Conformational Cycle of the ABC Transporter MsbA in Liposomes: Detailed Analysis Using Double Electron–Electron Resonance Spectroscopy

Abstract

Driven by the energy of ATP binding and hydrolysis, ATP binding cassette (ABC) 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 (DEER) 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 20-30Å closing motion while a 7-10Å 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. DEER 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. KeywordsABC transporter; MsbA; double electron-electron resonance (DEER); site-directed spin labeling; electron paramagnetic resonance Active transporters transduce various forms of energy into the mechanical work of substrate translocation. For the class of ATP binding cassettes (ABC) transporters, ATP energy drives protein conformational motions to carry molecules ranging from small ions to large polypeptides across membranes 1,2 . The spectrum of these movements, their amplitudes and *Corresponding author. Mailing address for Hassane S. Mchaourab: 741 Light Hall, 2215 Garland Ave., Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232, USA, hassane.mchaourab@vanderbilt.edu. ⊥ Current address: Dept. Scienze Chimiche, Universita' degli studi di Padova, Padova, Italy.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers tha...

Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET

“…These data were questioned as a detergent artifact mistaken for dynamic NBDs. DEER data of ABC transporters in liposomes and bicelles show broad lines for apo NBDs (Dong et al , 2005; Borbat et al , 2007; Zou et al , 2009; Bountra et al , 2017), further suggesting that in the absence of nucleotides the NBDs can have different degrees of disengagement. Upon nucleotide binding, the NBDs dimerize and display nearly identical distances across different transporters (Ward et al , 2007; Lin et al , 2015; Perez et al , 2015; Bountra et al , 2017).…”

“…All ABC transporters share a common architecture consisting of a transmembrane domain (TMD) for substrate recognition and transport, and a nucleotide‐binding domain (NBD) that converts the chemical energy of ATP into conformational changes for transport (Beis, 2015). The structures of several homodimeric (Dawson & Locher, 2006; Ward et al , 2007; Perez et al , 2015) and heterodimeric ABC transporters (Hohl et al , 2012; Noll et al , 2017) revealed distinct conformations and suggest, in combination with biophysical studies (e.g., EPR and NMR; Dong et al , 2005; Zou et al , 2009; Bountra et al , 2017; Timachi et al , 2017; Barth et al , 2018), that they undergo large conformational changes during transport. Their complex architecture is, however, a fundamental hurdle to fully understand the coupling between conformational changes, substrate binding, ATP binding and hydrolysis, and transport.…”

Structure, Dynamics, and Substrate-induced Conformational Changes of the Multidrug Transporter EmrE in Liposomes

“…Specifically, the tilt at the N terminus of TM2 provides direct access to the bilayer from which hydrophobic substrates may partition. A detailed global model of this conformational transition will also benefit from long range distance measurements (42).…”

PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

“…This EPR technique can accurately measure distances between paramagnetic centers in a range of 15 up to 170 Å (27,28) and has frequently been employed to study conformational changes in transporters and channels (29)(30)(31)(32)(33). Like most proteins, VcSiaP is diamagnetic, and therefore invisible for EPR.…”