Solution Structure of a Human Cystatin A Variant, Cystatin A^2-98 M65L, by NMR Spectroscopy. A Possible Role of the Interactions between the N- and C-Termini To Maintain the Inhibitory Active Form of Cystatin A

The solution structure of a human cystatin A variant, cystatin A^2-98 M65L, 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 ø and x¹ 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 a-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 A. There are some other significant differences, especially for the N-terminal and a-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 a-helices linked by a type II reverse turn were found, instead of the continuous single a-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 A^2-98 with that of the cystatin A^5-98 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.