Wild-Type and Met-65 → Leu Variants of Human Cystatin A Are Functionally and Structurally Identical

• Published in: Biochemistry (Easton) Vol. 39; no. 51; pp. 15783 - 15790
• Main Authors: Craven, C. Jeremy, Baxter, Nicola J, Murray, Ewan H, Hill, Nicola J, Martin, John R, Ylinenjärvi, Karin, Björk, Ingemar, Waltho, Jonathan P, Murray, Iain A
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 26.12.2000
• Subjects: Amino Acid Substitution - genetics; Animals; Chickens; Cystatins - antagonists & inhibitors; Cystatins - chemistry; Cystatins - genetics; Cystatins - physiology; Genetic Variation; Humans; Hydrogen-Ion Concentration; Kinetics; Leucine - genetics; Methionine - genetics; Mutagenesis, Insertional; Mutagenesis, Site-Directed; Nuclear Magnetic Resonance, Biomolecular; Papain - chemistry; Peptide Fragments - chemistry; Peptide Fragments - genetics; Polymerase Chain Reaction; Sequence Deletion; Sequence Homology, Amino Acid; Structure-Activity Relationship; Titrimetry
• DOI: 10.1021/bi0017069
• ISSN: 0006-2960

The solution structure of an N-terminally truncated and mutant form (M65L2-98) of the human cysteine protease inhibitor cystatin A has been reported that reveals extensive structural differences when compared to the previously published structure of full-length wild-type (WT) cystatin A. On the basis of the M65L2-98 structure, a model of the inhibitory mechanism of cystatin A was proposed wherein specific interactions between the N- and C-terminal regions of cystatin A are invoked as critical determinants of protease binding. To test this model and to account for the reported differences between the two structures, we undertook additional structural and mechanistic analyses of WT and mutant forms of human cystatin A. These show that modification at the C-terminus of cystatin A by the addition of nine amino acids has no effect upon the affinity of papain inhibition (K D = 0.18 ± 0.02 pM) and the consequences of such modification are not propagated to other parts of the structure. These findings indicate that perturbation of the C-terminus can be achieved without any measurable effect on the N-terminus or the proteinase binding loops. In addition, introduction of the methionine-65 → leucine substitution into cystatin A that retains the N-terminal methionine (M65L1-98) has no significant effect upon papain binding (K D = 0.34 ± 0.02 pM). Analyses of the structures of WT and M65L1-98 using 1H NMR chemical shifts and residual dipolar couplings in a partially aligning medium do not reveal any evidence of significant differences between the two inhibitors. Many of the differences between the published structures correspond to major violations by M65L2-98 of the WT constraints list, notably in relation to the position of the N-terminal region of the inhibitor, one of three structural motifs indicated by crystallographic studies to be involved in protease binding by cystatins. In the WT structure, and consistent with the crystallographic data, this region is positioned adjacent to another inhibitory motif (the first binding loop), whereas in M65L2-98 there is no proximity of these two motifs. As the NMR data for both WT9C and M65L1-98 are wholly consistent with the published structure of WT cystatin A and incompatible with that of M65L2-98, we conclude that the former represents the most reliable structural model of this protease inhibitor.