F o subcomplex of ATP synthase is a membrane-embedded rotary motor that converts proton motive force into mechanical energy. Despite a rapid increase in the number of high-resolution structures, the mechanism of tight coupling between proton transport and motion of the rotary c-ring remains elusive. Here, using extensive all-atom free energy simulations, we show how the motor’s directionality naturally arises from the interplay between intraprotein interactions and energetics of protonation of the c-ring. Notably, our calculations reveal that the strictly conserved arginine in the a-subunit (R176) serves as a jack-of-all-trades: it dictates the direction of rotation, controls the protonation state of the proton-release site, and separates the two proton-access half-channels. Therefore, arginine is necessary to avoid slippage between the proton flux and the mechanical output and guarantees highly efficient energy conversion. We also provide mechanistic explanations for the reported defective mutations of R176, reconciling the structural information on the F o motor with previous functional and single-molecule data.

Fo subcomplex of ATP synthase is an membrane-embedded rotary motor that converts proton motive force into mechanical energy. Despite a rapid increase in the number of high-resolution structures, the mechanism of tight coupling between proton transport and motion of the rotary c-ring remains elusive. Here, using extensive all-atom free energy simulations, we show how the motor's directionality naturally arises from the interplay between intra-protein interactions and energetics of protonation of the c-ring. Notably, our calculations reveal that the strictly conserved arginine in the a-subunit (R176) serves as a jack-of-all-trades: it dictates the direction of rotation, controls the protonation state of the proton-release site and separates the two proton-access half-channels. Therefore, arginine is necessary to avoid slippage between the proton flux and the mechanical output and guarantees highly efficient energy conversion. We also provide mechanistic explanations for the reported defective mutations of R176, reconciling the structural information on the Fo motor with previous functional and single-molecule data.

F 1 -ATPase is a motor protein that couples the rotation of its rotary γ subunit with ATP synthesis or hydrolysis. Single-molecule experiments indicate that nucleotide binding and release events occur almost simultaneously during the synthesis cycle, allowing the energy gain due to spontaneous binding of ADP to one catalytic β subunit to be directly harnessed for driving the release of ATP from another rather than being dissipated as heat. Here, we examine the unknown mechanism of this coupling that is critical for an exceptionally high mechanochemical efficiency of F 1 -ATPase by means of all-atom free-energy simulations. We find that nondissipative and kinetically fast progression of the motor in the synthesis direction requires a concerted conformational change involving the closure of the ADP-binding β subunit followed by the gradual opening of the ATP-releasing β subunit over the course of the 30 to 40° rotary substep of the γ subunit. This rotary substep, preceding the ATP-dependent metastable state, allows for the recovery of a large portion of the ADP binding energy in the conformation of ATP-bound β that gradually adopts the low-affinity conformation, captured also by the recent cryo-EM structure of this elusive state. The release of ATP from this nearly open conformation leads to its further opening, which enables the progression of the motor to the next catalytic metastable state. Our simulations explain this energy conversion mechanism in terms of intersubunit and ligand–protein interactions.

Excitatory amino acid transporters (EAAS) are important members of solute carrier 1 (SLC1) family, which prevent the neurotransmitter toxicity in the synaptic cleft by transporting the excess glutamate to the cytoplasm in glia and neuronal cells. Malfunction of this crucial family of proteins has been associated with numerous neurological disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease, epilepsy, cerebral stroke, dementia, Huntington's disease and malignant glioma. In continuation of the past decade efforts on understanding the structure and function of this family of integral membrane proteins we have investigated the dynamics of prokaryotic glutamate transporter homolog (Glt Ph ) that is a model system for mammalian EAATs. Here, we report on our latest technological advances based on highspeed atomic force microscopy (HS-AFM) to reach millisecond time resolution. We find that Glt Ph transport domain has faster than previously expected dynamics and occupies previously undetectable transport states.

No abstract