A Multiprotein Binding Interface in an Intrinsically Disordered Region of the Tumor Suppressor Protein Interferon Regulatory Factor-1

Abstract

The interferon-regulated transcription factor and tumor suppressor protein IRF-1 is predicted to be largely disordered outside of the DNA-binding domain. One of the advantages of intrinsically disordered protein domains is thought to be their ability to take part in multiple, specific but low affinity protein interactions; however, relatively few IRF-1-interacting proteins have been described. The recent identification of a functional binding interface for the E3-ubiquitin ligase CHIP within the major disordered domain of IRF-1 led us to ask whether this region might be employed more widely by regulators of IRF-1 function. Here we describe the use of peptide aptamer-based affinity chromatography coupled with mass spectrometry to define a multiprotein binding interface on IRF-1 (Mf2 domain; amino acids 106 -140) and to identify Mf2-binding proteins from A375 cells. Based on their function as known transcriptional regulators, a selection of the Mf2 domain-binding proteins (NPM1, TRIM28, and YB-1) have been validated using in vitro and cell-based assays. Interestingly, although NPM1, TRIM28, and YB-1 all bind to the Mf2 domain, they have differing amino acid specificities, demonstrating the degree of combinatorial diversity and specificity available through linear interaction motifs.

Mutations in the Nucleolar Phosphoprotein, Nucleophosmin, Promote the Expression of the Oncogenic Transcription Factor MEF/ELF4 in Leukemia Cells and Potentiates Transformation

“…As mentioned above, the association of Wt-NPM1 and IRF-1 inhibits the DNA binding of IRF-1. Narayan et al showed that IRF1 binds directly to Wt-NPM1 through a short linear motif in the nuclear localization sequence outside the DNA-binding domain (31). These results suggest that the inhibition of DNA binding by NPM1 may not be through simple interference with the DNA-binding domain of MEF/ELF4.…”

Differential Transit Peptide Recognition during Preprotein Binding and Translocation into Flowering Plant Plastids

“…Mechanistically, the ability of IDPs to assume different conformations when interacting with different binding partners (Kriwacki et al, 1996;Tompa et al, 2005;Uversky et al, 2008;Narayan et al, 2011) may explain how multiple interactions can be accommodated by the relatively short TPs during binding and translocation. The kinetics of favorable recognition between two components may be accelerated by increasing the encounter productivity using the fly-casting model (Shoemaker et al, 2000), which proposes that TPs are rapidly recognized regardless of whether the peptide binds to the receptor with one topological orientation or the opposite.…”

DNA-binding regulates site-specific ubiquitination of IRF-1

“…CHIP, a U-box E3 ligase, docks to a multi-protein-binding interface in the intrinsically disordered Mf2 domain of IRF-1 and this interaction is required for efficient modification of IRF-1 by CHIP in cells [16]. Interestingly, a domain with homology with the Mf2 is involved in ubiquitination of IRF-2 ( Figure 1A), a close relative of IRF-1, by the RING E3 ligase MDM2 [30], suggesting that the Mf2 may comprise a general docking site for IRF-1 and IRF-2 E3 ligases.…”

“…His-CHIP, His-UbcH5 and His-SET (pET-26b-SET was from J. Libermann and T. Tuschi via Addgene [18]) were purified using Ni-NTA (Ni 2 + -nitrilotriacetate) agarose (Qiagen) following the manufacturer's instructions. Kap-1 was purified as described previously [16] (pGEX-4T1-Kap-1 was a gift from Dr A. Ivanov, West Virginia University, Morgantown, West Virginia, U.S.A. [19]). Untagged p53 purified from insect cells was a gift from Dr Jennifer Fraser and Professor Ted Hupp (both from the University of Edinburgh, Edinburgh, Scotland, U.K.).…”

“…At 80 % confluence, cells were transfected using Attractene (Invitrogen) following the manufacturer's instructions. FLAG pull-down and half-life determination was carried out as described previously [13,16]. Immunoblotting was performed as described previously [17].…”