alexa Conformational cycle of the ABC transporter MsbA in liposomes: detailed analysis using double electron-electron resonance spectroscopy.


Journal of Physical Chemistry & Biophysics

Author(s): Zou P, Bortolus M, McHaourab HS

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Abstract Driven by the energy of ATP binding and hydrolysis, ATP-binding cassette transporters alternate between inward- and outward-facing conformations, allowing vectorial movement of substrates. Conflicting models have been proposed to describe the conformational motion underlying this switch in access of the transport pathway. One model, based on three crystal structures of the lipid flippase MsbA, envisions a large-amplitude motion that disengages the nucleotide-binding domains and repacks the transmembrane helices. To test this model and place the crystal structures in a mechanistic context, we use spin labeling and double electron-electron resonance spectroscopy to define the nature and amplitude of MsbA conformational change during ATP hydrolysis cycle. For this purpose, spin labels were introduced at sites selected to provide a distinctive pattern of distance changes unique to the crystallographic transformation. Distance changes in liposomes, induced by the transition from nucleotide-free MsbA to the highest energy intermediate, fit a simple pattern whereby residues on the cytoplasmic side undergo 20-30 A closing motion while a 7- to 10-A opening motion is observed on the extracellular side. The transmembrane helices undergo relative movement to create the outward opening consistent with that implied by the crystal structures. Double electron-electron resonance distance distributions reveal asymmetric backbone flexibility on the two sides of the transporter that correlates with asymmetric opening of the substrate-binding chamber. Together with extensive accessibility analysis, our results suggest that these structures capture features of the motion that couples ATP energy expenditure to work, providing a framework for the mechanism of substrate transport.
This article was published in J Mol Biol and referenced in Journal of Physical Chemistry & Biophysics

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