A multiprotein binding interface in an intrinsically disordered region of the tumor suppressor protein interferon regulatory factor-1.
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.
CRUK Interferon and Cell Signalling Group, Cell Signalling Unit, Edinburgh Cancer Research UK Centre, University of Edinburgh, Edinburgh EH4 2XR, United Kingdom.
Vikram
V
Narayan
Petr
P
Halada
Lenka
L
Hernychová
Yuh Ping
YP
Chong
Jitka
J
Žáková
Ted R
TR
Hupp
Borivoj
B
Vojtesek
Kathryn L
KL
Ball
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity / Q000379:methods; D004268:DNA-Binding Proteins / Q000737:chemistry; D005786:Gene Expression Regulation; D006801:Humans; D050856:Interferon Regulatory Factor-1 / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry; D000090243:Nucleophosmin; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012097:Repressor Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000076183:Tripartite Motif-Containing Protein 28; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D051840:Y-Box-Binding Protein 1
D004268:DNA-Binding Proteins; D050856:Interferon Regulatory Factor-1; C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D010455:Peptides; D012097:Repressor Proteins; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D000090243:Nucleophosmin; C101116:TRIM28 protein, human; D000076183:Tripartite Motif-Containing Protein 28; D044767:Ubiquitin-Protein Ligases
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