Identification And Characterization Of The Calmodulin-binding Domain Of Calcineurin In Cryptococcus Humicola

Ca2+ act as one of the most diverse and widespread second messenger in cellular processes. Calmodulin (CaM) is a small, ubiquitous protein and act as a calcium sensor that meditation a lot of Ca2+ signals. CaM is assumed to be involved in activation many enzymes such as nitric oxide synthases, phosphodiesterase, CaM-dependent protein kinases, CaM-dependent protein phosphatase.Calcineurin (CN) is a heterodimer consisting of a catalytic subunit A (CNA) and a regulatory subunit B (CNB). Although it is widely distributed, its primary sequence and structure is conserved among all eukaryotes. CN plays vital roles in T-cell activation, apoptosis, cardiac hypertrophy, and serves as a ubiquitous carrier of the intracellular calcium signal. CNA contains a catalytic domain, a CNB binding domain, a CaM-binding domain, and an autoinhibitory domain (AID). Previous studies suggested that CaM binds with CN in response to calcium stimulus. CaM binds to CaM-binding domains on CN, and then causes a conformational change that displaces an AID from the active site and activates its phosphatase activity.CaM binding sequences on CNA also called CaM recruitment signaling (CRS) motifs. A CaM target database (http://calcium.uhnres.utoronto.ca/ctdb) has been built based on CRS motifs structural and sequence information. The database can be used to predict whether a sequence contains CaM-binding domain, and to calculate average hydrophobic, average hydrophobic moment and average propensity for a short peptide. Many Ca2+ -dependent CRS motifs have been identified as 1-14 or 1-10 class, the numbers indicating the positions of bulky hydrophobic residues major binding with CaM. Studies on these CaM-binding proteins revealed the possibility of interacting residues on CNA, whereas the direct experimental evidence is lack.Here, we identified CNA, a catalytic subunit of CN, in Cryptococcus humicola. The cDNA of CNA encoded a 639-residue protein and the putative molecular mass was 71 kDa. A CaM-binding domain on CNA and key amino acids in this domain for interaction between CaM and CNA were identified using deletion and site-directed mutagenesis. In this work, we first submitted CNA sequence to CaM target database, predicted the CaM-binding domain, mutated the amino acids of CaM-binding domain, and then analyzed the binding level with CaM, and prove by experimentally. The main research results are as follows:1、 We submitted CNA sequence to CaM target database (http://calcium.Uhnres.utoronto. ca/ctdb) which includes almost all published CRS motif and obtained the putative CaM-binding domain sequence. The cDNA of CNA encodes a 71 kDa protein with a CaM-binding domain in its C-terminal region. The conserved hydrophobic residues,1439, 1443, V446, and V452, in the CaM-binding domain form a 1-5-8-14 motif, which probably involve in the interaction with CaM.1439 and F453 were predicted to be the anchor amino acids that possibly play an essential role in binding with CaM.2、In order to verify the location of the predicted calmodulin binding domain is correct. The bindings of the truncated proteins and mutant proteins with CaM were assayed using Far-Western blot analysis. To identify CaM-binding domain, we transferred CNA and its truncated proteins to nitrocellulose and verified their binding characteristics with CaM. The truncated mutant ACNAb (with deletion from S454 to A639) was detected to bind with CaM, while the truncated mutant ACNAa (with deletion from R436 to A639) which lacking the putative CaM-binding region was detected hardly binding with CaM. The results indicated that the region between R436 and S454 was responsible for the interaction with CaM.3、To determine which amino acid in CaM-binding domain was essential for the interaction with CaM, we mutated five conserved hydrophobic amino acids(I439,1443, V446, V452, and F453). The binding capacity of CNA with CaM was stronger than that of the mutant proteins, while the binding capacity of CNA1 and CNA2 were the smallest in these five mutants. This indicates that the conserved hydrophobic residues 1439 and 1443 were the important amino acids involved in binding with CaM.4、Phosphatase enzyme activity of CNA and its mutant proteins was measured by PNPP method. Of these, phosphatase enzyme activity of truncated mutant ACNAa proved to be smallest. CNA1 and CNA2 phosphatase enzyme activity is very low. This indicated that Ile439 and Ile443 is not just important in binding with CaM, but also affect the phosphatase enzyme activity.