Cellular homeostasis requires the energetically uphill transport of a wide range of molecules across membranes. Active transporters couple vectorial translocation of molecules against their concentration gradients to thermodynamically favorable processes such as ATP hydrolysis or ions moving down their electrochemical gradients. In prokaryotes, ATP energy powers the largest class of transporters, the ATP-binding cassette (ABC) transporters, to traffic a structurally and chemically diverse spectrum of molecules1
. Bidirectional transport is accomplished by two distinct subclasses of ABC transporters referred to as importers and exporters 2
. Both are invariably characterized by the presence of a conserved module, the ABC or nucleotide binding domain (NBD), that harnesses ATP energy for biological work. Substrate specificity is encoded in highly divergent transmembrane domains (TMD) that also provide the passageway in the membrane. Although importers and exporters have a common molecular architecture consisting minimally of two NBDs and two TMDs, the organization and packing of these four domains in the functional unit differ significantly2
. Bacterial ABC exporters are homodimers of molecules consisting of fused ABC and TMD whereas importers assemble from four independent subunits. A cognate protein delivers the substrate to ABC importers while exporters pick up their substrates from the inner leaflet of the membrane.
Transport entails the transduction of ATP energy to alternately expose the transport pathway to opposite sides of the membrane 3
. Crystallographic glimpses of ABC transporters 4–10
and analysis by spin labeling and EPR spectroscopy11–13
have led to models of the underlying molecular motion. The exporter-centric model is primarily based on studies of MsbA, the putative lipid A flippase from E. coli14
. MsbA crystallizes in three different conformations: two inward-facing and one outward-facing10
. In all three structures, the TMD consists of two wings each containing helices from both monomers. The cross-over helices hold the dimer together and impart a remarkable degree of conformational flexibility. The nucleotide-free (apo) structure is defined by a V-shaped chamber open to the cytoplasm and the inner leaflet of the bilayer. In this structure, referred to as the open apo, the two NBDs are disengaged and separated by 50Å. In contrast, when bound to the non-hydrolyzable ATP analog, AMPPNP, the structure is tightly packed at the cytoplasmic side where the NBDs form the canonical dimer15
. The TMD wings point away from each other in the outer leaflet of the bilayer resulting in an outward-facing conformation. The open-closed crystallographic transformation implies extensive molecular motion involving dissociation and association of the ATP dimer sandwich, swapping and repacking of helices in the transmembrane domain and rearrangements in the extracellular loops such that the inward and outward openings are mediated by different sets of helices. While the AMPPNP-bound closed conformation has been observed in two crystal structures of an MsbA homolog, Sav18664
, the rearrangements implied by the MsbA structures remain unique and at variance with the structural analysis of ABC importers captured in different intermediates. The importers structures suggest more subtle changes mediate the inward- to outward- facing transition wherein the two NBDs remain in contact throughout the cycle6
We have used spin labeling and EPR spectroscopy16
to obtain a complementary perspective on the nature and amplitude of MsbA conformational motion in a native-like environment without conformational selectivity subjected by lattice forces. In addition to testing conflicting models of transport, our analysis seeks to place the crystal structures in the context of the ATPase cycle. In previous work 12
, mobilities and accessibilities of spin labels along helices 2, 5 and 6 reported substantial changes in local environments upon ATP binding consistent with a transition from an inward- to an outward-facing conformation.
While a cursory, qualitative comparison of the EPR and the corrected crystal structures resolve earlier incongruencies brought about by incorrect helix assignment 17
, verification of the conformational changes deduced from the crystal structures requires a more detailed quantitative comparison. In this manuscript and the accompanying report18
, we expand the spin labeling analysis to develop a global perspective on the dynamic events driving the transport cycle. Here, residue accessibility and mobility along helices 3 and 4 and adjacent intracellular and extracellular segments are determined in the apo and high energy post-hydrolysis intermediates. Spin labels were introduced along the intracellular coupling helices, IH1 and IH2, that transmit the signal of ATP binding and hydrolysis. Spin labels along helix 3 which contact the transmembrane chamber are optimally located to report on the changes in accessibility during the ATPase cycle.