Structure of the ATP synthase catalytic complex (F(1)) from Escherichia coli in an autoinhibited conformation.

ATP synthase is a membrane-bound rotary motor enzyme that is critical for cellular energy metabolism in all kingdoms of life. Despite conservation of its basic structure and function, autoinhibition by one of its rotary stalk subunits occurs in bacteria and chloroplasts but not in mitochondria. The crystal structure of the ATP synthase catalytic complex (F(1)) from Escherichia coli described here reveals the structural basis for this inhibition. The C-terminal domain of subunit ɛ adopts a heretofore unknown, highly extended conformation that inserts deeply into the central cavity of the enzyme and engages both rotor and stator subunits in extensive contacts that are incompatible with functional rotation. As a result, the three catalytic subunits are stabilized in a set of conformations and rotational positions distinct from previous F(1) structures

Mutation of a single residue, β-glutamate-20, alters protein–lipid interactions of light harvesting complex II

It is well established that assembly of the peripheral antenna complex, LH2, is required for proper photosynthetic membrane biogenesis in the purple bacterium Rhodobacter sphaeroides. The underlying interactions are, as yet, not understood. Here we examined the relationship between the morphology of the photosynthetic membrane and the lipid–protein interactions at the LH2–lipid interface. The non-bilayer lipid, phosphatidylethanolamine, is shown to be highly enriched in the boundary lipid phase of LH2. Sequence alignments indicate a putative lipid binding site, which includes β-glutamate-20 and the adjacent carotenoid end group. Replacement of β-glutamate-20 with alanine results in significant reduction of phosphatidylethanolamine and concomitant raise in phosphatidylcholine in the boundary lipid phase of LH2 without altering the lipid composition of the bulk phase. The morphology of the LH2 housing membrane is, however, unaffected by the amino acid replacement. In contrast, simultaneous modification of glutamate-20 and exchange of the carotenoid sphaeroidenone with neurosporene results in significant enlargement of the vesicular membrane invaginations. These findings suggest that the LH2 complex, specifically β-glutamate-20 and the carotenoids' polar head group, contribute to the shaping of the photosynthetic membrane by specific interactions with surrounding lipid molecules

The dynamic stator stalk of rotary ATPases

Rotary ATPases couple ATP hydrolysis/synthesis with proton translocation across biological membranes and so are central components of the biological energy conversion machinery. Their peripheral stalks are essential components that counteract torque generated by rotation of the central stalk during ATP synthesis or hydrolysis. Here we present a 2.25-Å resolution crystal structure of the peripheral stalk from Thermus thermophilus A-type ATPase/synthase. We identify bending and twisting motions inherent within the structure that accommodate and complement a radial wobbling of the ATPase headgroup as it progresses through its catalytic cycles, while still retaining azimuthal stiffness necessary to counteract rotation of the central stalk. The conformational freedom of the peripheral stalk is dictated by its unusual right-handed coiled-coil architecture, which is in principle conserved across all rotary ATPases. In context of the intact enzyme, the dynamics of the peripheral stalks provides a potential mechanism for cooperativity between distant parts of rotary ATPases

ATP synthase: from single molecule to human bioenergetics

ATP synthase (FoF1) consists of an ATP-driven motor (F1) and a H+-driven motor (Fo), which rotate in opposite directions. FoF1 reconstituted into a lipid membrane is capable of ATP synthesis driven by H+ flux. As the basic structures of F1 (α3β3γδε) and Fo (ab2c10) are ubiquitous, stable thermophilic FoF1 (TFoF1) has been used to elucidate molecular mechanisms, while human F1Fo (HF1Fo) has been used to study biomedical significance. Among F1s, only thermophilic F1 (TF1) can be analyzed simultaneously by reconstitution, crystallography, mutagenesis and nanotechnology for torque-driven ATP synthesis using elastic coupling mechanisms. In contrast to the single operon of TFoF1, HFoF1 is encoded by both nuclear DNA with introns and mitochondrial DNA. The regulatory mechanism, tissue specificity and physiopathology of HFoF1 were elucidated by proteomics, RNA interference, cytoplasts and transgenic mice. The ATP synthesized daily by HFoF1 is in the order of tens of kilograms, and is primarily controlled by the brain in response to fluctuations in activity

DISCUSSIONS Thymic Involution in Ontogenesis: Role in Aging Program

Despite the important role of the thymus as the central organ of the immune system, aging is accompanied by thymic involution in most mammals In addition to physiological conditions that change throughout life and control age-related thymus development, random events can cause thymic involution as well as reversible temporal hypoplasia or hyperplasia of the thymus. Rapid reduction of thymic cellularity takes place in young patients who have experienced trauma, chemotherapy, and other forms of stress. Mechanisms that determine the process of involution appear to depend on factors inherent in thymic tissue, such as the local production of cytokines and chemoattractants that promote mobilization, growth, and differentiation of T-cells predecessors in the thymus and on external factors, such as the levels of endocrine hormones and mediators released by intrathymic neurons of the autonomic nervous system Abstract-In most mammals, involution of the thymus occurs with aging. In this issue of Biochemistry (Moscow) devoted to phenoptosis, A. V. Khalyavkin considered involution of a thymus as an example of the program of development and further -of proliferation control and prevention of tumor growth. However, in animals devoid of a thymus (e.g. naked mice), stimulation of carcinogenesis, but not its prevention was observed. In this report, we focus on the involution of the thymus as a manifestation of the aging program (slow phenoptosis). We also consider methods of reversal/arrest of this program at different levels of organization of life (cell, tissue, and organism) including surgical manipulations, hormonal effects, genetic techniques, as well as the use of conventional and mitochondria-targeted antioxidants. We conclude that programmed aging (at least on the model of age-dependent thymic atrophy) can be inhibited

EFFECT OF THE gammaM23-K MUTATION ON ATP SYNTHASE ACTIVATION BY THE PROTONMOTIVE FORCE AND ON COUPLING ATP HYDROLYSIS TO PROTON TRANSLOCATION

The single-site mutation M23-K in the gamma-subunit of the E.coli ATP synthase has been reported to perturb the energetic coupling between F1 and FO and to increase the transition state activation energy for ATP hydrolysis (1). We have introduced the homologous mutation gamma- M23-K in the ATP synthase of Rhodobacter capsulatus in order to study its phenotype by taking advantage of the photosynthetic system. In our hands, the most striking phenotypic difference to the wild-type complex was found in the ATP hydrolysis activation by the protonmotive force. The higher ATP hydrolysis rate is best observed when the activating protonmotive force is dissipated by addition of uncouplers. At low or zero protonmotive force, though, this activated state decays and can be measured only for a limited time. We have found that in the mutated enzyme, the half-life time of the light-activated state, measured under the above conditions, is reduced from 13 to 4 s. Moreover, after a burst of ATP synthesis triggered by a train of flashes, while the wild-type rapidly hydrolyzes the newly synthesized ATP, the mutant does not, indicating that stabilization of the inactive state is even higher at low ATP concentration. The efficiency of proton coupling could be modified, both in the wild type (2) and mutated strain according to the experimental conditions. While a large difference in efficiency could be detected in the M23K mutated strain as compared to the wild type under certain conditions, in other cases the difference was much more limited. A detailed analysis of the two phenotypes will be presented and discussed in relation to the modulation of proton pumping activity and the possible differences produced by the mutation. (1) Al-Shawi MK, Ketchum CJ, Nakamoto RK.. (1997) J. Biol. Chem. 272: 2300-6. (2) Turina P., Giovannini D., Gubellini F. and Melandri B.A. (2004) Biochemistry 43: 11126-34

A single-point mutation in the ATP synthase of Rb. capsulatus impairing the stability of the protonmotive forceactivated state

The single-point mutation gammaM23-K introduced in the ATP synthase of E. coli has been reported to perturb the coupling efficiency between ATP hydrolysis and proton pumping as measured with the ACMA assay (1). Supporting this conclusion, the ATP synthesis rate was more affected compared to wild-type than the ATP hydrolysis rate, by about threefold (2). In addition, a study of interaction between subunits indicated that, in the mutated complex, the epsilon subunit inhibition of ATPase activity was not relieved upon binding of F1 to the membrane as observed in the wild-type (2). With the aim of further investigating the uncoupling process in a photosynthetic system in which analysis of the kinetics of the phosphorylating proton fluxes is possible (3), we have introduced this same mutation in the ATP synthase of Rb. capsulatus.In this organism, ATP synthesis and hydrolysis rates were impaired to a similar extent, both to approximately 1/3 of wild-type. Analysis of phosphorylating proton fluxes and associated ATP synthesis in the mutated and wild-type enzymes has not revealed uncoupling.However, the protonmotive force-activated state (measured as the transient increase of the ATP hydrolysis rate upon addition of uncouplers to energised vesicles), decayed extremely fast compared to wild-type. In agreement with this finding, the coupled proton flux through FoF1 induced by a single flash, which is usually observed in the wild-type enzyme in the presence of ADP and Pi, was completely absent. We conclude that the gammaM23-K mutated ATP synthase of Rb. capsulatus is an excellent system for studying the mechanism of ATP synthase activation by the protonmotive force. References [1] K. Shin, R.K. Nakamoto, M. Maeda, M. Futai, J. Biol Chem. 267 (1992) 20835–20839. [2] M.K. Al-Shawi, C.J. Ketchum, R.K. Nakamoto, J. Biol Chem. 272 (1997) 2300–2306. [3] B.A. Feniouk, D.A. Cherepanov, W. Junge, A.Y. Mulkidjanian, Biochim. Biophys. Acta 506 (2001) 189–203