The Presence of the Iron-Sulfur Motif Is Important for the Conformational Stability of the Antiviral Protein, Viperin

Viperin, an antiviral protein, has been shown to contain a CX3CX2C 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

The presence of the iron-sulfur motif is important for the conformational stability of the antiviral protein, Viperin.

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

Unfolding transitions of WT and cysteine mutants of Viperin monitored by far- UV CD.

<p>Urea induced unfolding of the WT (black), the triple mutant (red), C83A mutant (blue), C87A mutant (dark green), and C90A mutant (magenta) of Viperin. The ellipticity measured at 223 nm is plotted against urea concentration. The data for WT and the cysteine mutants are fit to equation 1 assuming two state unfolding transitions. The experiments have been carried out in 20 mM phosphate buffer at pH 7.5.</p

Thermodynamic parameters of the equilibrium unfolding transitions of WT and cysteine mutants of Viperin.

<p>Thermodynamic parameters of the equilibrium unfolding transitions of WT and cysteine mutants of Viperin.</p

Fluorescence spectra of WT and cysteine mutants of Viperin in folded and unfolded condition.

<p>Fluorescence emission spectra of (a) the WT Viperin, (b) the triple mutant (c) C83A mutant, (d) C87A mutant and (e) C90A mutant (f) NATA in the absence (void circle) and presence (closed circle) of 10 M urea. Fluorescence experiments have been carried out in 20 mm phosphate buffer at pH 7.5. A red shift in the emission spectra is shown by a double headed arrow for the WT protein.</p

UV-visible absorption spectra of WT and cysteine mutants of Viperin.

<p>UV-visible absorption spectra of the WT Viperin (black), the triple cysteine mutant (cyan), C83A mutant (red), C87A mutant (blue), and C90A mutant (dark green). The experiments are performed in 20 mM sodium phosphate buffer at ph7.5. The WT protein exhibits two peaks at 325 nm and 410 nm (shown by arrows) which are characteristic of Fe-S cluster. They are found to be absent in the case of cysteine mutants.</p

Far-UV circular dichroism spectra of WT and cysteine mutants of Viperin.

<p>Far-UV CD spectra of the WT Viperin (____), the triple cysteine mutant (-.-.-.), C83A mutant (_ . . . _), C87A mutant (- - - -), C90A mutant (. . . .). The CD experiments have been carried out in 20 mM sodium phosphate buffer at ph7.5.</p