NMR investigation and secondary structure of domains I and II of rat brain calbindin D28k (1-93)

Calbindin D28k, a member of the troponin C superfamily of calcium- binding proteins, contains six putative EF hand domains but binds only four calcium-atoms: one at a binding site of very high affinity and three calcium-atoms at binding sites of lower affinity. The high- affinity site could be located within domain I while domains III, IV, and V bind calcium less tightly. The recombinant protein construct calb I-II (residues 1-93) comprising the first two EF hands affords a unique opportunity to study a pair of EF hands with one site binding calcium tightly and the second site empty. A series of heteronuclear 2D, 3D and 4D high-resolution NMR experiments were applied to calb I-II, and led to the complete assignment of the 1H, 13C and 15N resonances. The secondary structure of the protein was deduced from the size of the 3JHN-Halpha coupling constants, the chemical shift indices of 1Etaalpha, 13Calpha, 13C' and 13Cbeta nuclei and from an analysis of backbone NOEs observed in 3D and 4D NOESY spectra. Four major alpha- helices are identified: Ala13-Phe23, Gly33-Ala50, Leu54-Asp63, Val76- Leu90, while residues Ala2-Leu6 form a fifth, flexible helical segment. Two short beta-strands (Tyr30-Glu32, Lys72-Gly74) are found preceding helices B and D and are arranged in an anti-parallel interaction. Based on these data a structural model of calb I-II was constructed that shows that the construct adopts a tertiary structure related to other well-described calcium-binding proteins of the EF-hand family. Surprisingly, the protein forms a homodimer in solution, as was shown by its NMR characterization, size-exclusion chromatography and analytical ultra-centrifugation studies.