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The structural model derived from X-ray crystallography for unphosphorylated
wild-type chicken cystatin is compared with two chicken cystatin structures
derived from NMR spectroscopy: the phosphorylated wild-type and the genetically
engineered variant AEF-SIM-M29I-M89L. The comparison shows the same overall
fold, but also significant differences in structurally variable segments of the
polypeptide chain. The largest such segment, comprising residues 71 to 89, is a
region characteristic of the family 2 cystatin inhibitors which contains a
disulphide bridge (71-81) and the phosphorylation site (Ser80) discussed in the
accompanying article. In the crystal structure, the segment 71 to 76 is found as
a flexible loop, 77 to 85 as an alpha-helical segment, and 86 to 89 is
completely undefined. The solution NMR structures on the other hand are
disordered in the initial segment 72 to 80, have an extended conformation at 81
to 83 in contact with the beta-sheet, and clearly show a beta-turn at residues
87 to 90. The segment comprising residues 53 to 57, with smaller variability, is
of particular interest as the hairpin loop conserved throughout the cystatin
superfamily which binds to the cysteine proteinase. In most of the solution NMR
structures, this segment adopts a conformation more like that of stefin B, a
family 1 cystatin inhibitor, as was observed in the crystal structure of its
inhibitory complex with papain. The differences between the structures are
rationalized by an examination of the crystal contacts generated by hypothetical
crystal packing of the NMR structures. Additionally, the X-ray refinement shows
evidence of conformational disorder in the crystal. Joint refinement with NOE
restraints and reflection data does not produce a structure to satisfy the
restraints of both methods.
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