MSEPKAIDPKLST

1. X-ray crystallography (CaCl2; polyethylene glycol (PEG) 6000)

1. Kissinger CR, Parge HE, Knighton DR, Lewis CT, Pelletier LA, Tempczyk A, Kalish VJ, Tucker KD, Showalter RE, Moomaw EW, et al. "Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex." Nature. 1995; 378(6557): 641-4. PubMed: 8524402
   
   

Mass spectrometry combined with enzymatic digestion showed that the N-terminal residues were sensitive to proteolysis. (PMID 12357034)

DDELGSEEDGFDGATAAARKEVIRNKIRAIGKMARVFSVLREESESVLTLKGLTPTGMLP
SGVLSGGKQTLQSATVEAIEADEAIKGFSPQHKIT

1. X-ray crystallography (CaCl2; polyethylene glycol (PEG) 6000)

1. Kissinger CR, Parge HE, Knighton DR, Lewis CT, Pelletier LA, Tempczyk A, Kalish VJ, Tucker KD, Showalter RE, Moomaw EW, et al. "Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex." Nature. 1995; 378(6557): 641-4. PubMed: 8524402
   
   

The disordered region from 374-468 includes the CaM-binding domain predicted to lie between residues 390-414. As in the crystal structure of native CaN, the C-terminal portion of CaNA is disordered beyond the CaNB-binding helix (343-373), and electron density for the CaM-binding domain of CaNA is not visible. Mass spectrometry combined with enzymatic digestion showed that the calmodulin-binding region was removed by trypsin, indicating that this disordered region is sensitive to proteolysis. Calmodulin binds to this disordered region between the CnB-binding site and the autoinhibitory helix, and this segment takes on an alpha-helical conformation to pull the AI helix away from the catalytic site in order to activate the phosphatase activity. Calmodulin is a protein that wraps around a target helix in calcineurin upon binding. The ~20-residue target helix is located within a 95-residue disordered region that is not observed in crystal structures. The disorder allows the target helix to separate from the rest of the protein, thus permitting access for binding. For the calmodulin/target helix interaction, it is unknown whether the other ~75 disordered residues become ordered upon binding, but it is thought that this region takes on an alpha-helical conformation when calmodulin binds. The function of the disordered region is to make the helix accessible for calmodulin binding, and this region of disorder is essential to the regulation of calcineurin by calcium/calmodulin. Binding of calcium/calcineurin activates calcineurin's serine/threonine phosphatase activity.

AARKEVIRNKIRAIGKMARVFSVLR

1. X-ray crystallography (CaCl2; polyethylene glycol (PEG) 6000)

1. Kissinger CR, Parge HE, Knighton DR, Lewis CT, Pelletier LA, Tempczyk A, Kalish VJ, Tucker KD, Showalter RE, Moomaw EW, et al. "Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex." Nature. 1995; 378(6557): 641-4. PubMed: 8524402
   
   

SFEEAKGLDRINERMPPR

1. X-ray crystallography (CaCl2; polyethylene glycol (PEG) 6000)

1. Kissinger CR, Parge HE, Knighton DR, Lewis CT, Pelletier LA, Tempczyk A, Kalish VJ, Tucker KD, Showalter RE, Moomaw EW, et al. "Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex." Nature. 1995; 378(6557): 641-4. PubMed: 8524402
   
   

As in the crystal structure of native CaN, the C-terminal portion of CaNA is disordered beyond the CaNB-binding helix (343-373), which indicates that the autoinhibitory domain is disordered. However, this disordered region is present for PDB 1AUI. Unlike the native CaN crystal structure, the AI segment of CaN is not seen in the crystal structure of the FKBP12-FK506-CaN complex. This might indicate that the autoinhibitory complex is ordered when CaNA is in a complex with CaNB, but other evidence suggests that when the AI domain is either not in contact with the catalytic domain (as is the case with immunosuppressants) or when CaNA exists as a monomer, the AI domain may not be structured. Calmodulin binds to the disordered region between the CnB-binding site and the autoinhibitory helix, and this segment takes on an alpha-helical conformation to pull the AI helix away from the catalytic site in order to activate the phosphatase activity according to Jin and Harrison. When this occurs, the structure of the AI helix may change as well, leading to disorder. Further evidence needs to be found in order to indicate the state of the AI domain. For now, the AI domain will be considered disordered based on the statement made by Kissinger above and the information provided by Jin and Harrison in regards to proteolytic sensitivity. The autoinhibitory domain lies over the substrate-binding cleft in the catalytic domain and consists of two short alpha-helical regions plus five residues in an extended conformation. The helical regions restrict interaction with the substrate-binding cleft when the AI segment contacts the cleft. Mass spectrometry combined with enzymatic digestion showed that the autoinhibitory helix was removed by trypsin, indicating that this disordered region is sensitive to proteolysis.

RDAMPSDANLNSINKALTSETNGTDSNGSNSSNIQ

1. X-ray crystallography (CaCl2; polyethylene glycol (PEG) 6000)

1. Kissinger CR, Parge HE, Knighton DR, Lewis CT, Pelletier LA, Tempczyk A, Kalish VJ, Tucker KD, Showalter RE, Moomaw EW, et al. "Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex." Nature. 1995; 378(6557): 641-4. PubMed: 8524402
   
   

CaNA residues 487-521 are not visible in the electron density map of the crystal structure and are disordered.

TDRVVKAVPFPPSHRLTAKEVFDNDGKPRVDILKAHLMKEGRLEESVALRIITEGASILR
QEKNLLDIDAPVTVCGDIHGQFFDLMKLFEVGGSPANTRYLFLGDYVDRGYFSIECVLYL
WALKILYPKTLFLLRGNHECRHLTEYFTFKQECKIKYSERVYDACMDAFDCLPLAALMNQ
QFLCVHGGLSPEINTLDDIRKLDRFKEPPAYGPMCDILWSDPLEDFGNEKTQEHFTHNTV
RGCSYFYSYPAVCEFLQHNNLLSILRAHEAQDAGYRMYRKSQTTGFPSLITIFSAPNYLD
VYNNKAAVLKYENNVMNIRQFNCSPHPYWLPNFMDVFTWSLPFVGEKVTEMLVNVLNICS

1. X-ray crystallography (CaCl2; polyethylene glycol (PEG) 6000)

1. Kissinger CR, Parge HE, Knighton DR, Lewis CT, Pelletier LA, Tempczyk A, Kalish VJ, Tucker KD, Showalter RE, Moomaw EW, et al. "Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex." Nature. 1995; 378(6557): 641-4. PubMed: 8524402
   
   

1. Muramatsu T, Kincaid RL. "Molecular cloning of a full-length cDNA encoding the catalytic subunit of human calmodulin-dependent protein phosphatase (calcineurin A alpha)." Biochim Biophys Acta. 1993; 1178(1): 117-20. PubMed: 8392375
   
   

Catalytic subunit of calcineurin. Interacts with DP00565, Calcineurin subunit B type 1.