Solution structure of a human cystatin A variant, cystatin A2-98 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

Abstract

The solution structure of a human cystatin A variant, cystatin A2-98 M65L, which maintains the full inhibitory activity of the wild-type protein, was determined at pH 3.8 by sD/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 A for the backbone atoms and 1.05 +/- 0.11 A 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 A 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 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 in close 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 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of Solanum tuberosum Multicystatin and the Significance of Core Domains

“…The ;35-kD peptide is composed of three cystatin domains designated 5, 6, and 7 (Nissen et al, 2009) and remains resistant to further proteolysis by some unknown mechanism. Our previous work (Nissen et al, 2009) established the structural similarity of a single domain of PMC (PMC-2) with that of other cystatins, such as chicken egg white cystatin (CEW; Bode et al, 1988;Dieckmann et al, 1993), human stefin A (Martin et al, 1995;Tate et al, 1995;Jenko et al, 2003) and human stefin B (Stubbs et al, 1990), and rice (Oryza sativa) cystatin (Nagata et al, 2000).…”

Characterization of Solanum tuberosum Multicystatin and Its Structural comparison with Other Cystatins

“…High-resolution structures for some of the cystatins have been determined, including CEW cystatin (Bode et al, 1988;Dieckmann et al, 1993), human stefin A (Martin et al, 1995;Tate et al, 1995;Jenko et al, 2003), and human stefin B (Stubbs et al, 1990). To date, only one nuclear magnetic resonance structure for cystatin (oryzacystatin-1) from rice (Oryza sativa) is available (Nagata et al, 2000).…”

Design and Validation of a Neutral Protein Scaffold for the Presentation of Peptide Aptamers

“…SteA itself is a monomeric, single-chain, single-domain protein of 98 amino acid residues. 11,12 The structure of SteA has been solved, [13][14][15] facilitating the rational mutation of SteA into the stefin A triple mutant (STM) scaffold. The only known biological activity of cystatins is the inhibition of cathepsin activity, which allowed us to test exhaustively for residual biological activity of our engineered proteins.…”