Analysis of Ankyrin Repeats Reveals How a Single Point Mutation in RFXANK Results in Bare Lymphocyte Syndrome

Abstract

Ankyrin repeats are well-known structural modules that mediate interactions between a wide spectrum of proteins. The regulatory factor X with ankyrin repeats (RFXANK) is a subunit of a tripartite RFX complex that assembles on promoters of major histocompatibility complex class II (MHC II) genes. Although it is known that RFXANK plays a central role in the nucleation of RFX, it was not clear how its ankyrin repeats mediate the interactions within the complex and with other proteins. To answer this question, we modeled the RFXANK protein and determined the variable residues of the ankyrin repeats that should contact other proteins. Site-directed alanine mutagenesis of these residues together with in vitro and in vivo binding studies elucidated how RFXAP and CIITA, which simultaneously interact with RFXANK in vivo, bind to two opposite faces of its ankyrin repeats. Moreover, the binding of RFXAP requires two separate surfaces on RFXANK. One of them, which is located in the ankyrin groove, is severely affected in the FZA patient with the bare lymphocyte syndrome. This genetic disease blocks the expression of MHC II molecules on the surface of B cells. By pinpointing the interacting residues of the ankyrin repeats of RFXANK, the mechanism of this subtype of severe combined immunodeficiency was revealed.

New Functions of the Major Histocompatibility Complex Class II-Specific Transcription Factor RFXANK Revealed by a High-Resolution Mutagenesis Study

“…The C-terminal region containing the ARD (aa 84 to 260) is thus essential and sufficient for the function of RFXANK. These findings are in agreement with previous studies demonstrating the importance of the ARD of RFXANK (7,(32)(33)(34).…”

“…RFXANK has been shown to interact with CIITA (13,16,33,49). We therefore performed GST pull-down experiments to check whether this interaction might be lost in the P4 and T4 mutants.…”

Association of Transcription Factor YY1 with the High Molecular Weight Notch Complex Suppresses the Transactivation Activity of Notch

“…The ANK domain has been detected in many proteins, such as cyclin-dependent kinase inhibitors, signal transduction and transcriptional regulators, cytoskeletal organizers, developmental regulators, and toxins (55,56). This functional motif is a well known region for the interaction between proteins (38,57). Although only the ANK domain of N1IC is sufficient to associate with YY1, it alone cannot transactivate CBF1-dependent Notch signaling (Fig.…”

The ankyrin repeat as molecular architecture for protein recognition

“…This mutation may replace a contact residue and reduce the affinity for one of the protein-protein interaction partners (Table 1). Mutation L195P is located in the inner helix of the third repeat of RFXANK and presumably disrupts the helix, abolishing the interaction with binding partners RFX5 and RFXAP (Nekrep et al 2001). Taken together, these three proteins represent testable models of disease-related ankyrin repeat proteins that are amenable to further biophysical and structural characterization.…”

“…RFXANK forms a heterotrimeric complex with RFXAP and RFX5, which can then bind DNA and recruit CIITA to trigger transcription of MHCII genes (Masternak et al 1998;DeSandro et al 2000). In the absence of a high-resolution structure for RFXANK, modeling the C-terminal four ankyrin repeats onto the existing crystal structure of GABP-␤ and Swi6, followed by subsequent mutagenesis studies allowed Nekrep et al (2001) to identify residues participating in the protein-protein interaction interface. Interestingly, mutagenesis studies indicated that RFXAP and CIITA bind to opposite surfaces of RFXANK, and that RFXAP interacts along two different surfaces of the ankyrin repeats.…”