Conformational dynamics of the nucleotide binding domains and the power stroke of a heterodimeric ABC transporter
Abstract
Multidrug ATP binding cassette (ABC) exporters are ubiquitous ABC transporters that extrude cytotoxic molecules across cell membranes. Despite recent progress in structure determination of these transporters, the conformational motion that transduces the energy of ATP hydrolysis to the work of substrate translocation remains undefined. Here, we have investigated the conformational cycle of BmrCD, a representative of the heterodimer family of ABC exporters that have an intrinsically impaired nucleotide binding site. We measured distances between pairs of spin labels monitoring the movement of the nucleotide binding (NBD) and transmembrane domains (TMD). The results expose previously unobserved structural intermediates of the NBDs arising from asymmetric configuration of catalytically inequivalent nucleotide binding sites. The two-state transition of the TMD, from an inward- to an outward-facing conformation, is driven exclusively by ATP hydrolysis. These findings provide direct evidence of divergence in the mechanism of ABC exporters.
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Reviewing Editor
- Volker Dötsch, Goethe University, Germany
Version history
- Received: March 8, 2014
- Accepted: May 13, 2014
- Accepted Manuscript published: May 16, 2014 (version 1)
- Version of Record published: June 10, 2014 (version 2)
Copyright
© 2014, Mishra et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Further reading
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Activation of the extracellular signal-regulated kinase-2 (ERK2) by phosphorylation has been shown to involve changes in protein dynamics, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and NMR relaxation dispersion measurements. These can be described by a global exchange between two conformational states of the active kinase, named ‘L’ and ‘R,’ where R is associated with a catalytically productive ATP-binding mode. An ATP-competitive ERK1/2 inhibitor, Vertex-11e, has properties of conformation selection for the R-state, revealing movements of the activation loop that are allosterically coupled to the kinase active site. However, the features of inhibitors important for R-state selection are unknown. Here, we survey a panel of ATP-competitive ERK inhibitors using HDX-MS and NMR and identify 14 new molecules with properties of R-state selection. They reveal effects propagated to distal regions in the P+1 and helix αF segments surrounding the activation loop, as well as helix αL16. Crystal structures of inhibitor complexes with ERK2 reveal systematic shifts in the Gly loop and helix αC, mediated by a Tyr-Tyr ring stacking interaction and the conserved Lys-Glu salt bridge. The findings suggest a model for the R-state involving small movements in the N-lobe that promote compactness within the kinase active site and alter mobility surrounding the activation loop. Such properties of conformation selection might be exploited to modulate the protein docking interface used by ERK substrates and effectors.
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- Biochemistry and Chemical Biology
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