H-1, C-13, N-15 NUCLEAR-MAGNETIC-RESONANCE BACKBONE ASSIGNMENTS AND SECONDARY STRUCTURE OF HUMAN CALCINEURIN-B

The calmodulin-and calcium-stimulated protein phosphatase calcineurin, PP2B, consists of two subunits: calcineurin B, which binds Ca2+, and calcineurin A, which contains the catalytic site and a calmodulin binding site. Heteronuclear 3D and 4D NMR experiments were carried out on a recombinant human calcineurin B which is a 170-residue protein of molecular mass 19.3 kDa, uniformly labeled with N-15 and C-13. The nondenaturing detergent CHAPS was used to obtain a monomeric form of calcineurin B. Three-dimensional triple resonance experiments yielded complete sequential assignment of the backbone nuclei (H-1, C-13, and N-15). This assignment was verified by a 4D HN(COCA)NH experiment carried out with 50% randomly deuteriated and uniformly N-15- and C-13-enriched calcineurin B. The secondary structure of calcineurin B has been determined on the basis of the C-13alpha and C-13beta secondary chemical shifts, J(H(N)H(alpha)) couplings, and NOE connectivities obtained from 3D N-15-separated and 4D C-13/N-15-separated NOESY spectra. Calcineurin B has eight helices distributed in four EF-hand, helix-loop-helix [Kretsinger, R. H. (1980) CRC Crit. Rev. Biochem. 8, 119-174] calcium binding domains. The secondary structure of calcineurin B is highly homologous to that of calmodulin. In comparison to calmodulin, helices B and C are shorter while helix G is considerably longer. As was observed for calmodulin in solution, calcineurin B does not have a single long central helix; rather, helices D and E are separated by a six-residue sequence in a flexible nonhelical conformation.