Biomedical subjects

Stephan Wilkens

Publications and source records attributed to Stephan Wilkens.

12 recordsLinked to original sources

PubMed2006

Localization of subunit C (Vma5p) in the yeast vacuolar ATPase by immuno electron microscopy.

Vacuolar ATPases (V1V0 -ATPases) function in proton translocation across lipid membranes of subcellular compartments. We have used antibody labeling and electron microscopy to define the position of subunit C in the vacuolar ATPase from yeast. The data show that subunit C is binding at the interface of the ATPase and proton channel, opposite from another stalk density previously identified as subunit H [Wilkens S., Inoue T., and Forgac M. (2004) Three-dimensional structure of the vacuolar ATPase - Localization of subunit H by difference imaging and chemical cross-linking. J. Biol. Chem. 279, 41942-41949]. A picture of the vacuolar ATPase stalk domain is emerging in which subunits C and H are positioned to play a role in reversible enzyme dissociation and activity silencing.

Antibodies, Monoclonal↗

PubMed2005

Structural characterization of the interaction of the delta and alpha subunits of the Escherichia coli F1F0-ATP synthase by NMR spectroscopy.

A critical point of interaction between F(1) and F(0) in the bacterial F(1)F(0)-ATP synthase is formed by the alpha and delta subunits. Previous work has shown that the N-terminal domain (residues 3-105) of the delta subunit forms a 6 alpha-helix bundle [Wilkens, S., Dunn, S. D., Chandler, J., Dahlquist, F. W., and Capaldi, R. A. (1997) Nat. Struct. Biol. 4, 198-201] and that the majority of the binding energy between delta and F(1) is provided by the interaction between the N-terminal 22 residues of the alpha- and N-terminal domain of the delta subunit [Weber, J., Muharemagic, A., Wilke-Mounts, S., and Senior, A. E. (2003) J. Biol. Chem. 278, 13623-13626]. We have now analyzed a 1:1 complex of the delta-subunit N-terminal domain and a peptide comprising the N-terminal 22 residues of the alpha subunit by heteronuclear protein NMR spectroscopy. A comparison of the chemical-shift values of delta-subunit residues with and without alpha N-terminal peptide bound indicates that the binding interface on the N-terminal domain of the delta subunit is formed by alpha helices I and V. NOE cross-peak patterns in 2D (12)C/(12)C-filtered NOESY spectra of the (13)C-labeled delta-subunit N-terminal domain in complex with unlabeled peptide verify that residues 8-18 in the alpha-subunit N-terminal peptide are folded as an alpha helix when bound to delta N-terminal domain. On the basis of intermolecular contacts observed in (12)C/(13)C-filtered NOESY experiments, we describe structural details of the interaction of the delta-subunit N-terminal domain with the alpha-subunit N-terminal alpha helix.

Amino Acid Sequence↗

PubMed2005

Structure of dimeric mitochondrial ATP synthase: novel F0 bridging features and the structural basis of mitochondrial cristae biogenesis.

The F1F0-ATP synthase exists as a dimer in mitochondria, where it is essential for the biogenesis of the inner membrane cristae. How two ATP synthase complexes dimerize to promote cristae formation is unknown. Here we resolved the structure of the dimeric F1F0 ATP synthase complex isolated from bovine heart mitochondria by transmission electron microscopy. The structure of the ATP synthase dimer has an overall conic appearance that is consistent with the proposed role of the dimeric enzyme in mitochondrial cristae biogenesis. The ATP synthase dimer interface is formed by contacts on both the F0 and F1 domains. A cross-bridging protein density was resolved which connects the two F0 domains on the intermembrane space side of the membrane. On the matrix side of the complex, the two F1 moieties are connected by a protein bridge, which is attributable to the IF1 inhibitor protein.

Animals↗

PubMed2005

A structural model of the vacuolar ATPase from transmission electron microscopy.

Vacuolar ATPases (V-ATPases) are large, membrane bound, multisubunit protein complexes which function as ATP hydrolysis driven proton pumps. V-ATPases and related enzymes are found in the endomembrane system of eukaryotic organsims, the plasma membrane of specialized cells in higher eukaryotes, and the plasma membrane of prokaryotes. The proton pumping action of the vacuolar ATPase is involved in a variety of vital intra- and inter-cellular processes such as receptor mediated endocytosis, protein trafficking, active transport of metabolites, homeostasis and neurotransmitter release. This review summarizes recent progress in the structure determination of the vacuolar ATPase focusing on studies by transmission electron microscopy. A model of the subunit architecture of the vacuolar ATPase is presented which is based on the electron microscopic images and the available information from genetic, biochemical and biophysical experiments.

Animals↗

PubMed2005

Rotary molecular motors.

The F-, V-, and A-adenosine triphosphatases (ATPases) represent a family of evolutionarily related ion pumps found in every living cell. They either function to synthesize adenosine triphosphate (ATP) at the expense of an ion gradient or they act as primary ion pumps establishing transmembrane ion motive force at the expense of ATP hydrolysis. The A-, F-, and V-ATPases are rotary motor enzymes. Synthesis or hydrolysis of ATP taking place in the three catalytic sites of the membrane extrinsic domain is coupled to ion translocation across the single ion channel in the membrane-bound domain via rotation of a central part of the complex with respect to a static portion of the enzyme. This chapter reviews recent progress in the structure determination of several members of the family of F-, A-, and V-ATPases and our current understanding of the rotary mechanism of energy coupling.

Adenosine Triphosphatases↗

PubMed2005

Deletion of highly conserved arginine-rich RNA binding motif in cowpea chlorotic mottle virus capsid protein results in virion structural alterations and RNA packaging constraints.

The N-proximal region of cowpea chlorotic mottle virus (CCMV) capsid protein (CP) contains an arginine-rich RNA binding motif (ARM) that is also found in the CPs of other members of Bromoviridae and in other RNA binding proteins such as the Tat and Rev proteins of human immunodeficiency virus. To assess the critical role played by this motif during encapsidation, a variant of CCMV RNA3 (C3) precisely lacking the ARM region (C3/Delta919) of its CP gene was constructed. The biology and the competence of the matured CP derived in vivo from C3/Delta919 to assemble and package progeny RNA was examined in whole plants. Image analysis and computer-assisted three-dimensional reconstruction of wild-type and mutant virions revealed that the CP subunits bearing the engineered deletion assembled into polymorphic virions with altered surface topology. Northern blot analysis of virion RNA from mutant progeny demonstrated that the engineered mutation down-regulated packaging of all four viral RNAs; however, the packaging effect was more pronounced on genomic RNA1 and RNA2 than genomic RNA3 and its CP mRNA. In vitro assembly assays with mutant CP subunits and RNA transcripts demonstrated that the mutant CP is inherently not defective in packaging genomic RNA1 (53%) and RNA2 (54%), but their incorporation into virions was competitively inhibited by the presence of other viral RNAs. Northern blot analysis of RNA encapsidation in vivo of two distinct bromovirus RNA3 chimeras, constructed by exchanging CPs having the Delta919 deletion, demonstrated that the role of the conserved N-terminal ARM in recognizing and packaging specific RNA is distinct for each virus.

Amino Acid Motifs↗

PubMed2004

Three-dimensional structure of the vacuolar ATPase. Localization of subunit H by difference imaging and chemical cross-linking.

The structure of the proton-pumping vacuolar ATPase (V-ATPase) from bovine brain clathrin coated vesicles was analyzed by electron microscopy and single molecule image analysis. A three-dimensional structural model of the complex was calculated by the angular reconstitution method at a resolution of 27 A. Overall, the appearance of the V(0) and V(1) domains in the three-dimensional model of the intact bovine V-ATPase resembles the models of the isolated bovine V(0) and yeast V(1) domains determined previously. To determine the binding position of subunit H in the V-ATPase, electron microscopy and cysteine-mediated photochemical cross-linking were used. Difference maps calculated from projection images of intact bovine V-ATPase and a V-ATPase preparation in which the two H subunit isoforms were removed by treatment with cystine revealed less protein density at the bottom of the V(1) in the subunit H-depleted enzyme, suggesting that subunit H isoforms bind at the interface of the V(1) and V(0) domains. A comparison of three-dimensional models calculated for intact and subunit H-depleted enzyme indicated that at least one of the subunit H isoforms, although poorly resolved in the three-dimensional electron density, binds near the putative N-terminal domain of the a subunit of the V(0). For photochemical cross-linking, unique cysteine residues were introduced into the yeast V-ATPase B subunit at sites that were localized based on molecular modeling using the crystal structure of the mitochondrial F(1) domain. Cross-linking was performed using the photoactivatable sulfhydryl reagent 4-(N-maleimido)benzophenone. Cross-linking to subunit H was observed from two sites on subunit B (E494 and T501) predicted to be located on the outer surface of the subunit closest to the membrane. Results from both electron microscopy and cross-linking analysis thus place subunit H near the interface of the V(1) and V(0) domains and suggest a close structural similarity between the V-ATPases of yeast and mammals.

Animals↗

PubMed2003

Yeast V1-ATPase: affinity purification and structural features by electron microscopy.

V1-ATPase from the yeast Saccharomyces cerevisiae was purified via a FLAG affinity tag introduced into the N terminus of the G subunit. The preparation migrated as a single band in native gel electrophoresis and contained subunits ABCDEFGH (with subunit C present at substoichiometric amounts) as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The initial specific Ca-ATPase activity was approximately 6 micromol/min/mg. The structure of the yeast V1-ATPase was studied by electron microscopy of negatively stained and frozen hydrated samples. A 25-A resolution three-dimensional model of the complex was calculated from two-dimensional projections by the angular reconstitution technique. The model shows six elongated densities arranged in pseudo-3-fold symmetry around a large central cavity. At the top of the molecule, various protrusions can be seen. At the bottom of the complex, two large masses are visible that are connected to the main body of the molecule. Comparison of the yeast V1 structure with the structure of the intact V1V0-ATPase from bovine brain clathrin-coated vesicles (Wilkens, S., Vasilyeva, E., and Forgac, M. (1999) J. Biol. Chem. 274, 31804-31810) indicates that the structure of the isolated V1 from yeast is very similar to the structure of the V1 domain in the intact V-ATPase complex.

Chromatography, Affinity↗

PubMed2003

Subunit structure, function, and arrangement in the yeast and coated vesicle V-ATPases.

The vacuolar (H+)-ATPases (or V-ATPases) are ATP-dependent proton pumps that function both to acidify intracellular compartments and to transport protons across the plasma membrane. Acidification of intracellular compartments is important for such processes as receptor-mediated endocytosis, intracellular trafficking, protein processing, and coupled transport. Plasma membrane V-ATPases function in renal acidification, bone resorption, pH homeostasis, and, possibly, tumor metastasis. This review will focus on work from our laboratories on the V-ATPases from mammalian clathrin-coated vesicles and from yeast. The V-ATPases are composed of two domains. The peripheral V1 domain has a molecular mass of 640 kDa and is composed of eight different subunits (subunits A-H) of molecular mass 70-13 kDa. The integral V0 domain, which has a molecular mass of 260 kDa, is composed of five different subunits (subunits a, d, c, c', and c'') of molecular mass 100-17 kDa. The V1 domain is responsible for ATP hydrolysis whereas the V0 domain is responsible for proton transport. Using a variety of techniques, including cysteine-mediated crosslinking and electron microscopy, we have defined both the overall shape of the V-ATPase and the V0 domain as well as the location of various subunits within the complex. We have employed site-directed and random mutagenesis to identify subunits and residues involved in nucleotide binding and hydrolysis, proton translocation, and the coupling of these two processes. We have also investigated the mechanism of regulation of the V-ATPase by reversible dissociation and the role of different subunits in this process.

Adenosine Triphosphate↗

PubMed2003

The binding of maize DHN1 to lipid vesicles. Gain of structure and lipid specificity.

Dehydrins (DHNs; late embryogenesis abundant D-11) are a family of plant proteins induced in response to abiotic stresses such as drought, low temperature, and salinity or during the late stages of embryogenesis. Spectral and thermal properties of these proteins in purified form suggest that they are "intrinsically unstructured." However, DHNs contain at least one copy of a consensus 15-amino acid sequence, the "K segment," which resembles a class A2 amphipathic alpha-helical, lipid-binding domain found in other proteins such as apolipoproteins and alpha-synuclein. The presence of the K segment raises the question of whether DHNs bind lipids, bilayers, or phospholipid vesicles. Here, we show that maize (Zea mays) DHN DHN1 can bind to lipid vesicles that contain acidic phospholipids. We also observe that DHN1 binds more favorably to vesicles of smaller diameter than to larger vesicles, and that the association of DHN1 with vesicles results in an apparent increase of alpha-helicity of the protein. Therefore, DHNs, and presumably somewhat similar plant stress proteins in the late embryogenesis abundant and cold-regulated classes may undergo function-related conformational changes at the water/membrane interface, perhaps related to the stabilization of vesicles or other endomembrane structures under stress conditions.

Adaptation, Physiological↗

PubMed2002

Structure, subunit function and regulation of the coated vesicle and yeast vacuolar (H(+))-ATPases.

The vacuolar (H(+))-ATPases (or V-ATPases) are ATP-dependent proton pumps that function to acidify intracellular compartments in eukaryotic cells. This acidification is essential for such processes as receptor-mediated endocytosis, intracellular targeting of lysosomal enzymes, protein processing and degradation and the coupled transport of small molecules. V-ATPases in the plasma membrane of specialized cells also function in such processes as renal acidification, bone resorption and pH homeostasis. Work from our laboratory has focused on the V-ATPases from clathrin-coated vesicles and yeast vacuoles.Structurally, the V-ATPases are composed of two domains: a peripheral complex (V(1)) composed of eight different subunits (A-H) that is responsible for ATP hydrolysis and an integral complex (V(0)) composed of five different subunits (a, d, c, c' and c") that is responsible for proton translocation. Electron microscopy has revealed the presence of multiple stalks connecting the V(1) and V(0) domains, and crosslinking has been used to address the arrangement of subunits in the complex. Site-directed mutagenesis has been employed to identify residues involved in ATP hydrolysis and proton translocation and to study the topology of the 100 kDa a subunit. This subunit has been shown to control intracellular targeting of the V-ATPase and to influence reversible dissociation and coupling of proton transport and ATP hydrolysis.

Adenosine Triphosphate↗

PubMed2002

Projection structure of P-glycoprotein by electron microscopy. Evidence for a closed conformation of the nucleotide binding domains.

The structure of P-glycoprotein (Pgp) from mouse has been studied by electron microscopy and image analysis. Two-dimensional crystals of Pgp in a lipid bilayer were generated by reconstituting pure, detergent-solubilized protein containing a C-terminal six-histidine tag using the lipid monolayer technique. The crystals belong to plane group P1 with a = b = 104 +/- 2 A and gamma = 90 +/- 4 degrees. The projection structure of Pgp calculated at a resolution of 22 A shows two closely interacting protein domains that can be interpreted as the N- and C-terminal halves of the protein. The projection structure of Pgp is consistent with the recently published x-ray structure of MsbA, a lipid A flippase from Escherichia coli with high sequence homology to Pgp but only when the two MsbA subunits are rotated to bring their nucleotide binding domains together.

ATP Binding Cassette Transporter, Subfamily B, Mem↗