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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 ?-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.

SUBMITTER: Laird KM

PROVIDER: S-EPMC2949527 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Solution structure of the heterotrimeric complex between the interaction domains of RFX5 and RFXAP from the RFX gene regulatory complex.

Laird Kholiswa M KM Briggs LaTese L LL Boss Jeremy M JM Summers Michael F MF Garvie Colin W CW

Journal of molecular biology 20100821 1

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 localiz ...[more]

PMID: 20732328

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Project description:BACKGROUND: The myelin sheath is a multilamellar membrane structure wrapped around the axon, enabling the saltatory conduction of nerve impulses in vertebrates. Myelin basic protein, one of the most abundant myelin-specific proteins, is an intrinsically disordered protein that has been shown to bind calmodulin. In this study, we focus on a 19-mer synthetic peptide from the predicted calmodulin-binding segment near the C-terminus of human myelin basic protein. RESULTS: The interaction of native human myelin basic protein with calmodulin was confirmed by affinity chromatography. The binding of the myelin basic protein peptide to calmodulin was tested with isothermal titration calorimetry (ITC) in different temperatures, and Kd was observed to be in the low muM range, as previously observed for full-length myelin basic protein. Surface plasmon resonance showed that the peptide bound to calmodulin, and binding was accompanied by a conformational change; furthermore, gel filtration chromatography indicated a decrease in the hydrodynamic radius of calmodulin in the presence of the peptide. NMR spectroscopy was used to map the binding area to reside mainly within the hydrophobic pocket of the C-terminal lobe of calmodulin. The solution structure obtained by small-angle X-ray scattering indicates binding of the myelin basic protein peptide into the interlobal groove of calmodulin, while calmodulin remains in an extended conformation. CONCLUSION: Taken together, our results give a detailed structural insight into the interaction of calmodulin with a C-terminal segment of a major myelin protein, the myelin basic protein. The used 19-mer peptide interacts mainly with the C-terminal lobe of calmodulin, and a conformational change accompanies binding, suggesting a novel mode of calmodulin-target protein interaction. Calmodulin does not collapse and wrap around the peptide tightly; instead, it remains in an extended conformation in the solution structure. The observed affinity can be physiologically relevant, given the high abundance of both binding partners in the nervous system.

Dataset Information

Solution structure of the heterotrimeric complex between the interaction domains of RFX5 and RFXAP from the RFX gene regulatory complex.

Publications

Solution structure of the heterotrimeric complex between the interaction domains of RFX5 and RFXAP from the RFX gene regulatory complex.

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