1cyu
SOLUTION NMR STRUCTURE OF RECOMBINANT HUMAN CYSTATIN A UNDER THE CONDITION OF PH 3.8 AND 310K
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OverviewOverview
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)
About this StructureAbout this Structure
1CYU is a Single protein structure of sequence from Homo sapiens. Full crystallographic information is available from OCA.
ReferenceReference
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., Tate S, Ushioda T, Utsunomiya-Tate N, Shibuya K, Ohyama Y, Nakano Y, Kaji H, Inagaki F, Samejima T, Kainosho M, Biochemistry. 1995 Nov 14;34(45):14637-48. PMID:7578072
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