1H, 13C, 15N 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 15N and 13C. 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 (1H, 13C, and 15N). This assignment was verified by a 4D HN(COCA)NH experiment carried out with 50% randomly deuteriated and uniformly 15N- and 13C-enriched calcineurin B. The secondary structure of calcineurin B has been determined on the basis of the 13C alpha and 13C beta secondary chemical shifts, J(HNH alpha) couplings, and NOE connectivities obtained from 3D 15N-separated and 4D 13C/15N-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.
Anglister
J
J
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Grzesiek
S
S
Wang
A C
AC
Ren
H
H
Klee
C B
CB
Bax
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D019703:Calcineurin; D002118:Calcium / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000737:chemistry*; D002793:Cholic Acids; D003903:Deuterium; D006801:Humans; D006859:Hydrogen; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000737:chemistry*; D017433:Protein Structure, Secondary
D002148:Calmodulin-Binding Proteins; D002793:Cholic Acids; D046911:Macromolecular Substances; D006859:Hydrogen; D003903:Deuterium; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D002118:Calcium