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Calcineurin (CaN) is a calcium- and calmodulin-dependent protein serine/threonine phosphate which is critical for several important cellular processes, including T-cell activation. CaN is the target of the immunosuppressive drugs cyclosporin A and FK506, which inhibit CaN after forming complexes with cytoplasmic binding proteins (cyclophilin and FKBP12, respectively). We report here the crystal structures of full-length human CaN at 2.1 A resolution and of the complex of human CaN with FKBP12-FK506 at 3.5 A resolution. In the native CaN structure, an auto-inhibitory element binds at the Zn/Fe-containing active site. The metal-site geometry and active-site water structure suggest a catalytic mechanism involving nucleophilic attack on the substrate phosphate by a metal-activated water molecule. In the FKBP12-FK506-CaN complex, the auto-inhibitory element is displaced from the active site. The site of binding of FKBP12-FK506 appears to be shared by other non-competitive inhibitors of calcineurin, including a natural anchoring protein.

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Conformational analysis of the neurohypophyseal hormones oxytocin (OT) and arginine‐vasopressin (AVP) has been carried out using two different computational approaches and three force fields, namely by the Electrostatically Driven Monte Carlo (EDMC) method, with the Empirical Conformational Energy Program for Peptides (ECEPP/3) force field or with the ECEPP/3 force field plus a hydration‐shell model, and by simulated‐annealing molecular dynamics with the Consistent Valence Force Field (CVFF). The low‐energy conformations obtained for both hormones were classified using the minimal‐tree clustering algorithm and characterized according to the locations of β‐turns in the cyclic moieties. Calculations with the CVFF force field located conformations with a β‐turn at residues 3 and 4 as the lowest energy ones both for OT and for AVP. In the ECEPP/3 force field the lowest energy conformation of OT contained a β‐turn at residues 2 and 3, conformations with this location of the turn being higher in energy for AVP. The later difference can be attributed to the difference in the size of the side chain in position 3 of the sequences: the bulkier phenylalanine residue of AVP in combination with the bulky Tyr2 residue hinders the formation of a turn at residues 2 and 3. Conformations of OT and AVP with a turn at residues 3,4 were in the best agreement with the x‐ray structures of deaminooxytocin and pressinoic acid (the cyclic moiety of vasopressin), respectively, and with the nmr‐derived distance constraints. Generally, the low‐energy conformations obtained with the hydration‐shell model were in a better agreement with the experimental data than the conformations calculated in vacuo. It was found, however, that the obtained low‐energy conformations do not satisfy all of the nmr‐derived distance constraints and the nuclear Overhauser effect pattern observed in nmr studies can be fully explained only by assuming a dynamic equilibrium between conformations with β‐turns at residues 2.3, 3.4, and 4.5. The low‐energy structures of OT with a β‐turn at residues 2.3 have the disulfide ring conformations close to the model proposed recently for a potent bicyclic antagonist of OT [M.D. Shenderovich et al. (1994) Polish Journal of Chemistry, Vol. 25, pp. 921–927], although the native hormone differs from the bicyclic analogue by the conformation of the C‐terminal tripeptide. This finding confirms the hypothesis of different receptor‐bound conformations of agonists and antagonists of OT. © 1996 John Wiley & Sons, Inc.

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