title: Load-dependent destabilization of the γ-rotor shaft in FOF1 ATP synthase revealed by hydrogen/deuterium-exchange mass spectrometry.
abstract: FoF1 is a membrane-bound molecular motor that uses proton-motive force (PMF) to drive the synthesis of ATP from ADP and Pi. Reverse operation generates PMF via ATP hydrolysis. Catalysis in either direction involves rotation of the γε shaft that connects the α3β3 head and the membrane-anchored cn ring. X-ray crystallography and other techniques have provided insights into the structure and function of FoF1 subcomplexes. However, interrogating the conformational dynamics of intact membrane-bound FoF1 during rotational catalysis has proven to be difficult. Here, we use hydrogen/deuterium exchange mass spectrometry to probe the inner workings of FoF1 in its natural membrane-bound state. A pronounced destabilization of the γ C-terminal helix during hydrolysis-driven rotation was observed. This behavior is attributed to torsional stress in γ, arising from γ⋅⋅⋅α3β3 interactions that cause resistance during γ rotation within the apical bearing. Intriguingly, we find that destabilization of γ occurs only when FoF1 operates against a PMF-induced torque; the effect disappears when PMF is eliminated by an uncoupler. This behavior resembles the properties of automotive engines, where bearings inflict greater forces on the crankshaft when operated under load than during idling.
authors.affiliation: Department of Chemistry, University of Western Ontario, London, ON, Canada N6A 5B7;
authors.forename: Siavash
authors.initials: S
authors.lastname: Vahidi
authors.affiliation: Department of Biochemistry, University of Western Ontario, London, ON, Canada N6A 5C1.
authors.forename: Yumin
authors.initials: Y
authors.lastname: Bi
authors.affiliation: Department of Biochemistry, University of Western Ontario, London, ON, Canada N6A 5C1 sdunn@uwo.ca konerman@uwo.ca.
authors.forename: Stanley D
authors.initials: SD
authors.lastname: Dunn
authors.affiliation: Department of Chemistry, University of Western Ontario, London, ON, Canada N6A 5B7; Department of Biochemistry, University of Western Ontario, London, ON, Canada N6A 5C1 sdunn@uwo.ca konerman@uwo.ca.
authors.forename: Lars
authors.initials: L
authors.lastname: Konermann
mesh_terms: D018360:Crystallography, X-Ray; D013058:Mass Spectrometry / Q000379:methods*; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D006180:Proton-Translocating ATPases
keywords: conformational dynamics; destabilized helix; molecular bearing; molecular motor; rotational resistance