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

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 13 C, 15 N‐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. 45 Ca‐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 D 28k ) 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.