6 and thereby contribute to essential cellular processes, adaptive immunity and multidrug resistance7,8. Despite their importance, the coupling of nucleotide binding, hydrolysis and release to the conformational dynamics of these proteins remains poorly resolved, especially for heterodimeric and/or asymmetric ABC exporters that are abundant in humans. Here we present eight high-resolution cryo-EM structures that delineate the full functional cycle of an asymmetric ABC exporter in a lipid environment. Cryo-EM analysis under active turnover conditions reveals distinct inward-facing (IF) conformations-one of them with a bound peptide substrate-and previously undescribed asymmetric post-hydrolysis states with dimerized nucleotide-binding domains and a closed extracellular gate. By decreasing the rate of ATP hydrolysis, we could capture an outward-facing (OF) open conformation-an otherwise transient state vulnerable to substrate re-entry. The ATP-bound pre-hydrolysis and vanadate-trapped states are conformationally equivalent; both comprise co-existing OF conformations with open and closed extracellular gates. By contrast, the post-hydrolysis states from the turnover experiment exhibit asymmetric ATP and ADP occlusion after phosphate release from the canonical site and display a progressive separation of the nucleotide-binding domains and unlocking of the intracellular gate. Our findings reveal that phosphate release, not ATP hydrolysis, triggers the return of the exporter to the IF conformation. By mapping the conformational landscape during active turnover, aided by mutational and chemical modulation of kinetic rates to trap the key intermediates, we resolved fundamental steps of the substrate translocation cycle of asymmetric ABC transporters.

Dataset Information

Quantitative comparison of ABC membrane protein type I exporter structures in a standardized way.

Publications

Quantitative comparison of ABC membrane protein type I exporter structures in a standardized way.

Similar Datasets

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