Interferon (IFN) regulatory factor family member 4 (IRF4) is a transcription factor that serves specific roles in transcriptional regulation of IFN responsive genes and is indispensable in B- & T-cell differentiation. IRF4 like the other members of the family has two major domains- the N-terminal DNA binding domain (DBD) essential for its recognition and binding to the Interferon Stimulated Response Element DNA sequence and a C-terminal Interferon activation domain (IAD) thought to maintain IRF4 in an auto-inhibited inactive state and is also critical in its activation. A putative unstructured linker connects the DBD and IAD. Activation in most members of the IRF family requires phosphorylation to induce homo and hetero-dimerization. In contrast, IRF4 functions primarily through ternary complex formation involving different proteins including PU.1 and MyD88. The IRF4IAD has a C-terminal auto-inhibitory region (AIR) that has been proposed to physically impede the DBD from interacting with DNA in the absence of its binding partner.
To understand the activation mechanism in molecular detail we determined the crystal structure of the IAD of IRF4 and also performed small-angle X-ray scattering (SAXS) studies. Our data reveals that the surface electrostatics of IAD and presence of additional loops confers exclusivity to IRF4 in the IRF family. SAXS studies suggest that the AIR is structured and makes interactions with the putative linker. We also performed analytical ultracentrifugation studies, fluorescence anisotropy binding experiments and SAXS studies on full-length IRF4 as well as on constructs where the first 20 residues, exclusive to IRF4 or the AIR were removed. We observe that the first 20 residues are critical in decreasing the binding affinity of full-length IRF4 to DNA. In addition, the putative linker of IRF4 connecting the N- and C-termini appears to be a folded domain and interacts with AIR. Also, overall full-length IRF4 appears as an elongated molecule and the N- and the C-terminal domains are arranged on either ends of full-length IRF4. Moreover, there are no signs of huge conformational changes in the protein during the activation process. Taken together, based on our data we propose that there is no auto-inhibited state for IRF4. Furthermore, it is the binding affinity of full-length IRF4 that is increased in the presence of its binding partner most likely through modest conformational changes.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-4570 |
| Date | 01 January 2014 |
| Creators | Govinda Remesh, Soumya |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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