A combined computational and experimental approach reveals the structure of a C/EBP–Spi1 interaction required for IL1B gene transcription

We previously reported that transcription of the human IL1B gene, encoding the proinflammatory cytokine interleukin 1, depends on long-distance chromatin looping that is stabilized by a mutual interaction between the DNA-binding domains (DBDs) of two transcription factors: Spi1 proto-oncogene at the promoter and CCAAT enhancer– binding protein (C/EBP) at a far-upstream enhancer. We have also reported that the C-terminal tail sequence beyond the C/EBP leucine zipper is critical for its association with Spi1 via an exposed residue (Arg-232) located within a pocket at one end of the Spi1 DNA-recognition helix. Here, combining in vitro interaction studies with computational docking and molecular dynamics of existing X-ray structures for the Spi1 and C/EBP DBDs, along with the C/EBP C-terminal tail sequence, we found that the tail sequence is intimately associated with Arg-232 of Spi1. The Arg-232 pocket was computationally screened for small-molecule binding aimed at IL1B transcription inhibition, yielding L-arginine, a known anti-inflammatory amino acid, revealing a potential for disrupting the C/EBP–Spi1 interaction. As evaluated by ChIP, cultured lipopolysaccharide (LPS)-activated THP-1 cells incubated with L-arginine had significantly decreased IL1B transcription and reduced C/EBP’s association with Spi1 on the IL1B promoter. No significant change was observed in direct binding of either Spi1 or C/EBP to cognate DNA and in transcription of the C/EBP-dependent IL6 gene in the same cells. These results support the notion that disordered sequences extending from a leucine zipper can mediate protein–protein interactions and can serve as drug-gable targets for regulating gene promoter activity.