The Role of the βDELSEED-loop of ATP Synthase

Nelli Mnatsakanyan,Arathianand M Krishnakumar,Toshiharu Suzuki,Joachim Weber

doi:10.1074/jbc.m900374200

Nelli Mnatsakanyan, Arathianand M Krishnakumar + Show 2 more

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https://doi.org/10.1074/jbc.m900374200

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Abstract

ATP synthase uses a unique rotational mechanism to convert chemical energy into mechanical energy and back into chemical energy. The helix-turn-helix motif, termed "DELSEED-loop," in the C-terminal domain of the beta subunit was suggested to be involved in coupling between catalysis and rotation. Here, the role of the DELSEED-loop was investigated by functional analysis of mutants of Bacillus PS3 ATP synthase that had 3-7 amino acids within the loop deleted. All mutants were able to catalyze ATP hydrolysis, some at rates several times higher than the wild-type enzyme. In most cases ATP hydrolysis in membrane vesicles generated a transmembrane proton gradient, indicating that hydrolysis occurred via the normal rotational mechanism. Except for two mutants that showed low activity and low abundance in the membrane preparations, the deletion mutants were able to catalyze ATP synthesis. In general, the mutants seemed less well coupled than the wild-type enzyme, to a varying degree. Arrhenius analysis demonstrated that in the mutants fewer bonds had to be rearranged during the rate-limiting catalytic step; the extent of this effect was dependent on the size of the deletion. The results support the idea of a significant involvement of the DELSEED-loop in mechanochemical coupling in ATP synthase. In addition, for two deletion mutants it was possible to prepare an alpha(3)beta(3)gamma subcomplex and measure nucleotide binding to the catalytic sites. Interestingly, both mutants showed a severely reduced affinity for MgATP at the high affinity site.

Highlights

F1 has three catalytic nucleotide binding sites located on the ␤ subunits at the interface to the adjacent ␣ subunit
According to a model favored by several authors [6, 15, 16], binding of ATP to the low affinity catalytic site on ␤E and the subsequent closure of this site, accompanied by its conversion into the high affinity site, are responsible for driving the large (80 –90°) rotation substep during ATP hydrolysis, with the DELSEED-loop acting as a “pushrod.” A recent molecular dynamics [20] study supports this model and implicates mainly the region around several hydrophobic residues upstream of the DELSEED motif ( ␤I386 and ␤L387)3 as being responsible for making contact with ␥ during the large rotation substep
Overview of Deletion Mutants in the DELSEED-loop—Fig. 1B shows the helix-turn-helix structure known as the DELSEEDloop in the C-terminal domain of the ␤ subunit of ATP synthase

Summary

Introduction

F1 (or F1-ATPase) has three catalytic nucleotide binding sites located on the ␤ subunits at the interface to the adjacent ␣ subunit. When all three catalytic sites are occupied by nucleotide, the previously open ␤E subunit assumes an intermediate, half-closed (␤HC) conformation. It cannot close completely because of steric clashes with ␥ [11]. 17–19), binding of ATP (or, more precisely, MgATP) to the low affinity catalytic site on ␤E and the subsequent closure of this site, accompanied by its conversion into the high affinity site, are responsible for driving the large (80 –90°) rotation substep during ATP hydrolysis, with the DELSEED-loop acting as a “pushrod.” A recent molecular dynamics [20] study supports this model and implicates mainly the region around several hydrophobic residues upstream of the DELSEED motif ( ␤I386 and ␤L387) as being responsible for making contact with ␥ during the large rotation substep According to a model favored by several authors [6, 15, 16] (see Refs. 17–19), binding of ATP (or, more precisely, MgATP) to the low affinity catalytic site on ␤E and the subsequent closure of this site, accompanied by its conversion into the high affinity site, are responsible for driving the large (80 –90°) rotation substep during ATP hydrolysis, with the DELSEED-loop acting as a “pushrod.” A recent molecular dynamics [20] study supports this model and implicates mainly the region around several hydrophobic residues upstream of the DELSEED motif ( ␤I386 and ␤L387) as being responsible for making contact with ␥ during the large rotation substep

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