Accessing the global minimum conformation of stefin A dimer by annealing under partially denaturing conditions

Jerala, Roman; Žerovnik, Eva

doi:10.1006/jmbi.1999.3045

Cited by 70 publications

(102 citation statements)

References 44 publications

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“…The primary change with pH is that the excellent quality of NOESY and TOCSY spectra obtained at pH 5.5, as expected from a protein with such a short correlation time (4.5 ns), is severely degraded at pH 3.7. Recently, cystatin A has also been shown to undergo a welldefined dimerization reaction under conditions in which the native fold is strongly destabilized (30), in a manner similar to that reported previously for the type II inhibitor, cystatin C (31,32). However, the introduction of this structural perturbation is again insufficient to account for the structure reported for M65L .…”

Section: Discussionmentioning

confidence: 75%

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

et al. 2000

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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 previously published structure of full-length wild-type (WT) cystatin A. On the basis of the M65L(2-98) 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 (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 (1)H 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 M65L(2-98) 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 M65L(2-98) there is no proximity of these two motifs. As the NMR data for both WT9C and M65L(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 represents the most reliable structural model of this protease inhibitor.

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Section: Discussionmentioning

confidence: 75%

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

et al. 2000

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“…Reversible dimer formation at physiological salt concentrations and neutral pH was also detected with the C3S variant of human stefin B [31]. An irreversible domain-swapped dimer (DSD), similarly to stefin A [34,35] was observed on heating to predenaturational temperatures (E. Zerovnik, unpublished). It has been shown that amyloid-fibril formation by stefin B follows reduction of pH to 4.7 [29] but also to pH 3.3 [36], where native-like and molten globule intermediates, respectively, are populated [30].…”

Section: Introductionmentioning

confidence: 83%

“…6). There are 7 over 22 amino acid identities (31%) for stefin B(41-60) -Aβ and 6 over 20 amino acid (30%) for Aβ (22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40) -PrP(90-144). High similarity of Aβ1-42 and HA-fd (fusion domain of influenza hemagglutinin) was reported [67].…”

Section: Parallels With Stefin Bmentioning

confidence: 98%

Conformational Changes Preceding Amyloid-Fibril Formation of Amyloid- Beta, Prion Protein and Stefin B; Parallels in pH Dependence

Matsunaga¹,

Žerovnik²,

Yamada³

et al. 2005

MCRO

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Amyloid beta (A β) protein in Alzheimer's disease and scrapie prion protein (PrP sc ) in Scrapie is the key component of amyloid plaques in brain whereas stefin B is an intracellular cysteine proteinase inhibitor, broadly distributed in different tissue and recently reported to form amyloid fibrils in vitro. By reducing the pH to around 4.6, the native conformation of both polypeptides is changed into less ordered, metastable intermediates that are stabilised by formation of the more stable fibrils. In Aβ, the Glu at position 11 was found to be responsible for the conformational change at pH 4.6. Metal ions, including copper and zinc, could also induce conformational changes of Aβ at neutral pH. The acid modified Aβ conformer exhibited protease K resistance, preferential internalisation and accumulation in the human glial cells. In N-terminally truncated PrP90-231, spanning PrP residues 94-112 and the central region 146-154 incorporating Glu152 was identified as a significant participant for the conformational changes in acidic pH. In stefin B, reducing the pH to pH 3.3 results in another intermediate of the molten-globule type which also leads to amyloid fibril formation. Multiple sequence alignment revealed distinct similarities of Aβ (1-42) peptide, stefin B (13 to 61 residues) and prion fragment (90 to 144 residues).

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“…Domain-swapped dimers have been observed for both the mammalian prion protein (Knaus et al 2001), human cystatins and stefins (Jerala and Zerovnik 1999;Janowski et al 2001;Staniforth et al 2001), and oligomers were observed for a number of other amyloidogenic proteins (Serag et al 2001). The height of the first barrier in the reaction of amyloid fibril formation for the case of stefins is distinct from that measured in the case of synuclein (Uversky et al 2001) and HET prion (Sabate et al 2005), where a smaller E a of 22 kcal/mol was observed.…”

Section: Energetics Of Domain-swapping (And Amyloid Growth Initiation)mentioning

confidence: 97%

“…In the case of human stefins A and B we have determined the conditions under which they undergo domain-swapping, which proved to demand close to unfolding transition and annealing into the domain-swapped dimer (Jerala and Zerovnik 1999) from the metastable monomeric state. The fibrils of stefin B themselves are most likely built from a distorted domain swapped dimer as was shown by H/D exchange and NMR (Morgan et al 2008).…”

Section: Energetics Of Domain-swapping (And Amyloid Growth Initiation)mentioning

confidence: 99%

Oligomerization preceding amyloid fibril formation: a process in common to intrinsically disordered and globular proteins

Žerovnik

2011

Network: Computation in Neural Systems

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Neurodegenerative diseases present a big burden to society. At the molecular level many of them - if not all - show protein aggregation (as an epiphenomenon or as a cause). The knowledge on details of thermodynamics and kinetics as well as structure of the protein aggregates, especially the early and soluble oligomers, may help in designing inhibitors for early stages of such diseases. Here, a possible outlook on more general mechanism for their formation is discussed. The oligomers of amyloid forming proteins, which are present prior and during nucleation and amyloid fibril formation, are claimed to be toxic to cells. Oligomers of the globular proteins and the intrinsically disordered proteins (IDPs), form in vitro upon partial denaturation and renaturation, respectively. Often they form if the sample is heated or freeze-thawed for a few cycles. A question is asked if this does not highlight one important property in common to globular proteins and IDPs, namely, a high energetic barrier dividing such oligomers from the monomers. This also would imply existence of two populations of states, one, the monomer - being metastable - at least under the conditions, which promote fibril formation.

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Accessing the global minimum conformation of stefin A dimer by annealing under partially denaturing conditions

Cited by 70 publications

References 44 publications

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

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

Conformational Changes Preceding Amyloid-Fibril Formation of Amyloid- Beta, Prion Protein and Stefin B; Parallels in pH Dependence

Oligomerization preceding amyloid fibril formation: a process in common to intrinsically disordered and globular proteins

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