Characterization of Oligomers of Heterogeneous Size as Precursors of Amyloid Fibril Nucleation of an SH3 Domain: An Experimental Kinetics Study

Understanding the earliest molecular events during nucleation of the amyloid aggregation cascade is of fundamental significance to prevent amyloid related disorders. We report here an experimental kinetic analysis of the amyloid aggregation of the N47A mutant of the α-spectrin SH3 domain (N47A Spc-SH3) under mild acid conditions, where it is governed by rapid formation of amyloid nuclei. The initial rates of formation of amyloid structures, monitored by thioflavine T fluorescence at different protein concentrations, agree quantitatively with high-order kinetics, suggesting an oligomerization pre-equilibrium preceding the rate-limiting step of amyloid nucleation. The curves of native state depletion also follow high-order irreversible kinetics. The analysis is consistent with the existence of low-populated and heterogeneous oligomeric precursors of fibrillation that form by association of partially unfolded protein monomers. An increase in NaCl concentration accelerates fibrillation but reduces the apparent order of the nucleation kinetics; and a double mutant (K43A, N47A) Spc-SH3 domain, largely unfolded under native conditions and prone to oligomerize, fibrillates with apparent first order kinetics. On the light of these observations, we propose a simple kinetic model for the nucleation event, in which the monomer conformational unfolding and the oligomerization of an amyloidogenic intermediate are rapidly pre-equilibrated. A conformational change of the polypeptide chains within any of the oligomers, irrespective of their size, is the rate-limiting step leading to the amyloid nuclei. This model is able to explain quantitatively the initial rates of aggregation and the observed variations in the apparent order of the kinetics and, more importantly, provides crucial thermodynamic magnitudes of the processes preceding the nucleation. This kinetic approach is simple to use and may be of general applicability to characterize the amyloidogenic intermediates and oligomeric precursors of other disease-related proteins.     

Evolutionary and structural constraints influencing apolipoprotein A-I amyloid behavior

Apolipoprotein A-I (apoA-I) has a key function in the reverse cholesterol transport. However, aggregation of apoA-I single point mutants can lead to hereditary amyloid pathology. Although several studies have tackled the biophysical and structural consequences introduced by these mutations, there is little information addressing the relationship between the evolutionary and structural features that contribute to the amyloid behavior of apoA-I. We combined evolutionary studies, in silico mutagenesis and molecular dynamics (MD) simulations to provide a comprehensive analysis of the conservation and pathogenic role of the aggregation-prone regions (APRs) present in apoA-I. Sequence analysis demonstrated that among the four amyloidogenic regions described for human apoA-I, only two (APR1 and APR4) are evolutionary conserved across different species of Sarcopterygii. Moreover, stability analysis carried out with the FoldX engine showed that APR1 contributes to the marginal stability of apoA-I. Structural properties of full-length apoA-I models suggest that aggregation is avoided by placing APRs into highly packed and rigid portions of its native fold. Compared to silent variants extracted from the gnomAD database, the thermodynamic and pathogenic impact of amyloid mutations showed evidence of a higher destabilizing effect. MD simulations of the amyloid variant G26R evidenced the partial unfolding of the alpha-helix bundle with the concomitant exposure of APR1 to the solvent, suggesting an insight into the early steps involved in its aggregation. Our findings highlight APR1 as a relevant component for apoA-I structural integrity and emphasize a destabilizing effect of amyloid variants that leads to the exposure of this region.     

Immunoglobulin kappa light chain and its amyloidogenic mutants: a molecular dynamics study

AL amyloidosis and LCDD are pathological conditions caused by extracellural deposition of monoclonal Ig light chain variable domains. In the former case, deposits have a form of amyloid fibrils, in the latter, amorphous aggregates. 1REI kappa light chain variable domain and its two point mutants, R61N and D82I, were chosen for the analysis in this work. Wild 1REI does not create deposits in vitro, while R61N aggregates as amyloid fibrils and D82I creates amorphous aggregates. Both mutated residues create a conserved salt bridge; thus, substitution of any of them should decrease V(L) domain stability. For these three proteins, 5 ns MD simulations were conducted in temperatures of 300 K and 400 K, with protonated and unprotonated acidic residues, mimicking acidic and neutral experimental pH conditions (3 sets: N300, N400, and A400). The analysis of trajectories focused on characterization of changes in conformational behavior and stability of Ig kappa light chain variable domain caused by single aminoacid substitutions that were experimentally proved to enhance aggregation propensity, both in the form of amyloid and amorphous aggregates. Residue D82 turns out to be involved not only in R61-D82 but also in K45-D82 interaction, which was not observed in the X-ray structure, but frequently populated simulations of 1REI. The substitution D82I excludes both interactions, resulting in substantial destabilization (i.e., easier aggregation). Examination of behavior of edge regions of V(L) beta-sandwich reveals significant alterations in D82I mutant compared to wild 1REI, while relatively small changes occur in R61N. This suggests that mild and slow destabilization is the reason of the conversion of V(L) to partially folded amyloidogenic intermediate structure.     

A single mutation induces amyloid aggregation in the alpha-spectrin SH3 domain: analysis of the early stages of fibril formation

The Src-homology region 3 domain of chicken alpha-spectrin (Spc-SH3) is a small two-state folding protein, which has never been described to form amyloid fibrils under any condition investigated so far. We show here that the mutation of asparagine 47 to alanine at the distal loop, which destabilises similarly the native and folding transition states of the domain, induces the formation of amyloid fibrils under mild acid conditions. Amyloid aggregation of the mutant is enhanced by the increase in temperature, protein concentration and NaCl concentration. The early stages of amyloid formation have been monitored as a function of time and temperature using a variety of biophysical methods. Differential scanning calorimetry experiments under conditions of amyloid formation have allowed the identification of different thermal transitions corresponding to conformational and aggregation processes as well as to the high-temperature disaggregation and unfolding of the amyloid fibrils. Aggregation is preceded by a rapid conformational change in the monomeric domain involving about 40% of the global unfolding enthalpy, considerable change in secondary structure, large loss of tertiary structure and exposure of hydrophobic patches to the solvent. The conformational change is followed by formation of a majority of oligomeric species with apparent hydrodynamic radius between 2.5 nm and 10nm, depending on temperature, together with the appearance and progressive growth of protofibrillar aggregates. After these early aggregation stages, long and curved fibrils of up to several micrometers start to develop by elongation of the protofibrils. The calorimetric data indicate that the specific enthalpy of fibril disaggregation and unfolding is relatively low, suggesting a low density of interactions within the fibril structure as compared to the native protein and a main entropy contribution to the stability of the amyloid fibrils.     

Heparin‐induced amyloid fibrillation of β2‐microglobulin explained by solubility and a supersaturation‐dependent conformational phase diagram

Amyloid fibrils are fibrillar deposits of denatured proteins associated with amyloidosis and are formed by a nucleation and growth mechanism. We revisited an alternative and classical view of amyloid fibrillation: amyloid fibrils are crystal‐like precipitates of denatured proteins formed above solubility upon breaking supersaturation. Various additives accelerate and then inhibit amyloid fibrillation in a concentration‐dependent manner, suggesting that the combined effects of stabilizing and destabilizing forces affect fibrillation. Heparin, a glycosaminoglycan and anticoagulant, is an accelerator of fibrillation for various amyloidogenic proteins. By using β₂‐microglobulin, a protein responsible for dialysis‐related amyloidosis, we herein examined the effects of various concentrations of heparin on fibrillation at pH 2. In contrast to previous studies that focused on accelerating effects, higher concentrations of heparin inhibited fibrillation, and this was accompanied by amorphous aggregation. The two‐step effects of acceleration and inhibition were similar to those observed for various salts. The results indicate that the anion effects caused by sulfate groups are one of the dominant factors influencing heparin‐dependent fibrillation, although the exact structures of fibrils and amorphous aggregates might differ between those formed by simple salts and matrix‐forming heparin. We propose that a conformational phase diagram, accommodating crystal‐like amyloid fibrils and glass‐like amorphous aggregates, is important for understanding the effects of various additives.     

Elongation of the C-terminal domain of an anti-amyloid β single-chain variable fragment increases its thermodynamic stability and decreases its aggregation tendency

Amyloid β (Aβ) immunotherapy is considered a promising approach to Alzheimer disease treatment. In contrast to the use of complete antibodies, administration of single-chain variable fragments (scFv) has not been associated with either meningoencephalitis or cerebral hemorrhage. ScFv-h3D6 is known to preclude cytotoxicity of the Aβ_1–42 peptide by removing its oligomers from the amyloid pathway. As is the case for other scFv molecules, the recombinant production of scFv-h3D6 is limited by its folding and stability properties. Here, we show that its urea-induced unfolding pathway is characterized by the presence of an intermediate state composed of the unfolded V_L domain and the folded V_H domain, which suggests the V_L domain as a target for thermodynamic stability redesign. The modeling of the 3D structure revealed that the V_L domain, located at the C-terminal of the molecule, was ending before its latest β-strand was completed. Three elongation mutants, beyond V_L-K107, showed increased thermodynamic stability and lower aggregation tendency, as determined from urea denaturation experiments and Fourier-transform infrared spectroscopy, respectively. Because the mutants maintained the capability of removing Aβ-oligomers from the amyloid pathway, we expect these traits to increase the half-life of scFv-h3D6 in vivo and, consequently, to decrease the effective doses. Our results led to the improvement of a potential Alzheimer disease treatment and may be extrapolated to other class-I scFv molecules of therapeutic interest.     

Amino-terminal domain stability mediates apolipoprotein E aggregation into neurotoxic fibrils

The three isoforms of apolipoprotein (apo) E are strongly associated with different risks for Alzheimer's disease: apoE4>apoE3>apoE2. Here, we show at physiological salt concentrations and pH that native tetramers of apoE form soluble aggregates in vitro that bind the amyloid dyes thioflavin T and Congo red. However, unlike classic amyloid fibrils, the aggregates adopt an irregular protofilament-like morphology and are seemingly highly alpha-helical. The aggregates formed at substantially different rates (apoE4>apoE3>apoE2) and were significantly more toxic to cultured neuronal cells than the tetramer. Since the three isoforms have large differences in conformational stability that can influence aggregation and amyloid pathways, we tested the effects of mutations that increased or decreased stability. Decreasing the conformational stability of the amino-terminal domain of apoE increased aggregation rates and vice versa. Our findings provide a new perspective for an isoform-specific pathogenic role for apoE aggregation in which differences in the conformational stability of the amino-terminal domain mediate neurodegeneration.     

Structural characterization of the partially folded intermediates of an immunoglobulin light chain leading to amyloid fibrillation and amorphous aggregation

Immunoglobulin light chain deposition diseases involve various types of extracellular deposition of light chain variable domains, including amyloid fibrils and amorphous deposits. The decreased thermodynamic stability of the light chain is believed to be the major factor leading to fibrillation. However, the differences in the nature of the deposits among the light chain deposition diseases raise the question of whether the mechanisms leading to fibrillar or amorphous aggregation is different. In this study, we generated two partially folded intermediates of the light chain variable domain SMA in the presence of guanidine hydrochloride (GuHCl) and characterized their conformations. The more unfolded intermediate formed fibrils most rapidly, while the more native-like intermediate predominantly led to amorphous deposits. The results also show that the monomeric, rather than the dimeric state, was critical for fibrillation. The data also indicate that fibril elongation involves addition of a partially unfolded intermediate, rather than the native state. We postulate that a more highly unfolded intermediate is more suited to undergo the topological rearrangements necessary to form amyloid fibrils than a more structured one and that this also correlates with increased destabilization. In the case of light chain aggregation, it appears that more native-like intermediate conformations are more prone to form amorphous deposits.     

Characterization of the structure and the amyloidogenic properties of the Josephin domain of the polyglutamine-containing protein ataxin-3

Expansion of the polyglutamine (polyQ) region in the protein ataxin-3 is associated with spinocerebellar ataxia type 3, an inherited neurodegenerative disorder that belongs to the family of polyQ diseases. Increasing evidence indicates that protein aggregation and fibre formation play an important role in these pathologies. In a previous study, we determined the domain architecture of ataxin-3, suggesting that it comprises a globular domain, named Josephin, and a more flexible C-terminal region, that includes the polyQ tract. Here, we have characterised for the first time the biophysical properties of the isolated Josephin motif, showing that it is an autonomously folded unit and that it has no significant interactions with the C-terminal region. Study of its thermodynamic stability indicates that Josephin has an intrinsic tendency to aggregate and forms temperature-induced fibrils similar to those described for expanded ataxin-3. We show that, under destabilising conditions, the behaviours of the isolated Josephin domain and ataxin-3 are extremely similar. Our data therefore strongly suggest that the stability and aggregation properties of non-expanded ataxin-3 are determined by those of the Josephin domain, which is sufficient to reproduce the behaviour of the full-length protein. Our data support a mechanism in which the thermodynamic stability of ataxin-3 is governed by the properties of the Josephin domain, but the presence of an expanded polyQ tract increases dramatically the protein's tendency to aggregate.     

Deamidation destabilizes and triggers aggregation of a lens protein, betaA3-crystallin

Protein aggregation is a hallmark of several neurodegenerative diseases and also of cataracts. The major proteins in the lens of the eye are crystallins, which accumulate throughout life and are extensively modified. Deamidation is the major modification in the lens during aging and cataracts. Among the crystallins, the betaA3-subunit has been found to have multiple sites of deamidation associated with the insoluble proteins in vivo. Several sites were predicted to be exposed on the surface of betaA3 and were investigated in this study. Deamidation was mimicked by site-directed mutagenesis at Q42 and N54 on the N-terminal domain, N133 and N155 on the C-terminal domain, and N120 in the peptide connecting the domains. Deamidation altered the tertiary structure without disrupting the secondary structure or the dimer formation of betaA3. Deamidations in the C-terminal domain and in the connecting peptide decreased stability to a greater extent than deamidations in the N-terminal domain. Deamidation at N54 and N155 also disrupted the association with the betaB1-subunit. Sedimentation velocity experiments integrated with high-resolution analysis detected soluble aggregates at 15%-20% in all deamidated proteins, but not in wild-type betaA3. These aggregates had elevated frictional ratios, suggesting that they were elongated. The detection of aggregates in vitro strongly suggests that deamidation may contribute to protein aggregation in the lens. A potential mechanism may include decreased stability and/or altered interactions with other beta-subunits. Understanding the role of deamidation in the long-lived crystallins has important implications in other aggregation diseases.     

Relation between protein stability, evolution and structure, as probed by carboxylic acid mutations

Native proteins are marginally stable. Low thermodynamic stability may actually be advantageous, although the accumulation of neutral, destabilizing mutations may have also contributed to it. In any case, once marginal stability has been reached, it appears plausible that mutations at non-constrained positions become fixed in the course of evolution (due to random drift) with frequencies that roughly reflect the mutation effects on stability ("pseudo-equilibrium hypothesis"). We have found that all glutamate-->aspartate mutations in wild-type Escherichia coli thioredoxin are destabilizing, as well as most of the aspartate-->glutamate mutations. Furthermore, the effect of these mutations on thioredoxin thermodynamic stability shows a robust correlation with the frequencies of occurrence of the involved residues in several-hundred sequence alignments derived from a BLAST search. These results provide direct and quantitative experimental evidence for the pseudo-equilibrium hypothesis and should have general consequences for the interpretation of mutation effects on protein stability, as they suggest that residue environments in proteins may be optimized for stabilizing interactions to a remarkable degree of specificity. We also provide evidence that such stabilizing interactions may be detected in sequence alignments, and briefly discuss the implications of this possibility for the derivation of structural information (on native and denatured states) from comparative sequence analyses.     

Understanding the contribution of disulfide bridges to the folding and misfolding of an anti‐Aβ scFv

ScFv‐h3D6 is a single chain variable fragment that precludes Aβ peptide‐induced cytotoxicity by withdrawing Aβ oligomers from the amyloid pathway to the worm‐like pathway. Production of scFv molecules is not a straightforward procedure because of the occurrence of disulfide scrambled conformations generated in the refolding process. Here, we separately removed the disulfide bond of each domain and solved the scrambling problem; and then, we intended to compensate the loss of thermodynamic stability by adding three C‐terminal elongation mutations, previously described to stabilize the native fold of scFv‐h3D6. Such stabilization occurred through stabilization of the intermediate state in the folding pathway and destabilization of a different, β‐rich, intermediate state driving to worm‐like fibrils. Elimination of the disulfide bridge of the less stable domain, V_L, deeply compromised the yield and increased the aggregation tendency, but elimination of the disulfide bridge of the more stable domain, V_H, solved the scrambling problem and doubled the production yield. Notably, it also changed the aggregation pathway from the protective worm‐like morphology to an amyloid one. This was so because a partially unfolded intermediate driving to amyloid aggregation was present, instead of the β‐rich intermediate driving to worm‐like fibrils. When combining with the elongation mutants, stabilization of the partially unfolded intermediate driving to amyloid fibrils was the only effect observed. Therefore, the same mutations drove to completely different scenarios depending on the presence of disulfide bridges and this illustrates the relevance of such linkages in the stability of different intermediate states for folding and misfolding.     

Vortex-Induced Formation of Insulin Amyloid Superstructures Probed by Time-Lapse Atomic Force Microscopy and Circular Dichroism Spectroscopy

Misfolding and aggregation of proteins are multipathway processes that result in polymorphism of amyloid fibrils. While agitation is one of the most common means of inducing structural variants of fibrils (the so-called ‘amyloid strains’), there is as yet no mechanistic explanation for this effect. In this study, time-lapse atomic force microscopy has been employed to probe insulin fibrillation upon intensive agitation. At 60 °C, the initial stages of aggregation in agitated samples are similar to those in quiescent solutions; however, in vortexed samples, an abrupt and highly cooperative collapse of early filaments occurs, yielding twisted and laterally aligned aggregates with defined chiroptical properties. In the absence of any detectable birefringence and linear dichroism, the observed strong Cotton effect is attributed to twisted chiral amyloid superstructures. Early fibrils formed in agitated samples, but transferred to quiescent conditions before the collapse event, do not form the superstructures. On the other hand, mature insulin fibrils grown in quiescent samples and later subjected to rapid vortexing transform into clumps of finely broken fibers lacking the superstructural chirality and chiroptical properties of the continuously agitated samples. A generalized mechanism of inducing structural variants of amyloid fibrils by hydrodynamic forces favoring secondary nucleation events over elongation of fibrils is put forward. We propose that competition between low-aspect-ratio and high-aspect-ratio amyloidogenic pathways driven by fluid dynamics may play an important role in promoting distinct amyloid strains.     

The effect of β2-α2 loop mutation on amyloidogenic properties of the prion protein

Recent studies revealed that elk-like S170N/N174T mutation in mouse prion protein (moPrP), which results in an increased rigidity of β2-α2 loop, leads to a prion disease in transgenic mice. Here we characterized the effect of this mutation on biophysical properties of moPrP. Despite similar thermodynamic stabilities of wild type and mutant proteins, the latter was found to have markedly higher propensity to form amyloid fibrils. Importantly, this effect was observed even under fully denaturing conditions, indicating that the increased conversion propensity of the mutant protein is not due to loop rigidity but rather results from greater amyloidogenic potential of the amino acid sequence within the loop region of S170N/N174T moPrP.     

Mechanism of rescue of common p53 cancer mutations by second-site suppressor mutations

The core domain of p53 is extremely susceptible to mutations that lead to loss of function. We analysed the stability and DNA-binding activity of such mutants to understand the mechanism of second-site suppressor mutations. Double-mutant cycles show that N239Y and N268D act as 'global stability' suppressors by increasing the stability of the cancer mutants G245S and V143A-the free energy changes are additive. Conversely, the suppressor H168R is specific for the R249S mutation: despite destabilizing wild type, H168R has virtually no effect on the stability of R249S, but restores its binding affinity for the gadd45 promoter. NMR structural comparisons of R249S/H168R and R249S/T123A/H168R with wild type and R249S show that H168R reverts some of the structural changes induced by R249S. These results have implications for possible drug therapy to restore the function of tumorigenic mutants of p53: the function of mutants such as V143A and G245S is theoretically possible to restore by small molecules that simply bind to and hence stabilize the native structure, whereas R249S requires alteration of its mutant native structure.     

Stabilization of an immunoglobulin fold domain by an engineered disulfide bond at the buried hydrophobic region

We report for the first time the stabilization of an immunoglobulin fold domain by an engineered disulfide bond. In the llama single-domain antibody, which has human chorionic gonadotropin as its specific antigen, Ala49 and Ile70 are buried in the structure. A mutant with an artificial disulfide bond at this position showed a 10 degrees C higher midpoint temperature of thermal unfolding than that without the extra disulfide bond. The modified domains exhibited an antigen binding affinity comparable with that of the wild-type domain. Ala49 and Ile70 are conserved in camel and llama single-domain antibody frameworks. Therefore, domains against different antigens are expected to be stabilized by the engineered disulfide bond examined here. In addition to the effect of the loop constraints in the unfolded state, thermodynamic analysis indicated that internal interaction and hydration also control the stability of domains with disulfide bonds. The change in physical properties resulting from mutation often causes unpredictable and destabilizing effects on these interactions. The introduction of a hydrophobic cystine into the hydrophobic region maintains the hydrophobicity of the protein and is expected to minimize the unfavorable mutational effects.     

High-affinity fragment complementation of a fibronectin type III domain and its application to stability enhancement

The tenth fibronectin type III (FN3) domain of human fibronectin (FNfn10), a prototype of the ubiquitous FN3 domain, is a small, monomeric beta-sandwich protein. In this study, we have bisected FNfn10 in each loop to generate a total of six fragment pairs. We found that fragment pairs bisected at multiple loops of FNfn10 show complementation in vivo as tested with a yeast two-hybrid system. The dissociation constant of these fragment pairs determined in vitro were as low as 3 nM, resulting in one of the tightest fragment complementation systems reported so far. Furthermore, we show that the affinity of fragment complementation is correlated with the stability of the uncut parent protein. Exploring this correlation, we screened a yeast two-hybrid library of one fragment and identified mutations that suppress the effect of a destabilizing mutation in the other fragment. One of the identified mutations significantly increased the stability of the uncut wild-type protein, proving that fragment complementation can be used as a novel strategy for the selection of proteins with enhanced stability.     

Expanding the Repertoire of Amyloid Polymorphs by Co-polymerization of Related Protein Precursors

Amyloid fibrils can be generated from proteins with diverse sequences and folds. Although amyloid fibrils assembled in vitro commonly involve a single protein precursor, fibrils formed in vivo can contain more than one protein sequence. How fibril structure and stability differ in fibrils composed of single proteins (homopolymeric fibrils) from those generated by co-polymerization of more than one protein sequence (heteropolymeric fibrils) is poorly understood. Here we compare the structure and stability of homo and heteropolymeric fibrils formed from human β₂-microglobulin and its truncated variant ΔN6. We use an array of approaches (limited proteolysis, magic angle spinning NMR, Fourier transform infrared spectroscopy, and fluorescence) combined with measurements of thermodynamic stability to characterize the different fibril types. The results reveal fibrils with different structural properties, different side-chain packing, and strikingly different stabilities. These findings demonstrate how co-polymerization of related precursor sequences can expand the repertoire of structural and thermodynamic polymorphism in amyloid fibrils to an extent that is greater than that obtained by polymerization of a single precursor alone.