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

Abstract 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 (RFX5 N ) and a C-terminal domain of RFXAP (RFXAP C ). We previously presented evidence that RFXAP C is unstructured in the absence of RFX5 N but adopts a regular structure in the RFX5 N 2 –RFXAP C complex and that the RFX5 N 2 –RFXAP C complex binds RFXB with high affinity. We now report the structure of the RFX5 N 2 –RFXAP C complex, determined in solution by 15 N- and 13 C-edited NMR spectroscopy. RFX5 N consists of a long central helix flanked by two shorter helices. The central helices of the two RFX5 N 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 RFX5 N dimer is shaped like a staple. RFXAP C consists of two α-helices that form a V-shaped structure that packs within the RFX5 N 2 staple. Leucine residues in the leucine-rich region of RFX5 N (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 RFX5 N –RFXAP C interfaces (Leu62 and Leu66). The clustering of hydrophobic residues from different regions of RFXAP C suggests a potential binding site for RFXB.