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-TERMINI 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 phi and chi(1) 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 Angstrom for the backbone atoms and 1.05 +/- 0.11 Angstrom for all heavy atoms. The structure consists of five antiparallel beta-sheets and two short alpha-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 Angstrom 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 Angstrom. There are some other significant differences, especially for the N-terminal and alpha-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 inclose vicinity to the C-terminus, as revealed by the distinctive NOEs between those segments; (2) two discrete short alpha-helices linked by a type II reverse turn were found, instead of the continuous single alpha-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 H-1-N-15 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.