Wild-Type and Met-65 Leu Variants of Human Cystatin A Are Functionally and Structurally Identical
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- 作者：C. Jeremy Craven ; Nicola J. Baxter ; Ewan H. Murray ; Nicola J. Hill ; John R. Martin ; Karin Ylinenjä ; rvi ; Ingemar Bjö ; rk ; Jonathan P. Waltho ; and Iain A. Murray
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：December 26, 2000
- 年：2000
- 卷：39
- 期：51
- 页码：15783 - 15790
- 全文大小：237K
- 年卷期：v.39,no.51(December 26, 2000)
- ISSN：1520-4995
文摘
The solution structure of an N-terminally truncated and mutant form (M65L2-98) of the humancysteine protease inhibitor cystatin A has been reported that reveals extensive structural differences whencompared to the previously published structure of full-length wild-type (WT) cystatin A. On the basis ofthe M65L2-98 structure, a model of the inhibitory mechanism of cystatin A was proposed wherein specificinteractions between the N- and C-terminal regions of cystatin A are invoked as critical determinants ofprotease 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 cystatinA. These show that modification at the C-terminus of cystatin A by the addition of nine amino acids hasno effect upon the affinity of papain inhibition (KD = 0.18 ± 0.02 pM) and the consequences of suchmodification are not propagated to other parts of the structure. These findings indicate that perturbationof the C-terminus can be achieved without any measurable effect on the N-terminus or the proteinasebinding loops. In addition, introduction of the methionine-65 
leucine substitution into cystatin A thatretains the N-terminal methionine (M65L1-98) has no significant effect upon papain binding (KD = 0.34± 0.02 pM). Analyses of the structures of WT and M65L1-98 using 1H NMR chemical shifts and residualdipolar couplings in a partially aligning medium do not reveal any evidence of significant differencesbetween the two inhibitors. Many of the differences between the published structures correspond to majorviolations by M65L2-98 of the WT constraints list, notably in relation to the position of the N-terminalregion of the inhibitor, one of three structural motifs indicated by crystallographic studies to be involvedin protease binding by cystatins. In the WT structure, and consistent with the crystallographic data, thisregion is positioned adjacent to another inhibitory motif (the first binding loop), whereas in M65L2-98there is no proximity of these two motifs. As the NMR data for both WT9C and M65L1-98 are whollyconsistent with the published structure of WT cystatin A and incompatible with that of M65L2-98, weconclude that the former represents the most reliable structural model of this protease inhibitor.
leucine substitution into cystatin A thatretains the N-terminal methionine (M65L1-98) has no significant effect upon papain binding (KD = 0.34± 0.02 pM). Analyses of the structures of WT and M65L1-98 using 1H NMR chemical shifts and residualdipolar couplings in a partially aligning medium do not reveal any evidence of significant differencesbetween the two inhibitors. Many of the differences between the published structures correspond to majorviolations by M65L2-98 of the WT constraints list, notably in relation to the position of the N-terminalregion of the inhibitor, one of three structural motifs indicated by crystallographic studies to be involvedin protease binding by cystatins. In the WT structure, and consistent with the crystallographic data, thisregion is positioned adjacent to another inhibitory motif (the first binding loop), whereas in M65L2-98there is no proximity of these two motifs. As the NMR data for both WT9C and M65L1-98 are whollyconsistent with the published structure of WT cystatin A and incompatible with that of M65L2-98, weconclude that the former represents the most reliable structural model of this protease inhibitor.