PMID:19715704

Citation	Zou, P and McHaourab, HS (2009) Alternating access of the putative substrate-binding chamber in the ABC transporter MsbA. J. Mol. Biol. 393:574-85
Abstract	MsbA is an ATP-binding cassette transporter from Escherichia coli that is involved in trafficking lipid A across the inner membrane. ATP-binding cassette transporters harness the free energy of ATP binding and hydrolysis to drive the uphill translocation of substrates against their concentration gradients. A model of protein motion coupling energy input to work was inspired by crystallographic snapshots of MsbA. Central to this model is a switch in the accessibility of a transmembrane chamber, implicated in substrate binding, from an inward- to an outward-facing configuration. Here, we used spin labeling and electron paramagnetic resonance spectroscopy to systematically explore rearrangements in MsbA structure during the ATP hydrolysis cycle. Spin-label accessibility and local dynamics were determined in liposomes for the nucleotide-free intermediate and the transition state of ATP hydrolysis. The changes in the electron paramagnetic resonance parameters between these two intermediates fit a global pattern consistent with alternating access of the chamber. In the transition state of ATP hydrolysis, spin labels on the cytoplasmic side report increased dynamic restrictions and reduced water accessibility, while those on the extracellular side report increased water penetration. Furthermore, spin-label mobility and accessibility as well as their changes are consistent with those expected based on the crystal structures. The reversal in chamber hydration is likely to reduce the free energy of amphipathic substrate binding and promote translocation across the membrane.
Links	PubMed PMC2760602 Online version:10.1016/j.jmb.2009.08.051
Keywords	ATP-Binding Cassette Transporters/chemistry; ATP-Binding Cassette Transporters/metabolism; Adenosine Diphosphate/metabolism; Adenosine Triphosphatases/metabolism; Apoproteins/chemistry; Apoproteins/metabolism; Bacterial Proteins/chemistry; Bacterial Proteins/metabolism; Escherichia coli/chemistry; Models, Molecular; Mutant Proteins/metabolism; Protein Structure, Secondary; Spin Labels; Substrate Specificity
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
ECOBD:C6EI68		GO:0016887: ATPase activity	ECO:0000314:		F		Table 1 shows ATPase activity of MsbA mutants.	complete

References

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