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The three-dimensional solution structure of apo rabbit lung calcyclin has been
refined to high resolution through the use of heteronuclear NMR spectroscopy and
13C, 15N-enriched protein. Upon completing the assignment of virtually all of
the 15N, 13C and 1H NMR resonances, the solution structure was determined from a
combination of 2814 NOE-derived distance constraints, and 272 torsion angle
constraints derived from scalar couplings. A large number of critical
inter-subunit NOEs (386) were identified from 13C-select, 13C-filtered NOESY
experiments, providing a highly accurate dimer interface. The combination of
distance geometry and restrained molecular dynamics calculations yielded
structures with excellent agreement with the experimental data and high
precision (rmsd from the mean for the backbone atoms in the eight helices: 0.33
A). Calcyclin exhibits a symmetric dimeric fold of two identical 90 amino acid
subunits, characteristic of the S100 subfamily of EF-hand Ca(2+)-binding
proteins. The structure reveals a readily identified pair of putative sites for
binding of Zn2+. In order to accurately determine the structural features that
differentiate the various S100 proteins, distance difference matrices and
contact maps were calculated for the NMR structural ensembles of apo calcyclin
and rat and bovine S100B. These data show that the most significant variations
among the structures are in the positioning of helix III and in loops, the
regions with least sequence similarity. Inter-helical angles and distance
differences for the proteins show that the positioning of helix III of calcyclin
is most similar to that of bovine S100B, but that the helix interfaces are more
closely packed in calcyclin than in either S100B structure. Surprisingly large
differences were found in the positioning of helix III in the two S100B
structures, despite there being only four non-identical residues, suggesting
that one or both of the S100B structures requires further refinement.
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