X-Ray Crystal Structure of Proto-Oncogene Product c-Rel Bound to the CD28 Response Element of IL-2

Abstract

Background: The proto-oncogene product c-Rel is a Rel/NF-κB family transcription factor that plays a critical role in lymphoid cell development and mediates CD28-induced expression of interleukin 2 (IL-2). The CD28 response element (CD28RE) in the IL-2 enhancer is nonameric and similar to the κB DNA target sites recognized by p65 homodimers. Results: We have determined and refined the X-ray crystal structure of the c-Rel homodimer complexed to the CD28RE DNA site, 5′-AGAAATTCC-3′, to 2.85 Å resolution. The c-Rel homodimer binds CD28RE in a mode similar to that observed in the p65/IL-8 κB crystallographic complex. Binding studies reveal that the c-Rel homodimer recognizes the CD28RE with higher affinity as compared to other canonical κB sequences despite the nonconsensus A:T base pair at the 5′ end of the CD28RE. Preferential recognition of the CD28RE by c-Rel results from the direct contacts between the protein and the DNA as well as intrasubunit interactions between the βf-βg loop in the dimerization domain and the DNA-contacting loop L1 of the N-terminal domain. Not only do these loops have different conformations in other Rel/DNA crystallographic complexes, but they also contain two of the five oncogenic point mutations found in v-Rel. Conclusions: The current structure indicates that a non-DNA-contacting loop in the dimerization domain and the DNA-contacting loop L1 may play critical roles in defining affinity and specificity. Two amino acid changes in these segments may account for the differential DNA binding by v-Rel as compared to that of c-Rel.