Program Director/Principal Investigator (Last, First, Middle): Privalov, Peter L. 1R01 AI080828-01 ABSTRACT This project is to investigate the mechanism of activation of the interferon regulatory factor (particularly IRF-3) following virus infection. The activation of IRF, which is triggered by the phosphorylation of certain SerIThr residues, results in its dimerization and binding as a dimer to the target IFN-P DNA. However, phosphorylation increases the negative charge of this highly charged protein, i.e. the electrostatic repulsive forces between IRFs and between them and the negatively charged DNA are augmented, so it is unclear why they dimerise and bind to DNA. Moreover, the residues which are phosphorylated are located within the Cterminal dimerization domain (CTD) of this two- domain protein, that is connected with the N-terminal DNA-binding domain (NTD) by a long proline-rich linker, and it is unclear how changes in the C-terminal domain switch on the ability of the NTDs to bind DNA. The mechanism of IRF activation therefore represents a physical, rather than biological problem and its solution requires detailed physical investigation of every stage of this process. A key step in realizing large scale physical studies of IRF activation was the demonstration that the in vivo phosphorylation of SerIThr residues in the CTD can be mimicked by their substitution with the charge- bearer Asp/Glu residues. Use of these phosphomimetic mutants has opened the way for detailed studies of the mechanism of activation of IRFs, since it permit to reveal the particular role of these SerlThr residues in activation of IRFs, still a subject of much debate. The original project, which was intended for four years, aimed the detailed investigation by modem physical methods of all stages of IRF-3 activation and its association with target DNA sequences. The main questions which have to be answered are: (a) Phosphorylation of which residues is primarily responsible for activation of IRF-3 and what are the forces involved in its dimerization? (b) What is the state of the linker that connects the NTD with the CTD and how does it change upon IRF activation? (c) What is the stoichiometry of activated IRF-3 binding to the target DNA and what are the thermodynamic characteristics of their association, i.e. the Gibbs energy, enthalpy, entropy, in particular their electrostatic and non-electrostatic components? (d) Does binding of the activated dimeric IRF-3 to the target IFN-P DNA result in its bending? Some of these questions, namely (a) and (c) we solved during the time lasted since this project was applied to NIH and results were published in JMB, 385(2008)335-348. Now, during two years of the expected Bridge Grant, we will concentrate on the realization of the (b) and (d) points of our proposal.
|Shukla, Hem; Vaitiekunas, Paulius; Majumdar, Ananya K et al. (2012) The linker of the interferon response factor 3 transcription factor is not unfolded. Biochemistry 51:6320-7|