Wild-type and Met-65 -> Leu variants of human cystatin A are functionally and structurally identical

The solution structure of an N-terminally truncated and mutant form (M65L(2 -98)) of the human cysteine protease inhibitor cystatin A has been reported that reveals extensive structural differences when compared to the previou sly published structure of full-length wild-type (WT) cystatin A. On the ba sis of the M65L(2-98) structure, a model of the inhibitory mechanism of cys tatin A was proposed wherein specific interactions between the N- and C-ter minal regions of cystatin A are invoked as critical determinants of proteas e binding. To test this model and to account for the reported differences b etween the two structures, we undertook additional structural and mechanist ic analyses of WT and mutant forms of human cystatin A. These show that mod ification at the C-terminus of cystatin A by the addition of nine amino aci ds has no effect upon the affinity of papain inhibition (K-D = 0.18 +/- 0.0 2 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 o r the proteinase binding loops. In addition, introduction of the methionine -65 --> leucine substitution into cystatin A that retains the N-terminal me thionine (M65L(1-98)) has no significant effect upon papain binding (K-D = 0.34 +/- 0.02 pM). Analyses of the structures of WT and M65L(1-98) using H- 1 NMR chemical shifts and residual dipolar couplings in a partially alignin g medium do not reveal any evidence of significant differences between the two inhibitors. Many of the differences between the published structures co rrespond to major violations by M65L(2-98) of the WT constraints list, nota bly 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 consist ent with the crystallographic data, this region is positioned adjacent to a nother inhibitory motif (the first binding loop), whereas in M65L(2-98) the re is no proximity of these two motifs. As the NMR data for both WT9C and M 65L(1-98) are wholly consistent with the published structure of WT cystatin A and incompatible with that of M65L(2-98). We conclude that the former re presents the most reliable structural model of this protease inhibitor.