Metadynamics study of mutant human interferon-gamma forms (original) (raw)
Related papers
IN SILICO STUDIES ON THE STABILITY OF HUMAN INTERFERON-GAMMA MUTANT
Biotechnology & Biotechnological Equipment, 2012
Human interferon-gamma (hIFNγ) is a key cytokine in the realisation of cellular immunity. It accomplishes its biological activity upon binding to a specific cell receptor thus inducing the JAK/STAT1 signal transduction pathway. Two putative NLS sequences were pointed out to assist in the translocation of STAT1 into the nucleus. In order to employ mutational analysis for study the biological significance of the polybasic sequence Lys86-Lys87-Lys88 belonging to the upstream putative NLS, hIFNγ mutants with preserved structure and intact binding affinity to cell receptor need to be selected. To this end in silico studies of molecular stability of hIFNγ mutants was performed. The potential conformational changes in the structure of the mutant proteins were investigated employing molecular dynamics simulations. The free energy surface of Lys86 backbone torsion angles space in hIFNγ wild type and mutants was analyzed using metadynamic model. The obtained in silico results were verified by construction of selected mutant recombinant hIFNγ proteins, which were analysed for biological activity. To judge for the secondary structure of the mutants the affinity to the cell receptor was investigated. High correlation between results of the molecular dynamics simulations and biological data was obtained.
2018
A key prerequisite for a deeper understanding of biological processes at molecular level is a detailed description of the three-dimensional structure of interaction partners and their complexes. We adopted the IFN-γ complex as our model system. Even though IFN-γ is one of the key modulators of the immunity response, which has been studied intensively for more than 60 years, the structure of the accessory receptor chain and the understanding of the IFN-γ complex is still lacking. In this work we firstly discussed the binary system between IFN-γ and its high affinity receptor R1 which is structurally known. Using a new innovative methodology we focused on the modulation of the affinity between IFN-γ and its receptor R1. Our approach was based on the modulation of protein – protein stability by mutating cavities in the proteins ́ structure and increasing the affinity about seven-fold. Secondly, we crystallized and solved the structure of the IFN-γ receptor 2, the accessory receptor mol...
A homology model of human interferon α-2
Proteins: Structure, Function, and Genetics, 1993
An atomic coordinate five a-helix three-dimensional model is presented for human interferon 01-2 (HuIFNa2). The HuIFNa2 structure was constructed from murine interferon p (MuIFNp) by homology modeling using the STEREO and IMPACT programs. The HuIFNor2 model is consistent with its known biochemical and biophysical properties including epitope mapping. Lysine residues predicted to be buried in the model were primarily unreactive with succinimidyl-7-amino-4methylcoumarin-3-acetic acid (AMCA-NHS), a lysine modification agent, as shown by mass spectrometric analysis of tryptic digests. N-terminal sequence analysis of polypeptides generated by limited digestion of HuIFNor2 with endoproteinase Lys-C demonstrated rapid cleavage at K31, which is consistent with the presence of this residue in a loop in the proposed H u I F N d model. Based on this model structure potential receptor binding sites are identified. o 1993 wiley-Liss, hc.
A homology model of human interferon a-2
Proteins, 1993
An atomic coordinate five a-helix three-dimensional model is presented for human interferon 01-2 (HuIFNa2). The HuIFNa2 structure was constructed from murine interferon p (MuIFNp) by homology modeling using the STEREO and IMPACT programs. The HuIFNor2 model is consistent with its known biochemical and biophysical properties including epitope mapping. Lysine residues predicted to be buried in the model were primarily unreactive with succinimidyl-7-amino-4methylcoumarin-3-acetic acid (AMCA-NHS), a lysine modification agent, as shown by mass spectrometric analysis of tryptic digests. N-terminal sequence analysis of polypeptides generated by limited digestion of HuIFNor2 with endoproteinase Lys-C demonstrated rapid cleavage at K31, which is consistent with the presence of this residue in a loop in the proposed H u I F N d model. Based on this model structure potential receptor binding sites are identified. o 1993 wiley-Liss, hc.
Homology model of human interferon-α8 and its receptor complex
Protein Science, 2008
Human interferond (HuIFNa8), a type I interferon (IFN), is a cytokine belonging to the hematopoietic superfamily that includes human growth hormone (HGH). Recent data identified two human type I IFN receptor components. One component (p40) was purified from human urine by its ability to bind to immobilized type I IFN. A second receptor component (IFNAR), consisting of two cytokine receptor-like domains (D200 and D200'), was identified by expression cloning. Murine cells transfected with a gene encoding this protein were able to produce an antiviral response to human IFNa8. Both of these receptor proteins have been identified as members of the immunoglobulin superfamily of which H G H receptor is a member. The cytokine receptor-like structural motifs present in p40 and IFNAR were modeled based on the HGH receptor X-ray structure. Models of the complexes of HuIFNa8 with the receptor subunits were built by superpositioning the conserved C a backbone of the HuIFNa8 and receptor subunit models with HGH and its receptor complex. The HuIFNa8 model was constructed from the Ca coordinates of murine interferon-p crystal structure. Electrostatic potentials and hydrophobic interactions appear to favor the model of HuIFNa8 interacting with p40 at site 1 and the D200' domain of IFNAR at site 2 because there are regions of complementary electrostatic potential and hydrophobic interactions at both of the proposed binding interfaces. Some of the predicted receptor binding residues within HuIFNa8 correspond to functionally important residues determined previously for human IFNal, IFNa2, and IFNa4 subtypes by site-directed mutagenesis studies. The models predict regions of interaction between HuIFNa8 and each of the receptor proteins, and provide insights into interactions between other type I IFNs (IFN-a subtypes and IFN-p) and their respective receptor components.
Design and Testing of High-Affinity Mutants of Interferon Gamma Receptor 1
Biophysical Journal, 2013
We describe a computer-based protocol to design protein mutations increasing binding affinity between ligand and its receptor. The method was applied to mutate interferon-receptor 1 (IFN--Rx) to increase its affinity to natural ligand IFN-, protein important for innate immunity. We analyzed all four available crystal structures of the IFN--Rx/IFN-complex to identify 40 receptor residues forming the interface with IFN-. For these 40 residues, we performed computational mutation analysis by substituting each of the interface receptor residues by the remaining standard amino acids. The corresponding changes of the free energy were calculated by a protocol consisting of FoldX and molecular dynamics calculations. Based on the computed changes of the free energy and on sequence conservation criteria obtained by the analysis of 32 receptor sequences from 19 different species, we selected 14 receptor variants predicted to increase the receptor affinity to IFN-. These variants were expressed as recombinant proteins in Escherichia coli, and their affinities to IFN-were determined experimentally by surface plasmon resonance (SPR). The SPR measurements showed that the simple computational protocol succeeded in finding two receptor variants with affinity to IFN-increased about fivefold compared to the wild-type receptor.
Molecular basis for the antagonistic activity of an anti- interferon alpha receptor 1 antibody
mAbs, 2015
MEDI546 is an antagonist human monoclonal antibody that targets interferon alpha receptor 1 (IFNAR1). MEDI546 has been developed to treat autoimmune diseases and is currently in clinical trials. To decipher the molecular basis of its mechanism of action, we engaged in multiple epitope mapping approaches to determine how it interacts with IFNAR1 and antagonizes the receptor. We identified the epitope of MEDI546 using enzymatic fragmentation, phage-peptide library panning and mutagenesis approaches. Our studies revealed that MEDI546 recognizes the SD3 subdomain of IFNAR1 with the critical residue R(279). Further, we solved the crystal structure of MEDI546 Fab to a resolution of 2.3 Å. Guided by our epitope mapping studies, we then used in silico protein docking of the MEDI546 Fab crystal structure to IFNAR1 and characterized the corresponding mode of binding. We find that MEDI546 sterically inhibits the binding of IFN ligands to IFNAR1, thus blocking the formation of the ternary IFN/I...
Structural model for interferons
FEBS Letters, 1985
Secondary structures of leucocyte α1‐ and α2‐interferons and of fibroblast β‐interferon are calculated using the molecular theory of protein secondary structures. The common secondary structure calculated for α‐ and β‐interferons is used to predict the three‐dimensional structures of fragments 1–110 and 111–166 of the chains (which are supposed to be quasi‐independent domains). The predicted structure of the active domain I (1–110) is an ‘up‐and‐down’ tetrahelical complex (in which the second helix is shorter than the others and can be absent in α1‐interferon) similar to the mirror image of myohaemoerythrin. The predicted structure of domain II (111–166) is either a three‐stranded β‐sheet screened from one side by two α‐helices or a three‐helical complex (similar to that in the N‐domain of papain), the first structure being more consistent with the circular dichroism data of α‐interferon and its C‐end fragment.
Proceedings of the National Academy of Sciences, 2001
The pleiotropic activity of type I interferons has been attributed to the specific interaction of IFN with the cell-surface receptor components ifnar1 and ifnar2. To date, the structure of IFN has been solved, but not that of the receptor or the complex. In this study, the structure of the IFN-α2–ifnar2 complex was generated with a docking procedure, using nuclear Overhauser effect-like distance constraints obtained from double-mutant cycle experiments. The interaction free energy between 13 residues of the ligand and 11 of the receptor was measured by double-mutant cycles. Of the 100 pairwise interactions probed, five pairs of residues were found to interact. These five interactions were incorporated as distance constraints into the flexible docking programprodockby using fixed and movable energy-gradient grids attached to the receptor and ligand, respectively. Multistart minimization and Monte Carlo minimization docking of IFN-α2 onto ifnar2 converged to a well-defined average str...
Type I interferon structures: Possible scaffolds for the interferon-α receptor complex
Canadian Journal of Chemistry
The structures of several type I interferons (IFNs) are known. We review the structural information known for IFN alphas and compare them to other interferons and cytokines. We also review the structural information known or proposed for IFN-cell receptor complexes. However, the structure of the IFN -cell receptor -IFN receptor2 (IFNAR2) and IFN receptor1 (IFNAR1) complex has not yet been determined. This paper describes a structural model of human IFN-IFNAR2/IFNAR1 complex using human IFN-a 2b dimer as the ligand. Both the structures of recombinant human IFN-a 2b and IFN-b were determined by X-ray crystallography as zinc-mediated dimers. Our proposed model was generated using human IFN-a 2b dimer docked with IFNAR2/IFNAR1. We compare our model with the receptor complex models proposed for IFN-b and IFN-g to contrast similarities and differences. The mutual binding sites of human IFN-a 2b and IFNAR2/IFNAR1 complex are consistent with available mutagenesis studies.