NMR investigation and secondary structure of domains I and II of rat brain calbindin D(28k) (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 ³J(HN-Hα) coupling constants, the chemical shift indices of ¹H^a , ¹³Cα, ¹³C' and ¹³Cβ nuclei and from an analysis of backbone NOEs observed in 3D and 4D NOESY spectra. Four major α- helices are identified: Ala13-Phe23, Gly33-Ala50, Leu54-Asp63, Val76-Leu90, while residues Ala2-Leu6 form a fifth, flexible helical segment. Two short β-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.