Proposed 3-D Structure of a Critical Protein-Protein Interaction Required for Transcription of the IL1B Gene Coding for Human Interleukin 1β

Abstract

Abstract We previously reported that expression of the human IL1B gene coding for Interleukin 1β in LPS-activated macrophages requires long-range chromatin looping mediated by a mutual DNA binding domain (DBD) interaction between two transcription factors, Spi-1/PU.1 (Spi1) at the promoter and C/EBPβ at a far-upstream enhancer. We have now combined preexisting X-ray structures and advanced computational techniques with additional biochemical and cell-based studies in order to further investigate the structure of this critical interaction. In vitro binding was used to generate a surface interaction map for the two proteins that suggested an association similar to that of a C/EBPβ·Myb X-ray structure reported by others. The distinction is that, in addition to a C/EBPβ leucine zipper interaction similar to that with the Myb DBD, there is an interaction between a carboxyl-tail extension of one of the C/EBPβ leucine zipper monomers with Spi1. This is consistent with our previous report that the carboxyl-tail of C/EBPβ is critical for association with arginine 232 within a pocket at one end of the Spi1 DNA recognition helix. L-arginine, a known anti-inflammatory, was computationally identified to bind in this interaction pocket. In vivo studies of LPS-activated THP-1 macrophages treated with L-arginine decreased IL1B transcription in parallel with a reduction in C/EBPβ association with Spi1 on the IL1B promoter. No significant change in direct binding of Spi1 and C/EBPβ to DNA was observed. These results suggest that L-arginine inhibits IL1B gene expression by competitive inhibition of C/EBPβ binding to Spi1 in the absence of any effect on direct DNA binding for either factor, and support the predicted structure of the C/EBPβ·Spi1 interaction.