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

Abstract

This study characterizes the calcium‐bound CR I–II domain (residues 1–100) of rat calretinin (CR). CR, with six EF‐hand motifs, is believed to function as a neuronal intracellular calcium‐buffer and/or calcium‐sensor. The secondary structure of CR I–II, defined by standard NMR methods on 13C,15N‐labeled protein, contains four helices and two short interacting segments of extended structure between the calcium‐binding loops. The linker between the two helix–loop–helix, EF‐hand motifs is 12 residues long. Limited trypsinolysis at K60 (there are 10 other K/R residues in CR I–II) confirms that the linker of CR I–II is solvent‐exposed and that other potential sites are protected by regular secondary structure. 45Ca‐overlay of glutathione S‐transferase (GST)–CR(1–60) and GST–CR(61–100) fusion proteins confirm that both EF‐hands of CR I–II have intrinsic calcium‐binding properties. The primary sequence and NMR chemical shifts, including calcium‐sensitive glycine residues, also suggest that both EF‐hand loops of CR I–II bind calcium. NMR relaxation, analytical ultracentrifugation, chemical cross‐linking and NMR translation diffusion measurements indicate that CR I–II exists as a monomer. Calb I–II (the homologous domain of calbindin D28k) has the same EF‐hand secondary structures as CR I–II, except that helix B is three residues longer and the linker has only four residues [Klaus, W., Grzesiek, S., Labhardt, A. M., Buckwald, P., Hunziker, W., Gross, M. D. & Kallick, D. A. (1999) Eur. J. Biochem.262, 933–938]. In contrast, Calb I–II binds one calcium cation per monomeric unit and exists as a dimer. Despite close homology and similar secondary structures, CR I–II and Calb I–II probably have distinct tertiary structure features that suggest different cellular functions for the full‐length proteins.

Original article Morphological and quantitative analysis of cerebellar cortical cells in Alzheimer’s disease

“…Despite conflicting reports on the calretinin expression in the ganglionic layer, we observed no IHC reaction in Purkinje cells [41,46]. However, this situation can be explained by the fixation procedure and conduct of the material, which caused damage to the less stable calretinin domain in Purkinje cells (Table I) [40]. A positive reaction with the antibody against parvalbumin, without changed expression intensity in both study groups was observed in neurons of molecular layers in stellate cells, while the weakening of the reaction in the basket cells and Purkinje cells was found in the experimental group.…”

Molecular dynamics study of the structural basis of dysfunction and the modulation of reactive oxygen species generation by pathogenic mutants of human dihydrolipoamide dehydrogenase

“…These findings suggest that LADH might display some of its moonlighting functions as an independent enzyme (see also [25,26]). Protein-protein interactions and conditions used for crystallography often shift conformational equilibria of proteins and stabilize the thermodynamically favored conformation under the existing conditions [59][60][61][62][63]. This conformation may considerably deviate from the uncomplexed or the solution structure [64][65][66][67][68].…”

The continuing disappearance of “pure” Ca2+ buffers

“…Currently, no structural data (X-ray or NMR) is available for full-length CR, but NMR results were published on the N-terminal 100 amino acids of rat CR consisting of EF-hand domains 1 and 2 [172]. The two domains form a relatively tight structure, while the linker region between domains is exposed to solvent and accessible for proteolytic enzymes.…”