The solution structure of a human cystatin A variant, cystatin A2-98M65L, which maintains the full inhibitory activity of the wild-type protein, was determined at pH 3.8 by 2D/3D heteronuclear double- and triple-resonance NMR spectroscopy. The structure is based on a total of 1343 experimental restraints, comprising 1139 distance, 154 φ and Χ1 torsion angle restraints, and 50 distance constraints for 25 backbone hydrogen bonds. A total of 15 structures was calculated using the YASAP protocol with X-PLOR, and the atomic rms distribution about the mean coordinate positions for residues 8-93 was 0.55 ± 0.10 Å for the backbone atoms and 1.05 ± 0.11 Å for all heavy atoms. The structure consists of five antiparallel β-sheets and two short α-helices. Comparison with the X-ray structure of cystatin B in the papain complex shows that the conformation of the first binding loop is quite similar to that of cystatin A, with an rms deviation of 0.78 Å for the backbone atoms in the 43-53 region (cystatin A numbering). The second binding loop, however, is significantly different in the two structures, with an rms deviation greater than 2 Å. There are some other significant differences, especially for the N-terminal and α-helix regions. The overall structure of cystatin A is also compared with the recently reported NMR structure of the wild-type cystatin A (stefin A) at pH 5.5 (Martin et al., 1995) and reveals the following features that differ in our structure from the previous one: (1) the N-terminal segment, which was unstructured in the previous report, folds over in close vicinity to the C-terminus, as revealed by the distinctive NOEs between those segments; (2) two discrete short α-helices linked by a type II reverse turn were found, instead of the continuous single α-helix with a slight kink shown in the previous structure; (3) the second binding loop, which was not well converged in the previous study at pH 5.5, is determined very well in our structure. The effect of the N-terminal truncation on the cystatin A structure was examined by comparing the 1H-15N HSQC spectrum of cystatin A2-98 with that of the cystatin A5-98 variant, which lacks the anti-papain activity, revealing significant chemical shift differences in the residual N-terminal segment and the first binding loop, together with small shifts in the other parts. The results imply that the conformational changes in the first binding loop, induced by the N-terminal truncation, are responsible for the loss of inhibitory activity. A possible role of the N- and C-interterminal interactions in maintaining the active conformation of cystatin A is discussed.
|Number of pages||12|
|Publication status||Published - 1995|
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