Activation of bacterial F-ATPase by LDAO: deciphering the molecular mechanism

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Proton FOF1-ATP synthase catalyzes the formation of ATP from ADP and inorganic phosphate coupled with transmembrane proton transfer using the energy of the proton motive force (pmf). As pmf falls, the direction of the reaction is reversed and the enzyme generates pmf, transferring protons across the membrane using the energy of ATP hydrolysis. The ATPase activity of the enzyme can be suppressed by ADP in a non-competitive manner (ADP-inhibition), and in a number of bacteria can be inhibited by the conformational changes of subunit ε regulatory C-terminal domain. Lauryldimethylamine oxide (LDAO), a zwitterionic detergent, is known to attenuate both aforementioned inhibitory mechanisms, stimulating a significant increase in the enzyme's ATPase activity. For this reason, LDAO is sometimes used for the semi-quantitative estimation of these regulatory mechanisms. However, the binding site of LDAO on ATP synthase remains unknown. The mechanism by which the detergent counteracts ADP-inhibition and inhibition by the ε subunit is also unclear. We performed molecular docking and predicted that LDAO binding might occur at the catalytic sites of ATP synthase, whether empty or containing nucleotides. Molecular dynamics simulations showed that LDAO could affect the mobility of a loop in the β subunit (residues β404-415 in Escherichia coli ATP synthase) near the catalytic site. Mutagenesis of the β409 residue in E. coli enzyme and the corresponding β419 residue in Bacillus subtilis ATP synthase revealed that the side chain type of this residue indeed affects LDAO-dependent stimulation of ATPase activity. We also found that LDAO activates the enzyme more strongly in the presence of 100 mM sulfate compared to sulfate-free media. This phenomenon is likely due to the enhancement of ADP-inhibition of the enzyme by sulfate.

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S. Bruman

Lomonosov Moscow State University

Email: feniouk@belozersky.msu.ru

Faculty of Bioengineering and Bioinformatics

俄罗斯联邦, 119234 Moscow

V. Zubareva

Lomonosov Moscow State University; Lomonosov Moscow State University

Email: feniouk@belozersky.msu.ru

Faculty of Bioengineering and Bioinformatics, Belozersky Institute of Physico-Chemical Biology

俄罗斯联邦, 119234 Moscow; 119992 Moscow

T. Shugaeva

Lomonosov Moscow State University

Email: feniouk@belozersky.msu.ru

Faculty of Bioengineering and Bioinformatics

俄罗斯联邦, 119234 Moscow

A. Lapashina

Lomonosov Moscow State University; Lomonosov Moscow State University

Email: feniouk@belozersky.msu.ru

Faculty of Bioengineering and Bioinformatics, Belozersky Institute of Physico-Chemical Biology

俄罗斯联邦, 119234 Moscow; 119992 Moscow

B. Feniouk

Lomonosov Moscow State University; Lomonosov Moscow State University

编辑信件的主要联系方式.
Email: feniouk@belozersky.msu.ru

Faculty of Bioengineering and Bioinformatics, Belozersky Institute of Physico-Chemical Biology

俄罗斯联邦, 119234 Moscow; 119992 Moscow

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3. Fig. 1. The structure of the bacterial subcomplex F1 and the position of the possible binding site of LDAO. a is the alignment of the β subunit sequences; a fragment with a loop–shaped section covering the catalytic center is presented (see in the text; on the alignment, the residues composing this section are highlighted in a gray rectangle). The black border highlights the position occupied by residues homologous to Q412 in the enzyme of the bacterium C. thermarum. b – General view of the F1 C. thermarum subcomplex (the 5HKK structure from the PDB database is used); ADP bound in the catalytic center is colored red, and the approximate position of LDAO bound nearby is blue. The frame highlights the area of the catalytic center shown in panels b and G. b, d is an enlarged image of the area of the catalytic center in two conformations: with the loop β407–418 highlighted in yellow, in the "initial" (c) and "elevated" (d) positions

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4. Fig. 2. ATPase activity of F1 complexes of E. coli wild-type and with replacement of βV409Q, as well as B. subtilis wild-type and with replacement of βQ419V, depending on the concentration of ATP. Activity was measured in a conjugated ATP-regenerating system by the NADH oxidation rate (see "Materials and Methods")

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5. Fig. 3. ATPase activity of the wild-type BF1 complex and with βQ419V substitution, measured in the ATP-regenerating system. a, b – Activity in the presence of 1 mM of ATP, depending on the concentration of LDAO. The points correspond to activity values normalized to the activity of the same drug without LDAO; the curves reflect the average values for each LDAO concentration. The solid curves represent a wild–type enzyme, and the dotted curves represent an enzyme with the βQ419V mutation. a – Activity in the presence of Cl−. b – Comparison of activities in the presence of Cl− (taken from Graph a) and SO42−. b – Absolute activity (in units/ mg of protein; 1 unit corresponds to the hydrolysis of 1 mmol of ATP per minute) depending on the concentration of ATP. The black symbols represent measurements without LDAO (taken from Fig. 2), blank symbols – measurements in the presence of 3.9 mM (0.09%) LDAO

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6. Fig. 4. ATPase activity of EF1 (a, b) complexes and EFOF1 (c, d) liposome-reconstructed wild-type (×) complexes with βV409Q (+) replacement in the presence of LDAO detergent. a are representative curves for measuring EF1 ATPase activity. The reaction was started by adding ATP up to 1 mM (long arrows). Next, LDAO (small arrows) was added to the cuvette to the concentration indicated above the arrow (mM). The gray color shows the results of control experiments (ATP hydrolysis in a cuvette without LDAO and in a cuvette where LDAO was introduced before the start of the ATPase reaction at a concentration of 4 mM). b–d – The points on the graphs correspond to the EF1 (b) and EFOF1 (c, d) activities, normalized by the activity value of the corresponding complex without the addition of LDAO. The lines show the average values calculated from the given points. A mixture of valinomycin and nigericin up to 500 nM each was added as uncoupler (g). The absolute values of the activities corresponding to measurements without LDAO and with the addition of LDAO to 1 mM and 8 mM are shown in the Table. 2 and 3

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7. Fig. 5. ATPase activity of EF1 complexes and wild-type EFOF1 embedded in liposomes and with βV409Q (⚫) replacement in the presence of inorganic phosphate (Ph). The points on the graphs correspond to EF1 or EFOF1 activities, normalized by the activity value of the corresponding complex without the addition of phosphate. A mixture of valinomycin and nigericin up to 500 nM each was added as a uncoupler

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8. 6. ATPase activity of EF1 and EFOF1 complexes of wild type (×) and with βV409Q (+) substitution in the presence of sulfite (Na2SO3). The points on the graphs correspond to EF1 or EFOF1 activities, normalized by the activity value of the corresponding complex without the addition of sulfite. The lines show the average values calculated from the above points. The solid curves represent a wild–type enzyme, and the dotted curves represent an enzyme with the βV409Q mutation. A mixture of valinomycin and nigericin up to 500 nM each was added as a uncoupler

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