Observation of an unexpected third receptor molecule in the crystal structure of human interferon-γ receptor complex

AbstractBackground: Molecular interactions among cytokines and cytokine receptors form the basis of many cell-signaling pathways relevant to immune function. Interferon-γ (IFN-γ) signals through a multimeric receptor complex consisting of two different but structurally related transmembrane chains: the high-affinity receptor-binding subunit (IFN-γRα) and a species-specific accessory factor (AF-1 or IFN-γRβ). In the signaling complex, the two receptors probably interact with one another through their extracellular domains. Understanding the atomic interactions of signaling complexes enhances the ability to control and alter cell signaling and also provides a greater understanding of basic biochemical processes.Results: The crystal structure of the complex of human IFN-γ with the soluble, glycosylated extracellular part of IFN-γRα has been determined at 2.9 Å resolution using multiwavelength anomalous diffraction methods. In addition to the expected 2:1 complex, the crystal structure reveals the presence of a third receptor molecule not directly associated with the IFN-γ dimer. Two distinct intermolecular contacts, involving the edge strands of the C-terminal domains, are observed between this extra receptor and the 2:1 receptor–ligand complex thereby forming a 3:1 complex.Conclusions: The observed interactions in the 2:1 complex of the high-affinity cell-surface receptor with the IFN-γ cytokine are similar to those seen in a previously reported structure where the receptor chains were not glycosylated. The formation of β-sheet packing interactions between pairs of IFN-γRα receptors in these crystals suggests a possible model for receptor oligomerization of Rα and the structurally homologous Rβ receptors in the fully active IFN-γ signaling complex

Characterization of an engineered human purine nucleoside phosphorylase fused to an anti-her2/neu single chain Fv for use in ADEPT

Abstract Background Antibody Directed Enzyme Prodrug Therapy (ADEPT) can be used to generate cytotoxic agents at the tumor site. To date non-human enzymes have mainly been utilized in ADEPT. However, these non-human enzymes are immunogenic limiting the number of times that ADEPT can be administered. To overcome the problem of immunogenicity, a fully human enzyme, capable of converting a non-toxic prodrug to cytotoxic drug was developed and joined to a human tumor specific scFv yielding a fully human targeting agent. Methods A double mutant of human purine nucleoside phosphorylase (hDM) was developed which unlike the human enzyme can cleave adenosine-based prodrugs. For tumor-specific targeting, hDM was fused to the human anti-HER2/neu single chain Fv (scFv), C6 MH3B1. Enzymatic activity of hDM with its natural substrates and prodrugs was determined using spectrophotomeric approaches. A cell proliferation assay was used to assess the cytotoxicity generated following conversion of prodrug to drug as a result of enzymatic activity of hDM. Affinity of the targeting scFv, C6 MH3B1 fused to hDM to Her2/neu was confirmed using affinity chromatography, surface plasmon resonance, and flow-cytometry. Results In vitro hDM-C6 MH3B1 binds specifically to HER2/neu expressing tumor cells and localizes hDM to tumor cells, where the enzymatic activity of hDM-C6 MH3B1, but not the wild type enzyme, results in phosphorolysis of the prodrug, 2-fluoro-2'-deoxyadenosine to the cytotoxic drug 2-fluoroadenine (F-Ade) causing inhibition of tumor cell proliferation. Significantly, the toxic small drug diffuses through the cell membrane of HER2/neu expressing cells as well as cells that lack the expression of HER2/neu, causing a bystander effect. F-Ade is toxic to cells irrespective of their growth rate; therefore, both the slowly dividing tumor cells and the non-dividing neighboring stromal cells that support tumor growth should be killed. Analysis of potential novel MHCII binding peptides resulting from fusion of hDM to C6 MH3B1 and the two mutations in hDM, and of the structure of hDM compared to the wild-type enzyme suggests that hDM-C6 MH3B1 should exhibit minimal immunogenicity in humans. Conclusion hDM-C6 MH3B1 constitutes a novel human based protein that addresses some of the limitations of ADEPT that currently preclude its successful use in the clinic

Cloning, purification and characterisation of a recombinant purine nucleoside phosphorylase from Bacillus halodurans Alk36

A purine nucleoside phosphorylase from the alkaliphile Bacillus halodurans Alk36 was cloned and overexpressed in Escherichia coli. The enzyme was purified fivefold by membrane filtration and ion exchange. The purified enzyme had a Vmax of 2.03 × 10−9 s −1 and a Km of 206 μM on guanosine. The optimal pH range was between 5.7 and 8.4 with a maximum at pH 7.0. The optimal temperature for activity was 70°C and the enzyme had a half life at 60°C of 20.8 h

Hijacking of the Pleiotropic Cytokine Interferon-γ by the Type III Secretion System of Yersinia pestis

Yersinia pestis, the causative agent of bubonic plague, employs its type III secretion system to inject toxins into target cells, a crucial step in infection establishment. LcrV is an essential component of the T3SS of Yersinia spp, and is able to associate at the tip of the secretion needle and take part in the translocation of anti-host effector proteins into the eukaryotic cell cytoplasm. Upon cell contact, LcrV is also released into the surrounding medium where it has been shown to block the normal inflammatory response, although details of this mechanism have remained elusive. In this work, we reveal a key aspect of the immunomodulatory function of LcrV by showing that it interacts directly and with nanomolar affinity with the inflammatory cytokine IFNγ. In addition, we generate specific IFNγ mutants that show decreased interaction capabilities towards LcrV, enabling us to map the interaction region to two basic C-terminal clusters of IFNγ. Lastly, we show that the LcrV-IFNγ interaction can be disrupted by a number of inhibitors, some of which display nanomolar affinity. This study thus not only identifies novel potential inhibitors that could be developed for the control of Yersinia-induced infection, but also highlights the diversity of the strategies used by Y. pestis to evade the immune system, with the hijacking of pleiotropic cytokines being a long-range mechanism that potentially plays a key role in the severity of plague

Application of the PM6 semi-empirical method to modeling proteins enhances docking accuracy of AutoDock

<p>Abstract</p> <p>Background</p> <p>Molecular docking methods are commonly used for predicting binding modes and energies of ligands to proteins. For accurate complex geometry and binding energy estimation, an appropriate method for calculating partial charges is essential. AutoDockTools software, the interface for preparing input files for one of the most widely used docking programs AutoDock 4, utilizes the Gasteiger partial charge calculation method for both protein and ligand charge calculation. However, it has already been shown that more accurate partial charge calculation - and as a consequence, more accurate docking- can be achieved by using quantum chemical methods. For docking calculations quantum chemical partial charge calculation as a routine was only used for ligands so far. The newly developed Mozyme function of MOPAC2009 allows fast partial charge calculation of proteins by quantum mechanical semi-empirical methods. Thus, in the current study, the effect of semi-empirical quantum-mechanical partial charge calculation on docking accuracy could be investigated.</p> <p>Results</p> <p>The docking accuracy of AutoDock 4 using the original AutoDock scoring function was investigated on a set of 53 protein ligand complexes using Gasteiger and PM6 partial charge calculation methods. This has enabled us to compare the effect of the partial charge calculation method on docking accuracy utilizing AutoDock 4 software. Our results showed that the docking accuracy in regard to complex geometry (docking result defined as accurate when the RMSD of the first rank docking result complex is within 2 Å of the experimentally determined X-ray structure) significantly increased when partial charges of the ligands and proteins were calculated with the semi-empirical PM6 method.</p> <p>Out of the 53 complexes analyzed in the course of our study, the geometry of 42 complexes were accurately calculated using PM6 partial charges, while the use of Gasteiger charges resulted in only 28 accurate geometries. The binding affinity estimation was not influenced by the partial charge calculation method - for more accurate binding affinity prediction development of a new scoring function for AutoDock is needed.</p> <p>Conclusion</p> <p>Our results demonstrate that the accuracy of determination of complex geometry using AutoDock 4 for docking calculation greatly increases with the use of quantum chemical partial charge calculation on both the ligands and proteins.</p

Direct-method-aided phasing of MAD data

The direct methods of breaking the phase ambiguity intrinsic in one-wavelength anomalous scattering (OAS) data and MAD phasing are powerful methods in their own rights. In a different context, in addition to their success in phasing OAS data, direct methods can also be useful in the treatment of MAD data. The idea has been tested with the MAD data at 2.5 Å resolution from the protein human adenosine kinase [Mathews et al. (1998), Biochemistry, 37, 15607-15620]. The results showed that the incorporation of direct methods in MAD phasing led to a significant improvement of phases over those obtained from the conventional MAD phasing method alone, as indicated by improved map correlation coefficients (with the existing model), reduced phase errors by 4.5° and improved map connectivity.link_to_subscribed_fulltex