Jan Pieter Abrahams

The crystal structure of the nucleotide-free α3β3 subcomplex of F1-ATPase from the thermophilic Bacillus PS3 is a symmetric trimer

Abstract Background: F 1 -ATPase, an oligomeric assembly with subunit stoichiometry α 3 β 3 γ δ ϵ, is the catalytic component of the ATP synthase complex, which plays a central role in energy transduction in bacteria, chloroplasts and mitochondria. The crystal structure of bovine mitochondrial F 1 -ATPase displays a marked asymmetry in the conformation and nucleotide content of the catalytic β subunits. The α 3 β 3 subcomplex of F 1 -ATPase has been assembled from subunits of the moderately thermophilic Bacillus PS3 made in Escherichia coli , and the subcomplex is active but does not show the catalytic cooperativity of intact F 1 -ATPase. The structure of this subcomplex should provide new information on the conformational variability of F 1 -ATPase and may provide insights into the unusual catalytic mechanism employed by this enzyme. Results: The crystal structure of the nucleotide-free bacterial α 3 β 3 subcomplex of F 1 -ATPase, determined at 3.2 A resolution, shows that the oligomer has exact threefold symmetry. The bacterial β subunits adopt a conformation essentially identical to that of the nucleotide-free β subunit in mitochondrial F 1 -ATPase; the α subunits have similar conformations in both structures. Conclusions: The structures of the bacterial F 1 -ATPase α and β subunits are very similar to their counterparts in the mitochondrial enzyme, suggesting a common catalytic mechanism. The study presented here allows an analysis of the different conformations adopted by the α and β subunits and may ultimately further our understanding of this mechanism.

Structure of the E. coli signal recognition particle bound to a translating ribosome

The prokaryotic signal recognition particle (SRP) targets membrane proteins into the inner membrane. It binds translating ribosomes and screens the emerging nascent chain for a hydrophobic signal sequence, such as the transmembrane helix of inner membrane proteins. If such a sequence emerges, the SRP binds tightly, allowing the SRP receptor to lock on. This assembly delivers the ribosome-nascent chain complex to the protein translocation machinery in the membrane. Using cryo-electron microscopy and single-particle reconstruction, we obtained a 16 Å structure of the Escherichia coli SRP in complex with a translating E. coli ribosome containing a nascent chain with a transmembrane helix anchor. We also obtained structural information on the SRP bound to an empty E. coli ribosome. The latter might share characteristics with a scanning SRP complex, whereas the former represents the next step: the targeting complex ready for receptor binding. High-resolution structures of the bacterial ribosome and of the bacterial SRP components are available, and their fitting explains our electron microscopic density. The structures reveal the regions that are involved in complex formation, provide insight into the conformation of the SRP on the ribosome and indicate the conformational changes that accompany high-affinity SRP binding to ribosome nascent chain complexes upon recognition of the signal sequence.

Structure of β-antithrombin and the effect of glycosylation on antithrombin's heparin affinity and activity

Antithrombin is a member of the serpin family of protease inhibitors and the major inhibitor of the blood coagulation cascade. It is unique amongst the serpins in that it circulates in a conformation that is inactive against its target proteases. Activation of antithrombin is brought about by a conformational change initiated upon binding heparin or heparan sulphate. Two isoforms exist in the circulation, alpha-antithrombin and beta-antithrombin, which differ in the amount of glycosylation present on the polypeptide chain; beta-antithrombin lacks the carbohydrate present at Asn135 in alpha-antithrombin. Of the two forms, beta-antithrombin has the higher affinity for heparin and thus functions as the major inhibitor in vivo even though it is the less abundant form. The reason for the differences in heparin affinity between the alpha and beta-forms have been shown to be due to the additional carbohydrate changing the rate of the conformational change. Here, we describe the most accurate structures of alpha-antithrombin and alpha-antithrombin+heparin pentasaccharide reported to date (2.6A and 2.9A resolution, respectively, both re-refinements using old data), and the structure of beta-antithrombin (2.6A resolution). The new structures have a remarkable degree of ordered carbohydrate and include parts of the antithrombin chain not modeled before. The structures have allowed a detailed comparison of the conformational differences between the three. They show that the structural basis of the lower affinity for heparin of alpha-antithrombin over beta-antithrombin is due to the conformational change that occurs upon heparin binding being sterically hindered by the presence of the additional bulky carbohydrate at Asn135.

ATP-induced conformational changes of the nucleotide-binding domain of Na,K-ATPase.

The Na,K-ATPase hydrolyzes ATP to drive the coupled extrusion and uptake of Na+ and K+ ions across the plasma membrane. Here, we report two high-resolution NMR structures of the 213-residue nucleotide-binding domain of rat α1 Na,K-ATPase, determined in the absence and the presence of ATP. The nucleotide binds in the anti conformation and shows a relative paucity of interactions with the protein, reflecting the low-affinity ATP-binding state. Binding of ATP induces substantial conformational changes in the binding pocket and in residues located in the hinge region connecting the N- and P-domains. Structural comparison with the Ca-ATPase stabilized by the inhibitor thapsigargin, E2(TG), and the model of the H-ATPase in the E1 form suggests that the observed changes may trigger the series of events necessary for the release of the K+ ions and/or disengagement of the A-domain, leading to the eventual transfer of the γ-phosphate group to the invariant Asp369.

Ultra-Small Graphene Oxide Functionalized with Polyethylenimine (PEI) for Very Efficient Gene Delivery in Cell and Zebrafish Embryos

AbstractEfficient DNA delivery is essential for introducing new genes into living cells. However, effective virus-based systems carry risks and efficient synthetic systems that are non-toxic remain to be discovered. The bottle-neck in synthetic systems is cytotoxicity, caused by the high concentration of DNA-condensing compounds required for efficient uptake of DNA. Here we report a polyethyleneimine (PEI) grafted ultra-small graphene oxide (PEI-g-USGO) for transfection. By removing the free PEI and ensuring a high PEI density on small sized graphene, we obtained very high transfection efficiencies combined with very low cytotoxicity. Plasmid DNA could be transfected into mammalian cell lines with up to 95% efficiency and 90% viability. Transfection in zebrafish embryos was 90%, with high viability, compared to efficiencies of 30% or lower for established transfection technologies. This result suggests a novel approach to the design of synthetic gene delivery vehicles for research and therapy.

Recombinant apoptin multimers kill tumor cells but are nontoxic and epitope-shielded in a normal-cell-specific fashion

Apoptin, a protein derived from chicken anemia virus, induces apoptosis in human transformed or tumor cells but not in normal cells. When produced in bacteria as a recombinant fusion with maltose-binding protein (MBP-Apoptin), Apoptin forms a distinct, stable multimeric complex that is remarkably homogeneous and uniform. Here, using cytoplasmic microinjection, we showed that recombinant MBP-Apoptin multimers retained the characteristics of the ectopically expressed wild-type Apoptin; namely, the complexes translocated to the nucleus of tumor cells and induced apoptosis, whereas they remained in the cytoplasm of normal, primary cells and exerted no apparent toxic effect. In normal cells, MBP-Apoptin formed increasingly large, organelle-sized globular bodies with time postinjection and eventually lost the ability to be detected by immunofluorescence analysis. Costaining with an acidotrophic marker indicated that these globular structures did not correspond to lysosomes. Immunoprecipitation studies showed that MBP-Apoptin remained fully antibody-accessible regardless of buffer stringency when microinjected into tumor cells. In contrast, MBP-Apoptin in normal cells was only recoverable under stringent lysis conditions, whereas under milder conditions they became fully shielded with time on two epitopes spanning the entire protein. Further biochemical analysis showed that the long-term fate of Apoptin protein aggregates in normal cells was their eventual elimination. Our results provide the first example of a tumor-specific apoptosis-inducing aggregate that is essentially sequestered by factors or conditions present in the cytoplasm of healthy, nontransformed cells. This characteristic should reveal more about the cellular interactions of this viral protein as well as further enhance its safety as a potential tumor-specific therapeutic agent.

Visualization by Cryo-electron Microscopy of Genomic RNA that Binds to the Protein Capsid Inside Bacteriophage MS2

The icosahedrally symmetrized structure of bacteriophage MS2 as determined by cryo-electron microscopy (EM) reveals the presence of genomic RNA that attaches to coat-protein dimers. Earlier X-ray diffraction studies revealed similar interactions between the unique operator hairpin of the MS2 genomic RNA and the coat-protein dimer. This observation leads us to conclude that not only the operator, but also many other RNA sequences in the genome of MS2, are able to bind to the coat-protein dimer. A substantial number of potential coat-protein-dimer binding sites are present in the genome of MS2 that can account for the observed RNA densities in the EM map. Moreover, it appears that these stem-loop structures are able to bind in a similar fashion to the coat protein dimer as the wild-type operator hairpin. The EM map also shows additional density between the potential operator-binding sites, linking the RNA stem-loops together to form an icosahedral network around the 3 and 5-fold axes. This RNA network is bound to the inside of the MS2 capsid and probably influences both capsid stability and formation, supporting the idea that capsid formation and RNA packaging are intimately linked to each other.

Structural and functional aspects of C1-Inhibitor

C1-Inh is a serpin that inhibits serine proteases from the complement and the coagulation pathway. C1-Inh consists of a serpin domain and a unique N-terminal domain and is heavily glycosylated. Non-functional mutants of C1-Inh can give insight into the inhibitory mechanism of C1-Inh. This review describes a novel 3D model of C1-Inh, based on a newly developed homology modelling method. This model gives insight into a possible potentiation mechanism of C1-Inh and based on this model the essential residues for efficient inhibition by C1-Inh are discussed.

Inherent asymmetry of the structure of F1-ATPase from bovine heart mitochondria at 6.5 A resolution

ATP synthase, the assembly which makes ATP in mitochondria, chloroplasts and bacteria, uses transmembrane proton gradients generated by respiration or photosynthesis to drive the phosphorylation of ADP. Its membrane domain is joined by a slender stalk to a peripheral catalytic domain, F1‐ATPase. This domain is made of five subunits with stoichiometries of 3 alpha: 3 beta: 1 gamma: 1 delta: 1 epsilon, and in bovine mitochondria has a molecular mass of 371,000. We have determined the 3‐dimensional structure of bovine mitochondrial F1‐ATPase to 6.5 A resolution by X‐ray crystallography. It is an approximately spherical globule 110 A in diameter, on a 40 A stem which contains two alpha‐helices in a coiled‐coil. This stem is presumed to be part of the stalk that connects F1 with the membrane domain in the intact ATP synthase. A pit next to the stem penetrates approximately 35 A into the F1 particle. The stem and the pit are two examples of the many asymmetric features of the structure. The central element in the asymmetry is the longer of the two alpha‐helices in the stem, which extends for 90 A through the centre of the assembly and emerges on top into a dimple 15 A deep. Features with threefold and sixfold symmetry, presumed to be parts of homologous alpha and beta subunits, are arranged around the central rod and pit, but the overall structure is asymmetric. The central helix provides a possible mechanism for transmission of conformational changes induced by the proton gradient from the stalk to the catalytic sites of the enzyme.

Heterogeneous nucleation of three-dimensional protein nanocrystals.

Nucleation is the rate-limiting step in protein crystallization. Introducing heterogeneous substrates may in some cases lower the energy barrier for nucleation and thereby facilitate crystal growth. To date, the mechanism of heterogeneous protein nucleation remains poorly understood. In this study, the nucleating properties of fragments of human hair in crystallization experiments have been investigated. The four proteins that were tested, lysozyme, glucose isomerase, a polysaccharide-specific Fab fragment and potato serine protease inhibitor, nucleated preferentially on the hair surface. Macrocrystals and showers of tiny crystals of a few hundred nanometres thickness were obtained also under conditions that did not produce crystals in the absence of the nucleating agent. Cryo-electron diffraction showed that the nanocrystals diffracted to at least 4 A resolution. The mechanism of heterogeneous nucleation was studied using confocal fluorescent microscopy which demonstrated that the protein is concentrated on the nucleating surface. A substantial accumulation of protein was observed on the sharp edges of the hairs cuticles, explaining the strong nucleating activity of the surface.