Biochemical and structural characterization of the NFAT(c).calcineurin interaction

The nuclear factor of activated T cells (NFAT) regulates gene expression of cytokines during the immune response. Its transcriptional activity is regulated through multiple phosphorylations within the N-terminal regulatory domain. A rise in the intracellular Ca2+ concentration enables the phosphatase calcineurin to bind and dephosphorylate NFAT, inducing a transition from the transcriptionally inactive state found in the cytoplasm to a form that is capable of entering the nucleus and initiating transcription.;Since calcineurin is known to associate directly with NFAT, characterizing this interaction in detail would provide a step towards understanding the mechanism that regulates NFAT's phosphorylation-dependent nuclear entry. Circular dichroism spectroscopy and partial proteolysis show that the domain of NFAT that interacts with calcineurin is conformationally flexible and lacks a stable tertiary structure. This regulatory domain was systematically truncated to generate a series of mutants, which were then tested for the ability to bind calcineurin. The study yielded two short peptides that bound calcineurin independently and simultaneously. The binding affinity of the full-length regulatory domain for calcineurin was substantially higher than that of either peptide, presumably due to synergistic binding of the two sequences.;These binding sites are functionally important, for calcineurin does not dephosphorylate NFAT efficiently in their absence. Detailed analysis of the C-terminal calcineurin binding motif (CnBP-B) shows that its binding affinity for calcineurin is isoform-dependent. Hence, these two binding sites may collaborate together to determine the overall binding affinity of various isoforms and in turn provide a mechanism for selective activation of NFAT isoforms in vivo.;In search of a nonnatural competitive inhibitor of the NFAT·calcineurin interaction, we modified CnBP-B and its variant BPB2, which was screened for calcineurin binding from a randomized peptide library, by replacing three consecutive amino acids from each sequence with a small molecule (morph) containing a C=C bond as well as two isobutyl groups and two hydroxyl groups in stereochemically unique combinations. We tested the validity of this approach by synthesizing thirty-two morphed peptides and assaying them for calcineurin binding. The small number of distinct morphs currently available, however, prevented identification of a nonnatural peptide that inhibited the NFAT·calcineurin interaction.