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Major histocompatibility complex class II (MHCII) molecules have a central role in the mammalian adaptive immune response against infection. The level of the immune response is directly related to the concentration of MHCII molecules in the cell, which have a central role in initiating the immune response. MHCII molecules are therefore a potential target for the development of immunosuppressant drugs for the treatment of organ transplant rejection and autoimmune disease. The expression of MHCII molecules is regulated by a cell specific multiprotein complex. The RFX complex is the key DNA binding component of this complex. The RFX complex is composed of three proteins-RFX5, RFXAP, and RFXB-all of which are required for activation of expression of the MHCII genes. Little is currently known about the precise regions of the RFX proteins that are required for complex formation, or their structure. We have therefore identified the key regions of RFX5, RFXAP, and RFXB, which are required to form the RFX complex and have characterized the individual domains and the complexes they form using NMR and circular dichroism spectroscopy and isothermal titration calorimetry. Our results support a model for the assembly of the RFX complex in which the interaction between RFX5 and RFXAP promote folding of a poorly structured region ofRFXAP, which is required for high affinity binding of RFXB to the RFX5.RFXAP complex.