Structural studies on the Ca2+-binding domain of human nucleobindin (calnuc).
Nucleobindin, also known as calnuc, participates in Ca2+ storage in the Golgi, as well as in other biological processes that involve DNA-binding and protein-protein interactions. We have determined the three-dimensional solution structure of the Ca(2+)-binding domain of nucleobindin by NMR showing that it consists of two EF-hand motifs. The NMR structure indicates that the phi and psi angles of residues in both motifs are very similar, despite the noncanonical sequence of the C-terminal EF-hand, which contains an arginine residue instead of the typical glycine at the sixth position of the 12-residue loop. The relative orientation of the alpha-helices in the N-terminal EF-hand falls within the common arrangement found in most EF-hand structures. In contrast, the noncanonical EF-hand deviates from the average orientation. The two helix-loop-helix moieties are in the open conformation characteristic of the Ca(2+)-bound state. We find that both motifs bind Ca2+ with apparent dissociation constants of 47 and 40 microM for the noncanonical and the canonical EF-hand, respectively. The Ca(2+)-binding domain of nucleobindin is unstructured in the absence of Ca2+ and folds upon Ca2+ addition. NMR relaxation data and structural studies of the folded domain indicate that it undergoes slow dynamics, suggesting that it is floppier and less compact than a globular domain.
Laboratory of Biophysical Chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, 50 Center Drive, Bethesda, Maryland 20892, USA.
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D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D020832:EF Hand Motifs; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D000080866:Nucleobindins; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013816:Thermodynamics
D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; D009587:Nitrogen Isotopes; D000080866:Nucleobindins; D010446:Peptide Fragments; D002118:Calcium