Solution Structure of the Heterotrimeric Complex between the Interaction Domains of RFX5 and RFXAP from the RFX Gene Regulatory Complex

The mammalian immune response is mediated by a heterotetrameric transcriptional control complex, called regulatory factor X (RFX), that regulates the expression of major histocompatibility complex class II genes. RFX comprises three proteins: RFX5 (two copies), RFXAP, and RFXB, and mutations and deletions that prevent the assembly of the RFX complex have been linked to a severe immunodeficiency disorder. Two RFX5 molecules and one RFXAP molecule assemble in the cytoplasm prior to nuclear localization, a process mediated by an N-terminal “dimerization domain” of RFX5 (RFX5N) and a C-terminal domain of RFXAP (RFXAPC). We previously presented evidence that RFXAPC is unstructured in the absence of RFX5N but adopts a regular structure in the RFX5N2–RFXAPC complex and that the RFX5N2–RFXAPC complex binds RFXB with high affinity. We now report the structure of the RFX5N2–RFXAPC complex, determined in solution by 15N- and 13C-edited NMR spectroscopy. RFX5N consists of a long central helix flanked by two shorter helices. The central helices of the two RFX5N molecules form an antiparallel coiled coil, and the flanking helices pack at the ends of the long helices in a perpendicular arrangement such that the RFX5N dimer is shaped like a staple. RFXAPC consists of two α-helices that form a V-shaped structure that packs within the RFX5N2 staple. Leucine residues in the leucine-rich region of RFX5N (62-LYLYLQL-68) that are critical for major histocompatibility complex class II gene expression in vivo contribute to both the dimer (Leu64 and Leu68) and the RFX5N–RFXAPC interfaces (Leu62 and Leu66). The clustering of hydrophobic residues from different regions of RFXAPC suggests a potential binding site for RFXB.