Structural and biochemical characterization of neuronal calretinin domain I-II (residues 1-100). Comparison to homologous calbindin D28k domain I-II (residues 1-93).
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.
Palczewska
M
M
Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, Warsaw, Poland.
Groves
P
P
Ambrus
A
A
Kaleta
A
A
Kövér
K E
KE
Batta
G
G
Kuźnicki
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D064092:Calbindin 1; D064032:Calbindin 2; D064026:Calbindins; D002118:Calcium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D064030:S100 Calcium Binding Protein G / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
C576500:Calb1 protein, rat; C576512:Calb2 protein, rat; D064092:Calbindin 1; D064032:Calbindin 2; D064026:Calbindins; D009419:Nerve Tissue Proteins; D064030:S100 Calcium Binding Protein G; D002118:Calcium