NMR investigation and secondary structure of domains I and II of rat brain calbindin D_28k (1–93)

Abstract

Calbindin D 28k , 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 1 H, 13 C and 15 N resonances. The secondary structure of the protein was deduced from the size of the 3 J HN-Ha coupling constants, the chemical shift indices of 1 H a , 13 C a , 13 C H and 13 C b nuclei and from an analysis of backbone NOEs observed in 3D and 4D NOESY spectra. Four major a-helices are identified: Ala13±Phe23, Gly33±Ala50, Leu54±Asp63, Val76±Leu90, while residues Ala2±Leu6 form a fifth, flexible helical segment. Two short b-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.Keywords: calbindin D 28k ; calcium binding protein; EF hand; NMR spectroscopy; secondary structure.Calbindin D 28k is an intracellular calcium binding protein of molecular mass 28 kDa that may be involved in transcellular calcium transport and may modulate effects occurring in response to changes in intracellular calcium concentrations. It was originally purified from chicken intestine [1] and is also expressed in kidney cells. The presence of the protein in numerous additional avian and mammalian tissues suggests an important physiological function [2]. The finding that it is particularly abundant in specific brain regions has led to studies investigating its particular role in calcium homeostasis [3,4].Calbindin D 28k belongs to a family of calcium-binding proteins including many well-known proteins such as calbindin D 9k , troponin C, S100B, calmodulin and calretinin. They are structurally characterized by one or more pairs of a helix-loophelix motif called the EF hand [5] in which five hydrophilic residues and one water molecule bind one calcium atom in a pentagonal bipyramidal coordination via oxygen atoms. The calcium-binding loop is flanked by two a-helices which have a number of residues with hydrophobic side chains that interact with a second EF hand domain forming a hydrophobic core. An approximate twofold symmetry axis relates the pair of EF hand domains....

Structural and biochemical characterization of neuronal calretinin domain I–II (residues 1–100)

“…6. The chemical shifts agree with biochemical data that only certain calcium‐binding sites of the reference proteins are filled [31,50–56].…”

“…Klaus et al . described the N‐terminal region as containing a pre‐A helix and noted that it is poorly formed [31]. This region was not detected in the NMR spectra of CR I–II (Fig.…”

“…It is difficult to localize the biochemical properties of CR and Calb to particular domains but this information can be obtained by studying protein fragments. For example, the structure of the calcium‐bound first domain of Calb, comprising the first two EF‐hands (Calb I–II, residues 1–93), was recently reported [31]. Calb I–II binds a single calcium and forms a homodimer [31].…”

Three functional facets of calbindin D-28k

“…A detailed NMR analysis of CB structure (Kojetin et al, 2006), completing earlier work (Klaus et al, 1999; Berggård et al, 2000, 2002a,b; Venters et al, 2003; Venyaminov et al, 2004; Vanbelle et al, 2005), confirmed that upon Ca 2+ binding CB adopts discrete hydrophobic states but also has exposed hydrophobic surfaces in its apo-form. In addition, the regions mediating the interactions with RanBPM, IMPase, Caspase-3 and also its pro-domain were mapped.…”

Calbindin D28k EF-Hand Ligand Binding and Oligomerization: Four High-Affinity SitesThree Modes of Action

“…The backbone was fixed, and the calbindin D 28k side chains were optimized using Swiss PDB Modeller (44). covalently linked, the EF1-EF2 fragment is folded (25), suggesting that it forms a pair within the intact protein. The EF1-EF2 and EF1-EF2-EF3 fragments form homodimers in solution (25,26), suggesting that EF1 and/or EF2 form additional contacts within the intact protein.…”