The Crystal Structure of Yeast Fatty Acid Synthase, a Cellular Machine with Eight Active Sites Working Together

SummaryIn yeast, the whole metabolic pathway for making 16- and 18-carbon fatty acids is carried out by fatty acid synthase, a 2.6 megadalton molecular-weight macromolecular assembly containing six copies of all eight catalytic centers. We have determined its crystal structure, which illuminates how this enzyme is initially activated and then carries out multiple steps of synthesis in each of six sterically isolated reaction chambers. Six of the catalytic sites are in the wall of the assembly facing an acyl carrier protein (ACP) bound to the ketoacyl synthase domain. Two-dimensional diffusion of substrates to the catalytic sites may be achieved by the electrostatically negative ACP swinging to each of the six electrostatically positive catalytic sites. The phosphopantetheinyl transferase domain lies outside the shell of the assembly, inaccessible to ACP that lies inside, suggesting that the attachment of the pantetheine arm to ACP must occur before complete assembly of the complex

On the Explanation of the Paramagnetic Meissner Effect in Superconductor/Ferromagnet Heterostructures

An increase of the magnetic moment in superconductor/ferromagnet (S/F) bilayers V(40nm)/F [F$=$Fe(1,3nm), Co(3nm), Ni(3nm)] was observed using SQUID magnetometry upon cooling below the superconducting transition temperature Tc in magnetic fields of 10 Oe to 50 Oe applied parallel to the sample surface. A similar increase, often called the paramagnetic Meissner effect (PME), was observed before in various superconductors and superconductor/ferromagnet systems. To explain the PME effect in the presented S/F bilayers a model based on a row of vortices located at the S/F interface is proposed. According to the model the magnetic moment induced below Tc consists of the paramagnetic contribution of the vortex cores and the diamagnetic contribution of the vortex-free region of the S layer. Since the thickness of the S layer is found to be 3-4 times less than the magnetic field penetration depth, this latter diamagnetic contribution is negligible. The model correctly accounts for the sign, the approximate magnitude and the field dependence of the paramagnetic and the Meissner contributions of the induced magnetic moment upon passing the superconducting transition of a ferromagnet/superconductor bilayer

A 9 Å Resolution X-Ray Crystallographic Map of the Large Ribosomal Subunit

AbstractThe 50S subunit of the ribosome catalyzes the peptidyl-transferase reaction of protein synthesis. We have generated X-ray crystallographic electron density maps of the large ribosomal subunit from Haloarcula marismortui at various resolutions up to 9 Å using data from crystals that diffract to 3 Å. Positioning a 20 Å resolution EM image of these particles in the crystal lattice produced phases accurate enough to locate the bound heavy atoms in three derivatives using difference Fourier maps, thus demonstrating the correctness of the EM model and its placement in the unit cell. At 20 Å resolution, the X-ray map is similar to the EM map; however, at 9 Å it reveals long, continuous, but branched features whose shape, diameter, and right-handed twist are consistent with segments of double-helical RNA that crisscross the subunit

Screening of Human Tumor Antigens for CD4+ T Cell Epitopes by Combination of HLA-Transgenic Mice, Recombinant Adenovirus and Antigen Peptide Libraries

BACKGROUND: As tumor antigen-specific CD4+ T cells can mediate strong therapeutic anti-tumor responses in melanoma patients we set out to establish a comprehensive screening strategy for the identification of tumor-specific CD4+ T cell epitopes suitable for detection, isolation and expansion of tumor-reactive T cells from patients. METHODS AND FINDINGS: To scan the human melanoma differentiation antigens TRP-1 and TRP-2 for HLA-DRB1*0301-restricted CD4+ T cell epitopes we applied the following methodology: Splenocytes of HLA-DRB1*0301-transgenic mice immunized with recombinant adenovirus encoding TRP-1 (Ad5.TRP-1) or TRP-2 (Ad5.TRP-2) were tested for their T cell reactivity against combinatorial TRP-1- and TRP-2-specific peptide libraries. CD4+ T cell epitopes thus identified were validated in the human system by stimulation of peripheral blood mononuclear cells (PBMC) from healthy donors and melanoma patients. Using this strategy we observed that recombinant Ad5 induced strong CD4+ T cell responses against the heterologous tumor antigens. In Ad5.TRP-2-immunized mice CD4+ T cell reactivity was detected against the known HLA-DRB1*0301-restricted TRP-2(60-74) epitope and against the new epitope TRP-2(149-163). Importantly, human T cells specifically recognizing target cells loaded with the TRP-2(149-163)-containing library peptide or infected with Ad5.TRP-2 were obtained from healthy individuals, and short term in vitro stimulation of PBMC revealed the presence of epitope-reactive CD4+ T cells in melanoma patients. Similarly, immunization of mice with Ad5.TRP-1 induced CD4+ T cell responses against TRP-1-derived peptides that turned out to be recognized also by human T cells, resulting in the identification of TRP-1(284-298) as a new HLA-DRB1*0301-restricted CD4+ T cell epitope. CONCLUSIONS: Our screening approach identified new HLA-DRB1*0301-restricted CD4+ T cell epitopes derived from melanoma antigens. This strategy is generally applicable to target antigens of other tumor entities and to different HLA class II molecules even without prior characterization of their peptide binding motives

Magnetic proximity effects in V/Fe superconductor/ferromagnet single bilayer revealed by waveguide-enhanced polarized neutron reflectometry

Polarized neutron reflectometry is used to study the magnetic proximity effect in a superconductor/ferromagnet (SC/FM) system of composition Cu(32nm)/V(40nm)/Fe(1nm)/MgO. In contrast to previous studies, here a single SC/FM bilayer, is studied and multilayer artefacts are excluded. The necessary signal enhancement is achieved by waveguide resonance, i.e. preparing the V(40nm)/Fe(1nm) SC/FM bilayer sandwiched by the highly reflective MgO substrate and Cu top layer, respectively . A new magnetic state of the system was observed at temperatures below 0.7 TC. manifested in a systematic change in the height and width of the waveguide resonance peak. Upon increasing the temperature from 0.7 TC to TC, a gradual decay of this state is observed, accompanied by a 5% growth of the diffuse scattering. According to theoretical studies, such behavior is the result of the magnetic proximity effect. Due to the presence of the thin FM layer the superconducting electrons are polarized and, as a result, near the SC/FM interface an additional magnetic layer appears in the SC with thickness comparable to ksi, the coherence length of the superconductor.Comment: Submitted to the Journal of Superconductivity and Novel Magnetism. 11 pages, 6 figures

Crystal structure of the C-terminal domain of DENR

© 2020 The Authors The density regulated protein (DENR) forms a stable heterodimer with malignant T-cell-amplified sequence 1 (MCT-1). DENR-MCT-1 heterodimer then participates in regulation of non-canonical translation initiation and ribosomal recycling. The N-terminal domain of DENR interacts with MCT-1 and carries a classical tetrahedral zinc ion-binding site, which is crucial for the dimerization. DENR-MCT-1 binds the small (40S) ribosomal subunit through interactions between MCT-1 and helix h24 of the 18S rRNA, and through interactions between the C-terminal domain of DENR and helix h44 of the 18S rRNA. This later interaction occurs in the vicinity of the P site that is also the binding site for canonical translation initiation factor eIF1, which plays the key role in initiation codon selection and scanning. Sequence homology modeling and a low-resolution crystal structure of the DENR-MCT-1 complex with the human 40S subunit suggests that the C-terminal domain of DENR and eIF1 adopt a similar fold. Here we present the crystal structure of the C-terminal domain of DENR determined at 1.74 Å resolution, which confirms its resemblance to eIF1 and advances our understanding of the mechanism by which DENR-MCT-1 regulates non-canonical translation initiation and ribosomal recycling

Phase Separation in a ternary DPPC DOPC POPC System with Reducing Hydration

The maintenance of plasma membrane structure is vital for the viability of cells. Disruption of this structure can lead to cell death. One important example is the macroscopic phase separation observed during dehydration associated with desiccation and freezing, often leading to loss of permeability and cell death. It has previously been shown that the hybrid lipid 1 palmitoyl 2 oleoyl sn glycero 3 phosphocholine POPC can act as a line active component in ternary lipid systems, inhibiting macroscopic phase separation and stabilising membrane microdomains in lipid vesicles [1]. The domain size is found to decrease with increasing POPC concentration until complete mixing is observed. However, no such studies have been carried out at reduced hydration. To examine if this phase separation is unique to vesicles in excess water, we have conducted studies on several binary and ternary model membrane systems at both reduced hydration powder type samples and oriented membrane stacks and in excess water supported lipid bilayers at 0.2 mol fraction POPC, in the range where microdomain stabilisation is reported. Differential scanning calorimetry DSC and Fourier transform infrared spectroscopy FTIR are used to map phase transition temperatures, with X ray and neutron scattering providing details of the changes in lipid packing and phase information within these boundaries. Atomic force microscopy AFM is used to image bilayers on a substrate in excess water. In all cases, macroscopic phase separation was observed rather than microdomain formation at this molar ratio. Thus POPC does not stabilise microdomains under these conditions, regardless of the type of model membrane, hydration or temperature. Thus we conclude that the driving force for separation under these conditions overcomes any linactant effects of the hybrid lipi

Utilization of a deoxynucleoside diphosphate substrate by HIV reverse transcriptase

Background: Deoxynucleoside triphosphates (dNTPs) are the normal substrates for DNA sysnthesis is catalyzed by polymerases such as HIV-1 reverse transcriptase (RT). However, substantial amounts of deoxynucleoside diphosphates (dNDPs) are also present in the cell. Use of dNDPs in HIV-1 DNA sysnthesis could have significant implications for the efficacy of nucleoside RT inhibitors such as AZT which are first line therapeutics fro treatment of HIV infection. Our earlier work on HIV-1 reverse transcriptase (RT) suggested that the interaction between the γ phosphate of the incoming dNTP and RT residue K65 in the active site is not essential for dNTP insertion, implying that this polymerase may be able to insert dNPs in addition to dNTPs. Methodology/Principal Findings: We examined the ability of recombinant wild type (wt) and mutant RTs with substitutions at residue K65 to utilize a dNDP substrate in primer extension reactions. We found that wild type HIV-1 RT indeed catalyzes incorporation of dNDP substrates whereas RT with mutations of residue K645 were unable to catalyze this reaction. Wild type HIV-1 RT also catalyzed the reverse reaction, inorganic phosphate-dependent phosphorolysis. Nucleotide-mediated phosphorolytic removal of chain-terminating 3′-terminal nucleoside inhibitors such as AZT forms the basis of HIV-1 resistance to such drugs, and this removal is enhanced by thymidine analog mutations (TAMs). We found that both wt and TAM-containing RTs were able to catalyze Pi-mediated phosphorolysis of 3′-terminal AZT at physiological levels of Pi with an efficacy similar to that for ATP-dependent AZT-excision. Conclusion: We have identified two new catalytic function of HIV-1 RT, the use of dNDPs as substrates for DNA synthesis, and the use of Pi as substrate for phosphorolytic removal of primer 3′-terminal nucleotides. The ability to insert dNDPs has been documented for only one other DNA polymerase The RB69 DNA polymerase and the reverse reaction employing inorganic phosphate has not been documented for any DNA polymerase. Importantly, our results show that Pi-mediated phosphorolysis can contribute to AZT resistance and indicates that factors that influence HIV resistance to AZT are more complex than previously appreciated. © 2008 Garforth et al

Neutron reflectometry studies of Gd/Nb and Cu30Ni70/Nb superlattices

We performed a comparative study of magnetic proximity effects in [Gd(5nm)/Nb(25nm)]12 and [Cu30Ni70(6nm)/Nb(27nm)]12 superlattices of S/F type by means of transport measurements and neutron scattering. Transport measurements have shown that Gd/Nb and CuNi/Nb superlattices shows 3D and 2D type of superconductivity respectively. In the case of proximity coupled Gd/Nb superconductor the effective thickness of the superconducting region, 300nm is enough to expel significant amount of applied magnetic field which was detected by neutron scattering. In decoupled CuNi/Nb superlattice thickness of every superconducting layer is only 27nm which is not enough to expel applied magnetic field. Our study shows how neutron reflectometry can be applied to study proximity coupling in superconducting/ferromagnet heterostructures

Human topoisomerase IIα uses a two-metal-ion mechanism for DNA cleavage

The DNA cleavage reaction of human topoisomerase IIα is critical to all of the physiological and pharmacological functions of the protein. While it has long been known that the type II enzyme requires a divalent metal ion in order to cleave DNA, the role of the cation in this process is not known. To resolve this fundamental issue, the present study utilized a series of divalent metal ions with varying thiophilicities in conjunction with DNA cleavage substrates that replaced the 3′-bridging oxygen of the scissile bond with a sulfur atom (i.e. 3′-bridging phosphorothiolates). Rates and levels of DNA scission were greatly enhanced when thiophilic metal ions were included in reactions that utilized sulfur-containing substrates. Based on these results and those of reactions that employed divalent cation mixtures, we propose that topoisomerase IIα mediates DNA cleavage via a two-metal-ion mechanism. In this model, one of the metal ions makes a critical interaction with the 3′-bridging atom of the scissile phosphate. This interaction greatly accelerates rates of enzyme-mediated DNA cleavage, and most likely is needed to stabilize the leaving 3′-oxygen