title: The presence of the iron-sulfur motif is important for the conformational stability of the antiviral protein, Viperin.
abstract: Viperin, an antiviral protein, has been shown to contain a CX(3)CX(2)C motif, which is conserved in the radical S-adenosyl-methionine (SAM) enzyme family. A triple mutant which replaces these three cysteines with alanines has been shown to have severe deficiency in antiviral activity. Since the crystal structure of Viperin is not available, we have used a combination of computational methods including multi-template homology modeling and molecular dynamics simulation to develop a low-resolution predicted structure. The results show that Viperin is an α-β protein containing iron-sulfur cluster at the center pocket. The calculations suggest that the removal of iron-sulfur cluster would lead to collapse of the protein tertiary structure. To verify these predictions, we have prepared, expressed and purified four mutant proteins. In three mutants individual cysteine residues were replaced by alanine residues while in the fourth all the cysteines were replaced by alanines. Conformational analyses using circular dichroism and steady state fluorescence spectroscopy indicate that the mutant proteins are partially unfolded, conformationally unstable and aggregation prone. The lack of conformational stability of the mutant proteins may have direct relevance to the absence of their antiviral activity.
authors.affiliation: Structural Biology and Bioinformatics, Indian Institute of Chemical Biology, Council for Scientific and Industrial Research, Kolkata, West Bengal, India.
authors.forename: Shubhasis
authors.initials: S
authors.lastname: Haldar
authors.forename: Simantasarani
authors.initials: S
authors.lastname: Paul
authors.forename: Nidhi
authors.initials: N
authors.lastname: Joshi
authors.forename: Anindya
authors.initials: A
authors.lastname: Dasgupta
authors.forename: Krishnananda
authors.initials: K
authors.lastname: Chattopadhyay
mesh_terms: D020816:Amino Acid Motifs; D000998:Antiviral Agents / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D019295:Computational Biology; D003545:Cysteine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions / Q000187:drug effects; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism*; D008297:Male; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D017510:Protein Folding / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014270:Trifluoroethanol / Q000494:pharmacology; D000090123:Viperin Protein
chemical_list: D000998:Antiviral Agents; D003545:Cysteine; D007506:Iron-Sulfur Proteins; D050505:Mutant Proteins; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011506:Proteins; D014270:Trifluoroethanol; C444427:RSAD2 protein, human; D000090123:Viperin Protein