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A functionally important hydrogen-bonding network at the betaDP/alphaDP interface of ATP synthase.

ABSTRACT: ATP synthase uses a unique rotary mechanism to couple ATP synthesis and hydrolysis to transmembrane proton translocation. The F1 subcomplex has three catalytic nucleotide binding sites, one on each beta subunit, at the interface to the adjacent alpha subunit. In the x-ray structure of F1 (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the three catalytic beta/alpha interfaces differ in the extent of inter-subunit interactions between the C termini of the beta and alpha subunits. At the closed betaDP/alphaDP interface, a hydrogen-bonding network is formed between both subunits, which is absent at the more open betaTP/alphaTP interface and at the wide open betaE/alphaE interface. The hydrogen-bonding network reaches from betaL328 (Escherichia coli numbering) and betaQ441 via alphaQ399, betaR398, and alphaE402 to betaR394, and ends in a cation/pi interaction between betaR394 and alphaF406. Using mutational analysis in E. coli ATP synthase, the functional importance of the betaDP/alphaDP hydrogen-bonding network is demonstrated. Its elimination results in a severely impaired enzyme but has no pronounced effect on the binding affinities of the catalytic sites. A possible role for the hydrogen-bonding network in coupling of ATP synthesis/hydrolysis and rotation will be discussed.

SUBMITTER: Mao HZ

PROVIDER: S-EPMC2529006 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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A functionally important hydrogen-bonding network at the betaDP/alphaDP interface of ATP synthase.

Mao Hui Z HZ Abraham Christopher G CG Krishnakumar Arathianand M AM Weber Joachim J

The Journal of biological chemistry 20080625 36

ATP synthase uses a unique rotary mechanism to couple ATP synthesis and hydrolysis to transmembrane proton translocation. The F1 subcomplex has three catalytic nucleotide binding sites, one on each beta subunit, at the interface to the adjacent alpha subunit. In the x-ray structure of F1 (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the three catalytic beta/alpha interfaces differ in the extent of inter-subunit interactions between the C termini o ...[more]

PMID: 18579516

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Project description:Ankyrin repeat (AR) proteins possess a distinctive modular and repetitive architecture, and their global folds are maintained by short-range interactions in terms of the primary sequence. In this work, we extended our previous study on the highly conserved TPLH tetrapeptide and investigated the impact of a solvent-exposed histidine residue on the pH-dependent stability of gankyrin, providing further insight into the contribution of the TPLH motif to the tertiary fold of AR proteins. Consisting of seven ARs, gankyrin has five histidine residues in TPLH motifs or its variants, all of which adopt a H(?2)-tautermeric form and are shielded from solvent. By truncating the C-terminal ankyrin repeat (AR7), we exposed H177 in the (174)TPLH(177) of AR6 (the second C-terminal AR) to an aqueous environment. We showed that this truncated gankyrin mutant, namely, Gank(1-201), was well-folded at a neutral pH with a slightly lower stability with respect to gankyrin wild type (WT). However, unlike gankyrin WT, the stability of Gank(1-201) was markedly decreased together with a loss of conformation at a pH slightly below 6.0. It was rationalized that the protonation of the H177 imidazole ring triggered the disruption of the TPLH-mediated hydrogen-bonding network, which in turn led to the loss of conformation and stability. These results together with the work on Q210H mutant nicely explain that the C-terminal AR7 has a (207)TPLQ(210) variant and are in support of the notion that a string of TPLH/variant, which may arguably act like a zip lock to the elongated AR proteins via intra-/inter-repeat hydrogen-bonding, is important in maintaining the tertiary fold. Additionally, we made rational mutagenesis to introduce extra surface charge on AR7 of gankyrin and demonstrated that G214E and I219D mutations increased the stability of gankyrin while the function remained intact. Taken together, our results indicate that the TPLH-mediated hydrogen-bonding network is important for the conformation and stability of human gankyrin, and the C-terminal AR contributes to the conformational stability of gankyrin (AR proteins in general) through shielding this TPLH network from solvent as well as making the surface area more accessible to solvent.

Dataset Information

A functionally important hydrogen-bonding network at the betaDP/alphaDP interface of ATP synthase.

Publications

A functionally important hydrogen-bonding network at the betaDP/alphaDP interface of ATP synthase.

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