Biomedical subjects

Achim Stocker

Publications and source records attributed to Achim Stocker.

7 recordsLinked to original sources

PubMed2006

Inhibition of ATP hydrolysis by thermoalkaliphilic F1Fo-ATP synthase is controlled by the C terminus of the epsilon subunit.

The F(1)F(o)-ATP synthases of alkaliphilic bacteria exhibit latent ATPase activity, and for the thermoalkaliphile Bacillus sp. strain TA2.A1, this activity is intrinsic to the F(1) moiety. To study the mechanism of ATPase inhibition, we developed a heterologous expression system in Escherichia coli to produce TA2F(1) complexes from this thermoalkaliphile. Like the native F(1)F(o)-ATP synthase, the recombinant TA2F(1) was blocked in ATP hydrolysis activity, and this activity was stimulated by the detergent lauryldimethylamine oxide. To determine if the C-terminal domain of the epsilon subunit acts as an inhibitor of ATPase activity and if an electrostatic interaction plays a role, a TA2F(1) mutant with either a truncated epsilon subunit [i.e., TA2F(1)(epsilon(DeltaC))] or substitution of basic residues in the second alpha-helix of epsilon with nonpolar alanines [i.e., TA2F(1)(epsilon(6A))] was constructed. Both mutants showed ATP hydrolysis activity at low and high concentrations of ATP. Treatment of the purified F(1)F(o)-ATP synthase and TA2F(1)(epsilon(WT)) complex with proteases revealed that the epsilon subunit was resistant to proteolytic digestion. In contrast, the epsilon subunit of TA2F(1)(epsilon(6A)) was completely degraded by trypsin, indicating that the C-terminal arm was in a conformation where it was no longer protected from proteolytic digestion. In addition, ATPase activity was not further activated by protease treatment when compared to the untreated control, supporting the observation that epsilon was responsible for inhibition of ATPase activity. To study the effect of the alanine substitutions in the epsilon subunit in the entire holoenzyme, we reconstituted recombinant TA2F(1) complexes with F(1)-stripped native membranes of strain TA2.A1. The reconstituted TA2F(o)F(1)(epsilon(WT)) was blocked in ATP hydrolysis and exhibited low levels of ATP-driven proton pumping consistent with the F(1)F(o)-ATP synthase in native membranes. Reconstituted TA2F(o)F(1)(epsilon(6A)) exhibited ATPase activity that correlated with increased ATP-driven proton pumping, confirming that the epsilon subunit also inhibits ATPase activity of TA2F(o)F(1).

Adenosine Triphosphate↗

PubMed2005

Purification, crystallization, and properties of F1-ATPase complexes from the thermoalkaliphilic Bacillus sp. strain TA2.A1.

Recently, we reported the cloning of the atp operon encoding for the F(1)F(0)-ATP synthase from the extremely thermoalkaliphilic bacterium Bacillus sp. strain TA2.A1. In this study, the genes encoding the F(1) moiety of the enzyme complex were cloned from the atp operon into the vector pTrc99A and expressed in Escherichia coli in two variant complexes, F(1)-wt consisting of subunits alpha(3)beta(3)gammadeltaepsilon and F(1)Deltadelta lacking the entire delta-subunit as a prerequisite for overproduction and crystallization trials. Both F(1)-wt and F(1)Deltadelta were successfully overproduced in E. coli and purified in high yield and purity. F(1)Deltadelta was crystallized by micro-batch screening yielding three-dimensional crystals that diffracted to a resolution of 3.1A using a synchrotron radiation source. After establishing cryo and dehydrating conditions, a complete set of diffraction data was collected from a single crystal. No crystals were obtained with F(1)-wt. Data processing of diffraction patterns showed that F(1)Deltadelta crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell parameters of a=121.70, b=174.80, and c=223.50A, alpha, beta, gamma=90.000. The asymmetric unit contained one molecule of bacterial F(1)Deltadelta with a corresponding volume per protein weight (V(M)) of 3.25A(3) Da(-1) and a solvent content of 62.1%. Silver staining of single crystals of F(1)Deltadelta analyzed by SDS-PAGE revealed four bands alpha, beta, gamma, and epsilon with identical M(r)-values as those found in the native F(1)F(0)-ATP synthase isolated from strain TA2.A1 membranes. ATPase assays of F(1)Deltadelta crystals exhibited latent ATP hydrolytic activity that was highly stimulated by lauryldimethylamine oxide, a hallmark of the native enzyme.

Bacillus↗

PubMed2004

Molecular determinants of heritable vitamin E deficiency.

Tocopherol transfer protein (TTP) is a key regulator of vitamin E homeostasis. TTP is presumed to function by transporting the hydrophobic vitamin between cellular compartments, thus facilitating its secretion to the extracellular space. Indeed, recombinant TTP demonstrates marked ability to facilitate tocopherol transfer between lipid bilayers. We report the biochemical characterization of six missense mutations TTP(1) that are found in human AVED patients. We expressed the H101Q, A120T, R192H, R59W, E141K, and R221W TTP mutants in Escherichia coli, and purified the proteins to homogeneity. We then characterized TTP and its mutant counterparts with respect to their affinity for RRR-alpha-tocopherol and to their ability to catalyze tocopherol transfer between membranes. We observe the R59W, E141K, and R221W mutations, associated with the severe, early-onset version of AVED, are impaired in tocopherol binding and transfer activity. Surprisingly, despite the profound clinical effect of the R59W, E141K, and R221W mutations in vivo, their impact on TTP activity in vitro is quite benign (2-3-fold reduction in transfer kinetics). Furthermore, mutations associated with milder forms of the AVED disease, while causing pronounced perturbations in tocopherol homeostasis in vivo, are remarkably similar to the wild-type protein in the tocopherol transfer assays in vitro. Our data indicate that tocopherol transfer activity in vitro does not properly recapitulate the physiological functions of TTP. These findings suggest the possibility that the AVED syndrome may not arise from an inability of TTP to bind or to transfer alpha tocopherol, but rather from defects in other activities of the protein.

Amino Acid Substitution↗

PubMed2004

Molecular mechanisms of vitamin E transport.

Vitamin E is the most important lipid-soluble antioxidant in humans. Specific tocopherol-binding proteins favor the retention of the most potent vitamin E homologue, RRR-alpha-tocopherol (RRR-alpha-T) in man. The crystal structures of both the ligand-charged and the apo-forms of human alpha-tocopherol transfer protein (alpha-TTP) and of human supernatant protein factor (SPF) have been solved. The renewed interest in the biological function of tocopherol binders is based on the discovery of ataxia with vitamin E deficiency, a neurological disorder that is caused by genetic defects of the alpha-TTP gene and/or vitamin E deficiency. The analysis of the crystal structure of alpha-TTP provides the molecular basis of vitamin E retention in man. SPF has been reported to enhance cholesterol biosynthesis by facilitating the conversion of squalene to lanosterol. Nevertheless, the physiological role of SPF as well as its ligand specificity is not known. Investigations on the substrate specificity of SPF have uncovered binding of RRR-alpha-tocopherylquinone (RRR-alpha-TQ). RRR-alpha-TQ represents the major physiological oxidation product of RRR-alpha-T. The three-dimensional overlay of the ligand-charged structures of SPF and alpha-TTP indicates that ligand specificity in both proteins is mostly modulated by side-chain variations rather than by the backbone. Recent reports point towards the in vivo reduction of RRR-alpha-TQ to RRR-alpha-TQH(2) and its protective role in low-density lipoprotein oxidation. On the basis of these reports, it is proposed that SPF may enhance cholesterol biosynthesis indirectly by mediating the transfer of RRR-alpha-TQ to low-density lipoprotein, thus reducing oxidation of low-density lipoprotein and its subsequent cellular uptake by scavenger receptors.

Animals↗

PubMed2003

Supernatant protein factor in complex with RRR-alpha-tocopherylquinone: a link between oxidized Vitamin E and cholesterol biosynthesis.

The vast majority of monomeric lipid transport in nature is performed by lipid-specific protein carriers. This class of proteins can enclose cognate lipid molecules in a hydrophobic cavity and transport them across the aqueous environment. Supernatant protein factor (SPF) is an enigmatic representative of monomeric lipid transporters belonging to the SEC14 family. SPF stimulates squalene epoxidation, a downstream step of the cholesterol biosynthetic pathway, by an unknown mechanism. Here, we present the three-dimensional crystal structure of human SPF in complex with RRR-alpha-tocopherylquinone, the major physiological oxidation product of RRR-alpha-tocopherol, at a resolution of 1.95A. The structure of the complex reveals how SPF sequesters RRR-alpha-tocopherylquinone (RRR-alpha-TQ) in its protein body and permits a comparison with the recently solved structure of human alpha-tocopherol transfer protein (alpha-TTP) in complex with RRR-alpha-tocopherol. Recent findings have shown that RRR-alpha-TQ is reduced in vivo to RRR-alpha-TQH(2), the latter has been suggested to protect low-density lipoprotein (LDL) particles from oxidation. Hence, the antioxidant function of the redox couple RRR-alpha-TQ/RRR-alpha-TQH(2) in blocking LDL oxidation may reduce cellular cholesterol uptake and thus explain how SPF upregulates cholesterol synthesis.

Antioxidants↗

PubMed2003

The molecular basis of vitamin E retention: structure of human alpha-tocopherol transfer protein.

Alpha-tocopherol transfer protein (alpha-TTP) is a liver protein responsible for the selective retention of alpha-tocopherol from dietary vitamin E, which is a mixture of alpha, beta, gamma, and delta-tocopherols and the corresponding tocotrienols. The alpha-TTP-mediated transfer of alpha-tocopherol into nascent VLDL is the major determinant of plasma alpha-tocopherol levels in humans. Mutations in the alpha-TTP gene have been detected in patients suffering from low plasma alpha-tocopherol and ataxia with isolated vitamin E deficiency (AVED). The crystal structure of alpha-TTP reveals two conformations. In its closed tocopherol-charged form, a mobile helical surface segment seals the hydrophobic binding pocket. In the presence of detergents, an open conformation is observed, which probably represents the membrane-bound form. The selectivity of alpha-TTP for RRR-alpha-tocopherol is explained from the van der Waals contacts occurring in the lipid-binding pocket. Mapping the known mutations leading to AVED onto the crystal structure shows that no mutations occur directly in the binding pocket.

Ataxia↗

PubMed2002

Crystal structure of the human supernatant protein factor.

Supernatant protein factor (SPF) promotes the epoxidation of squalene catalyzed by microsomes. Several studies suggest its in vivo role in the cholesterol biosynthetic pathway by a yet unknown mechanism. SPF belongs to a family of lipid binding proteins called CRAL_TRIO, which include yeast phosphatidylinositol transfer protein Sec14 and tocopherol transfer protein TTP. The crystal structure of human SPF at a resolution of 1.9 A reveals a two domain topology. The N-terminal 275 residues form a Sec14-like domain, while the C-terminal 115 residues consist of an eight-stranded jelly-roll barrel similar to that found in many viral protein structures. The ligand binding cavity has a peculiar horseshoe-like shape. Contrary to the Sec14 crystal structure, the lipid-exchange loop is in a closed conformation, suggesting a mechanism for lipid exchange.

Carrier Proteins↗