A model of the three-dimensional structure of human interferon responsive factor 1 and its modifications upon...

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Interferon responsive factor 1 (IRF-1) is a transcription factor with crucial biological roles.
• IRF-1 activity is modulated by protein interactions and post-translational modifications like phosphorylation.
• Specific kinases, TBK-1 and IKK-ε, target a 5'-SXXXSXS-3' motif in IRF-1.

Purpose of the Study:

• To determine a 3D model of human IRF-1.
• To investigate the structural and functional effects of phosphorylation and aspartate mutations at key residues (215, 219, 221) of IRF-1.
• To elucidate the mechanism of phosphorylation-induced damping of IRF-1 activity.

Main Methods:

• Multi-template comparative modeling to generate IRF-1 3D structures.
• Molecular dynamics simulations to analyze protein structure, electrostatics, and hydrophobicity.
• Reporter gene assays to assess IRF-1's synergistic activity with TNF-α stimulation.

Main Results:

• 3D models of wild-type and modified IRF-1 (phosphorylation or S215/219/221D mutations) were generated.
• Modifications induced structural rigidification with minor changes in surface electrostatics and hydrophobicity.
• Aspartate mutations (S221D, S215D/S219D/S221D) impaired IRF-1's ability to synergize with TNF-α in IFN promoter activation.
• The degree of negative charge introduced by modifications correlated with the observed functional impairment.

Conclusions:

• Phosphorylation and aspartate mutations at residues 215, 219, and 221 of IRF-1 lead to structural rigidification.
• This structural change negatively impacts IRF-1's transcriptional activity, particularly its synergy with TNF-α.
• The findings provide insights into the structural mechanism underlying phosphorylation-mediated regulation of IRF-1 function.