Rosemary A. Staniforth

Three-dimensional domain swapping in the folded and molten-globule states of cystatins, an amyloid-forming structural superfamily

Cystatins, an amyloid‐forming structural superfamily, form highly stable, domain‐swapped dimers at physiological protein concentrations. In chicken cystatin, the active monomer is a kinetic trap en route to dimerization, and any changes in solution conditions or mutations that destabilize the folded state shorten the lifetime of the monomeric form. In such circumstances, amyloidogenesis will start from conditions where a domain‐swapped dimer is the most prevalent species. Domain swapping occurs by a rearrangement of loop I, generating the new intermonomer interface between strands 2 and 3. The transition state for dimerization has a high level of hydrophobic group exposure, indicating that gross conformational perturbation is required for domain swapping to occur. Dimerization also occurs when chicken cystatin is in its reduced, molten‐globule state, implying that the organization of secondary structure in this state mirrors that in the folded state and that domain swapping is not limited to the folded states of proteins. Although the interface between cystatin‐fold units is poorly defined for cystatin A, the dimers are the appropriate size to account for the electron‐dense regions in amyloid protofilaments.

The stability and hydrophobicity of cytosolic and mitochondrial malate dehydrogenases and their relation to chaperonin-assisted folding

mMDH and cMDH are structurally homologous enzymes which show very different responses to chaperonins during folding. The hydrophilic and stable cMDH is bound by cpn60 but released by MG‐ATP alone, while the hydrophobic and unstable mMDH requires both Mg‐ATP and cpn 10. Citrate equalises the stability of the native state of the two proteins but has no effect on the co‐chaperonin requirement, implying that hydrophobicity, and not stability, is the determining factor. The yield and rate of folding of cMDH is unaffected while that of mMDH is markedly increased by the presence of cpn60, cpn10 and Mg‐ATP. In 200 mM orthophosphate, chaperonins do not enhance the rate of folding of mMDH, but in low phosphate concentrations chaperonin‐assisted folding is 3–4‐times faster.

Amyloid fibril formation by human stefin B: influence of pH and TFE on fibril growth and morphology

As shown before, human stefin B (cystatin B) populates two partly unfolded species, a native-like state at pH 4.8 and a structured molten globule state at pH 3.3 (high ionic strength), from each of which amyloid fibrils grow. Here, we show that the fibrils obtained at pH 3.3 differ from those at pH 4.8 and that those obtained at pH 3.3 (protofibrils) do not transform readily to mature fibrils. In addition we show that amorphous aggregates are also a source of fibrils. The kinetics of amyloid fibril formation at different trifluoroethanol (TFE) concentrations were measured. TFE accelerates fibril growth at predenaturational concentrations of the alcohol. At concentrations higher than 10%, the fibrillar yield decreases proportionately as the population of an all α-helical, denatured form of the protein increases. At an optimum TFE concentration, the lag and the growth phases are observed, similarly to some other amyloidogenic proteins. Morphology of the protein species at the beginning and the end of the reactions was observed using atomic force microscopy and transmission electron microscopy. Final fibril morphologies differ depending on solvent conditions.

Mechanisms of amyloid fibril formation – focus on domain‐swapping

Conformational diseases constitute a group of heterologous disorders in which a constituent host protein undergoes changes in conformation, leading to aggregation and deposition. To understand the molecular mechanisms of the process of amyloid fibril formation, numerous in vitro and in vivo studies, including model and pathologically relevant proteins, have been performed. Understanding the molecular details of these processes is of major importance to understand neurodegenerative diseases and could contribute to more effective therapies. Many models have been proposed to describe the mechanism by which proteins undergo ordered aggregation into amyloid fibrils. We classify these as: (a) templating and nucleation; (b) linear, colloid‐like assembly of spherical oligomers; and (c) domain‐swapping. In this review, we stress the role of domain‐swapping and discuss the role of proline switches.

Amyloid fibril formation by human stefins: Structure, mechanism & putative functions.

Many questions in the field of protein aggregation to amyloid fibrils remain open. In this review we describe predominantly in vitro studies of oligomerization and amyloid fibril formation by human stefins A and B. In human stefin B amyloidogenesis in vitro we have observed some general and many specific properties of its prefibrillar oligomers and amyloid fibrils. One characteristic feature in common to stefins and cystatins (and possibly some other amyloid proteins) is domain-swapping. In addition to solution structure of the domain-swapped dimer of stefin A, we recently have determined 3D structure of stefin B tetramer, which proved to be composed from two domain-swapped dimers, whose interaction occurs by a proline switch in the loop surrounding the conserved Pro 74. Studying the mechanism of fibril formation by stefin B, we found that the nucleation and fibril elongation reactions have energies of activation (E(a)s) in the range of proline isomerisation, strongly indicating importance of the Pro at site 74 and/or other prolines in the sequence. Correlation between toxicity of the prefibrillar oligomers and their interaction with acidic phospholipids was demonstrated. Stefin B was shown to interact with amyloid-beta peptide of Alzheimers disease in an oligomer specific manner, both in vitro and in the cells. It also has been shown that endogenous stefin B (with E at site 31) but especially the EPM1 mutant R68X and Y31-stefin B variant, and to a lesser extent EPM1 mutant G4R, are prone to form aggregates in cells.

Modulation of Contact Order Effects in the Two-State Folding of Stefins A and B

It is well established that contact order and folding rates are correlated for small proteins. The folding rates of stefins A and B differ by nearly two orders of magnitude despite sharing an identical native fold and hence contact order. We break down the determinants of this behavior and demonstrate that the modulation of contact order effects can be accounted for by the combined contributions of a framework-like mechanism, characterized by intrinsic helix stabilities, together with nonnative helical backbone conformation and nonnative hydrophobic interactions within the folding transition state. These contributions result in the formation of nonnative interactions in the transition state as evidenced by the opposing effects on folding rate and stability of these proteins.

A Method for the Reversible Trapping of Proteins in Non-Native Conformations

High-dilution equilibrium macrocyclization is developed as a general approach to trapping proteins in a non-native state with a synthetic cross-linking agent. The approach is illustrated using the N-terminal domain of phosphoglycerate kinase and a synthetic reagent containing two maleimide groups, for selective attachment to cysteines introduced onto the protein surface through mutagenesis, and an aromatic disulfide that can be chemically or photochemically cleaved. Following functionalization of the cysteine residues, thiol-disulfide exchange chemistry under strongly unfolding conditions was used to achieve intramolecular cyclization and a high yield of the cross-linked protein. (1)H NMR, CD, and fluorescence spectroscopies indicate that the conformation of the cross-linked protein is non-native. Chemical cleavage of the aromatic disulfide cross-link by a reducing agent results in the acquisition of a nativelike conformation for the reduced protein. Thus, the cross-link acts as a reversible switch of protein folding.

The role of initial oligomers in amyloid fibril formation by human stefin B.

Oligomers are commonly observed intermediates at the initial stages of amyloid fibril formation. They are toxic to neurons and cause decrease in neural transmission and long-term potentiation. We describe an in vitro study of the initial steps in amyloid fibril formation by human stefin B, which proved to be a good model system. Due to relative stability of the initial oligomers of stefin B, electrospray ionization mass spectrometry (ESI MS) could be applied in addition to size exclusion chromatography (SEC). These two techniques enabled us to separate and detect distinguished oligomers from the monomers: dimers, trimers, tetramers, up to decamers. The amyloid fibril formation process was followed at different pH and temperatures, including such conditions where the process was slow enough to detect the initial oligomeric species at the very beginning of the lag phase and those at the end of the lag phase. Taking into account the results of the lower-order oligomers transformations early in the process, we were able to propose an improved model for the stefin B fibril formation.

Limited Proteolysis Reveals That Amyloids from the 3D Domain-Swapping Cystatin B Have a Non-Native β-Sheet Topology.

3D domain-swapping proteins form multimers by unfolding and then sharing of secondary structure elements, often with native-like interactions. Runaway domain swapping is proposed as a mechanism for folded proteins to form amyloid fibres, with examples including serpins and cystatins. Cystatin C amyloids cause a hereditary form of cerebral amyloid angiopathy whilst cystatin B aggregates are found in cases of Unverricht-Lundborg Syndrome, a progressive form of myoclonic epilepsy. Under conditions that favour fibrillisation, cystatins populate stable 3D domain-swapped dimers both in vitro and in vivo that represent intermediates on route to the formation of fibrils. Previous work on cystatin B amyloid fibrils revealed that the α-helical region of the protein becomes disordered and identified the conservation of a continuous 20-residue elongated β-strand (residues 39-58), the latter being a salient feature of the dimeric 3D domain-swapped structure. Here we apply limited proteolysis to cystatin B amyloid fibrils and show that not only the α-helical N-terminal of the protein (residues 1-35) but also the C-terminal of the protein (residues 80-98) can be removed without disturbing the underlying fibril structure. This observation is incompatible with previous models of cystatin amyloid fibrils where the β-sheet is assumed to retain its native antiparallel arrangement. We conclude that our data favour a more generic, at least partially parallel, arrangement for cystatin β-sheet structure in mature amyloids and propose a model that remains consistent with available data for amyloids from either cystatin B or cystatin C.

Mapping local structural perturbations in the native state of stefin B (cystatin B) under amyloid forming conditions

Unlike a number of amyloid-forming proteins, stefins, and in particular stefin B (cystatin B) form amyloids under conditions where the native state predominates. In order to trigger oligomerization processes, the stability of the protein needs to be compromised, favoring structural re-arrangement however, accelerating fibril formation is not a simple function of protein stability. We report here on how optimal conditions for amyloid formation lead to the destabilization of dimeric and tetrameric states of the protein in favor of the monomer. Small, highly localized structural changes can be mapped out that allow us to visualize directly areas of the protein which eventually become responsible for triggering amyloid formation. These regions of the protein overlap with the Cu (II)-binding sites which we identify here for the first time. We hypothesize that in vivo modulators of amyloid formation may act similarly to painstakingly optimized solvent conditions developed in vitro. We discuss these data in the light of current structural models of stefin B amyloid fibrils based on H-exchange data, where the detachment of the helical part and the extension of loops were observed.