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.     

Conformational change, aggregation and fibril formation induced by detergent treatments of cellular prion protein

The conversion of protease-sensitive prion protein (PrP-sen) to a high beta-sheet, protease-resistant and often fibrillar form (PrP-res) is a central event in transmissible spongiform encephalopathies (TSE) or prion diseases. This conversion can be induced by PrP-res itself in cell-free conversion reactions. The detergent sodium N-lauroyl sarkosinate (sarkosyl) is a detergent that is widely used in PrP-res purifications and is known to stimulate the PrP-res-induced conversion reaction. Here we report effects of sarkosyl and other detergents on recombinant hamster PrP-sen purified from mammalian cells under oxidizing conditions that maintain the single native disulfide bond. Low concentrations of sarkosyl (0.001-0.1%) induced aggregation of PrP-sen molecules, increased light scattering, altered fluorescence excitation and emission spectra, and enhanced the proportion of beta-sheet secondary structure according to circular dichroism and infrared spectroscopies. An enhancement of beta-sheet content was also seen with 0.001% sodium dodecyl sulfate (SDS) but not several other types of detergents. Electron microscopy revealed that sarkosyl induced the formation of both amorphous and fibrillar aggregates. The fibrils appeared to be constructed from spherical bead-like protofibrils. Neither TSE infectivity nor the characteristic partial proteinase K resistance of PrP-res was detected in the sarkosyl-induced PrP aggregates. We conclude that certain anionic detergents can disrupt the conformation of PrP-sen and induce high beta-sheet aggregates that are distinct from scrapie-associated PrP-res in terms of protease-resistance, infrared spectrum and infectivity. These results reinforce the idea that not all high-beta aggregates of PrP are equivalent to the pathologic form, PrP-res.     

Probing the mechanism of amyloidogenesis through a tandem repeat of the PI3-SH3 domain suggests a generic model for protein aggregation and fibril formation

Aggregation of the SH3 domain of the PI3 kinase, both as a single domain and as a tandem repeat in which the C terminus of one domain is linked to the N terminus of another by a flexible linker of ten glycine/serine residues, has been studied under a range of conditions in order to investigate the mechanism of protein aggregation and amyloid formation. The tandem repeat was found to form amyloid fibrils much more readily than the single domain under the acidic conditions used here, and the fibrils themselves have higher morphological homogeneity. The folding-unfolding transition of the PI3-SH3 domain shows two-state behaviour and is pH dependent; at pH 3.6, which is near the pH mid-point for folding and only slightly below the isoelectric point of the protein, both the single domain and the tandem repeat spontaneously form broad distributions of soluble oligomers without requirement for nucleation. Under prolonged incubation under these conditions, the oligomers convert into thin, curly fibrils that interact with thioflavin-T, suggesting that they contain an organised beta-sheet structure. Under more acidic conditions (pH 2.0) where the proteins are fully denatured and carry a positive net charge, long, straight fibrils are formed in a process having a pronounced lag phase. The latter was found to be reduced dramatically by the addition of oligomers exceeding a critical size of approximately 20 molecules. The results suggest that the process of aggregation of these SH3 domains can take place by a variety of mechanisms, ranging from downhill formation of relatively amorphous species to nucleated formation of highly organised structures, the relative importance of which varies greatly with solution conditions. Comparison with the behaviour of other amyloidogenic systems suggests that the general mechanistic features outlined here are likely to be common to at least a wide variety of peptides and proteins.     

Crystallographic structure of the SH3 domain of the human c-Yes tyrosine kinase: loop flexibility and amyloid aggregation

SH3 domains from the Src family of tyrosine kinases represent an interesting example of the delicate balance between promiscuity and specificity characteristic of proline-rich ligand recognition by SH3 domains. The development of inhibitors of therapeutic potential requires a good understanding of the molecular determinants of binding affinity and specificity and relies on the availability of high quality structural information. Here, we present the first high-resolution crystal structure of the SH3 domain of the c-Yes oncogen. Comparison with other SH3 domains from the Src family revealed significant deviations in the loop regions. In particular, the n-Src loop, highly flexible and partially disordered, is stabilized in an unusual conformation by the establishment of several intramolecular hydrogen bonds. Additionally, we present here the first report of amyloid aggregation by an SH3 domain from the Src family.     

Controlling amyloid fibril formation by partial stirring

Many proteins undergoe self‐assembly into fibrillar structures known as amyloid fibrils. During the self‐assembly process, related structures known as spherulites can be formed. Herein we report a facile method where the balance between amyloid fibrils and spherulites can be controlled by stirring of the reaction mixture during the initial stages of the self‐assembly process. Moreover, we report how this methodology can be used to prepare non‐covalently functionalized amyloid fibrils. By stirring the reaction mixture continuously or for a limited time during the lag phase, the fibril length, and hence the propensity to form liquid crystalline phases, can be influenced. This phenomena is utilized in order to prepare films consisting of aligned protein fibrils incorporating the laser dye Nile red. The resulting films display polarized Nile red fluorescence. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 249–259, 2016.     

Partly folded states of members of the lysozyme/lactalbumin superfamily: a comparative study by circular dichroism spectroscopy and limited proteolysis

The partly folded states of protein members of the lysozyme (LYS)/alpha-lactalbumin (LA) superfamily have been analyzed by circular dichroism (CD) measurements and limited proteolysis experiments. Hen, horse, dog, and pigeon LYSs and bovine LA were used in the present study. These are related proteins of 123- to 129-amino-acid residues with similar three-dimensional structures but low similarity in amino acid sequences. Moreover, notable differences among them reside in their calcium-binding properties and capability to adopt partly folded states or molten globules in acid solution (A-state) or on depletion of calcium at neutral pH (apo-state). Far- and near-UV CD measurements revealed that although the structures of hen and dog LYS are rather stable in acid at pH 2.0 or at neutral pH in the absence of calcium, conformational transitions to various extents occur with all other LYS/LA proteins herewith investigated. The most significant perturbation of tertiary structure in acid was observed with bovine LA and LYS from horse milk and pigeon egg-white. Pepsin and proteinase K were used as proteolytic probes, because these proteases show broad substrate specificity, and therefore, their sites of proteolysis are dictated not by the specific amino acid sequence of the protein substrate but by its overall structure and dynamics. Although hen LYS at pH 2.0 was fully resistant to proteolysis by pepsin, the other members of the LYS/LA superfamily were cleaved at different rates at few sites of the polypeptide chain and thus producing rather large protein fragments. The apo-form of bovine LA, horse LYS, and pigeon LYS were attacked by proteinase K at pH 8.3, whereas dog and hen LYSs were resistant to proteolysis when reacted under identical experimental conditions. Briefly, it has been found that the proteolysis data correlate well with the extent of conformational transitions inferred from CD spectra and with existing structural informations regarding the proteins herewith investigated, mainly derived from NMR and hydrogen exchange measurements. The sites of initial proteolytic cleavages in the LYS variants occur at the level of the beta-subdomain (approximately chain region 34-57), in analogy to those observed with bovine LA. Proteolysis data are in agreement with the current view that the molten globule of the LYS/LA proteins is characterized by a structured alpha-domain and a largely disrupted beta-subdomain. Our results underscore the utility of the limited proteolysis approach for analyzing structure and dynamics of proteins, even if adopting an ensemble of dynamic states as in the molten globule.     

Protein engineering as a strategy to avoid formation of amyloid fibrils

The activation domain of human procarboxypeptidase A2 (ADA2h) aggregates following thermal or chemical denaturation at acidic pH. The aggregated material contains well-defined ordered structures with all the characteristics of the fibrils associated with amyloidotic diseases. Variants of ADA2h containing a series of mutations designed to increase the local stability of each of the two helical regions of the protein have been found to have a substantially reduced propensity to form fibrils. This arises from a reduced tendency of the denatured species to aggregate rather than from a change in the overall stability of the native state. The reduction in aggregation propensity may result from an increase in the stability of local relative to longer range interactions within the polypeptide chain. These findings show that the intrinsic ability of a protein to form amyloid can be altered substantially by protein engineering methods without perturbing significantly its overall stability or activity. This suggests new strategies for combating diseases associated with the formation of aggregated proteins and for the design of novel protein or peptide therapeutics.     

Stability and folding of the SH3 domain of bruton's tyrosine kinase

Bruton's tyrosine kinase (BTK) plays an important role in B cell development. Deletion of C-terminal 14 amino acids of the SH3 domain of BTK results in X-linked agammaglobulinemia (XLA), an inherited disease. We report here on the stability and folding of SH3 domain of BTK. Peptides corresponding to residues 216–273 (58 residues) and 216–259 (44 residues) of BTK SH3 domain were synthesized by solid phase methods; the first peptide constitutes the entire SH3 domain of BTK while the latter peptide lacks 14 amino acid residues of the C-terminal. The 58 amino acid peptide forms mainly a β-barrel type folding unit. Although small and lacking disulfide bonds, this peptide is extremely stable to thermal denaturation. Based on circular dichroism measurements, its melting temperature was found to be high, 82°C at pH 6.0. However, the Gibbs free energy (ΔG) of the intrinsic stability and thermodynamic spontaneity of unfolding were found to be low, 2.6 kcal/mol by Gdn·HCl denaturation experiments, as compared to 12 kcal/mol obtained for larger single domain proteins, indicating poor stability of SH3 domain. Addition of 500 mM of Na₂SO₄ increased the free energy change ΔG to 4.0 kcal/mol, suggesting an ionic strength effect. The truncated peptide fails to fold correctly and adopts random coil conformation in contrast to 58 amino acid β-barrel peptide, which exhibits high thermal stability but normal or low stability at ambient temperature. These results, to our knowledge the first to delineate the importance of C-terminal in structural integrity of SH3 domains, indicate also that improper folding and/or poor stability of mutant SH3 domain in BTK likely causes XLA. Proteins 28:465–471 © 1996 Wiley-Liss, Inc.     

Interaction between Btk TH and SH3 domain

Several mechanisms are involved in the regulation of cellular signaling. Bruton tyrosine kinase (Btk) of the Tec family contains in the Tec homology (TH) domain a proline-rich region (PRR) capable of interacting with several SH3 domains. The Btk has the SH3 domain adjacent to the TH domain. CD and fluorescence spectroscopy were used to study the binding of two peptides corresponding to segments in the PRR to the Btk SH3 domain. The peptide for the N-terminal half of the PRR binds specifically, whereas the other peptide had hardly any affinity. The TH domain has about four times lower affinity to the SH3 domain than the peptide, 17.0 vs 3.9 microM. The interaction was further tested with an SH3 domain construct that contained the PRR. The two peptides cannot compete for the binding to the extended protein and the TH domain has two times lower affinity to the extended SH3 domain. The intra- or intermolecular interaction between the TH and SH3 domain might have regulatory function also in the other Tec family members.     

Insights into the origin of the tendency of the PI3-SH3 domain to form amyloid fibrils

The SH3 domain of the p85alpha subunit of phosphatidylinositol 3 kinase has been found to form amyloid fibrils in vitro under acidic conditions. PI3-SH3 is peculiar due to a large insertion of 15 amino acid residues in the n-Src loop when compared with more canonical members of the family. Spectrin-SH3 (SPC-SH3) with a shorter loop does not form fibrils under any of our conditions tested. Thus, it could be that the longer loop could play a role in amyloid formation. To investigate this we have engineered two chimeras containing the common core of the PI3-SH3 and SPC-SH3 with an exchanged n-Src loop. Thermodynamic and kinetic analyses show that the two chimeras are less stable than the parent proteins, but useful for our comparative purposes they have similar stability. Neither stability, nor folding rates, or pH transition can be invoked as being responsible for the amyloid formation in the PI3-SH3 domain. Substitution of the long n-Src loop in PI3-SH3 by that of SPC-SH3 does not prevent fibril formation. The SPC-SH3 with the PI3-SH3 n-Src loop is in an A-state at low pH and forms beta-sheet amorphous aggregates, but not amyloid fibrils. Thus, we conclude that, for a protein to form ordered fibrils, a delicate balance between solubility of non-native states to allow efficient nucleation and the formation of amorphous aggregates, must be achieved. It is the amino acid residue sequence of the protein and probably its parts that play a determinant role in shifting this balance in one direction or the other.     

Protein stabilization by specific binding of guanidinium to a functional arginine-binding surface on an SH3 domain

Guanidinium hydrochloride (GuHCl) at low concentrations significantly stabilizes the Fyn SH3 domain. In this work, we have demonstrated that this stabilizing effect is manifested through a dramatic (five- to sixfold) decrease in the unfolding rate of the domain with the folding rate being affected minimally. This behavior contrasts to the effect of NaCl, which stabilizes this domain by accelerating the folding rate. These data imply that the stabilizing effect of GuHCl is not predominantly ionic in nature. Through NMR studies, we have identified a specific binding site for guanidinium, and we have determined a dissociation constant of 90 mM for this interaction. The guanidinium-binding site overlaps with a functionally important arginine-binding pocket on the domain surface, and we have shown that GuHCl is a specific inhibitor of the peptide-binding activity of the domain. A different SH3 domain possessing a similar arginine-binding pocket is also thermodynamically stabilized by GuHCl. These data suggest that many proteins that normally interact with arginine-containing ligands may also be able to specifically interact with guanidinium. Thus, some caution should be used when using GuHCl as a denaturant in protein folding studies. Since arginine-mediated interactions are often important in the energetics of protein-protein interactions, our observations could be relevant for the design of small molecule inhibitors of protein-protein interactions.     

The role of protein stability, solubility, and net charge in amyloid fibril formation

Ribonuclease Sa and two charge-reversal variants can be converted into amyloid in vitro by the addition of 2,2,2-triflouroethanol (TFE). We report here amyloid fibril formation for these proteins as a function of pH. The pH at maximal fibril formation correlates with the pH dependence of protein solubility, but not with stability, for these variants. Additionally, we show that the pH at maximal fibril formation for a number of well-characterized proteins is near the pI, where the protein is expected to be the least soluble. This suggests that protein solubility is an important determinant of fibril formation.     

Domain swapping and amyloid fibril conformation

For several different proteins an apparent correlation has been observed between the propensity for dimerization by domain-swapping and the ability to aggregate into amyloid-like fibrils. Examples include the disease-related proteins β₂-microglobulin and transthyretin. This has led to proposals that the amyloid-formation pathway may feature extensive domain swapping. One possible consequence of such an aggregation pathway is that the resulting fibrils would incorporate structural elements that resemble the domain-swapped forms of the protein and, thus, reflect certain native-like structures or domain-interactions. In magic angle spinning solid-state NMR-based and other structural studies of such amyloid fibrils, it appears that many of these proteins form fibrils that are not native-like. Several fibrils, instead, have an in-register, parallel conformation, which is a common amyloid structural motif and is seen, for instance, in various prion fibrils. Such a lack of native structure in the fibrils suggests that the apparent connection between domain-swapping ability and amyloid-formation may be more subtle or complex than may be presumed at first glance.     

Nucleation and the transition state of the SH3 domain

We present a verified computational model of the SH3 domain transition state (TS) ensemble. This model was built for three separate SH3 domains using experimental phi-values as structural constraints in all-atom protein folding simulations. While averaging over all conformations incorrectly considers non-TS conformations as transition states, quantifying structures as pre-TS, TS, and post-TS by measurement of their transmission coefficient ("probability to fold", or p(fold)) allows for rigorous conclusions regarding the structure of the folding nucleus and a full mechanistic analysis of the folding process. Through analysis of the TS, we observe a highly polarized nucleus in which many residues are solvent-exposed. Mechanistic analysis suggests the hydrophobic core forms largely after an early nucleation step. SH3 presents an ideal system for studying the nucleation-condensation mechanism and highlights the synergistic relationship between experiment and simulation in the study of protein folding.     

Conformational characterization of human angiogenin by limited proteolysis

The primary structure of angiogenin is 33% identical to that of bovine pancreatic ribonuclease (RNase), but the enzymatic activities of the two proteins differ markedly. Similarly, their susceptibilities to limited proteolysis differ as well. In contrast to RNase, angiogenin totally resists proteolysis by subtilisin. Indeed, among 16 proteases examined, only endoprotease Lys-C, trypsin, and pepsin are able to cleave angiogenin. Even with prolonged incubation, endoprotease Lys-C selectively cleaves the Lys-60-Asn-61 bond; the product retains full ribonucleolytic activity. Initially, trypsin also cleaves this same bond, but with time it causes extensive degradation. Pepsin, atpH 2, cleaves the Phe-9-Leu-10 bond, to give angiogenin (10–123), which displays 15% of the native activity toward ribosomal RNA (rRNA). The susceptibility to proteolysis and/or the sites of cleavage of angiogenin and bovine RNase differ markedly despite their structural homology. These differences are considered in terms of the amino acid sequences of the two proteins.     

Molten globule of bovine alpha-lactalbumin at neutral pH induced by heat,trifluoroethanol, and oleic acid: a comparative analysis by circular dichroism spectroscopy and limited proteolysis

The calcium-depleted form of alpha-lactalbumin (alpha-LA) at neutral pH can be induced to adopt a partly folded state or molten globule upon moderate heating, by dissolving the protein in aqueous TFE or by adding oleic acid. This last folding variant of the protein, named HAMLET, can induce apoptosis in tumor cells. The aim of the present work was to unravel from circular dichroism (CD) measurements and proteolysis experiments structural features of the molten globule of apo-alpha-LA at neutral pH. CD spectra revealed that the molten globule of apo-alpha-LA can be obtained upon mild heating at 45 degrees C, as well as at room temperature in the presence of 15% TFE or by adding to the protein solution 7.5 equivalents of oleic acid. Under these various conditions the far- and near-UV CD spectra of apo-alpha-LA are essentially identical to those of the most studied molten globule of alpha-LA at pH 2.0 (A-state). Proteolysis of the 123-residue chain of apo-alpha-LA by proteinase K at 4 degrees C occurs slowly as an all-or-none process leading to small peptides only. At 37 degrees C, proteinase K preferentially cleaves apo-alpha-LA at peptide bonds Ser34-Gly35, Gln39-Ala40, Gln43-Asn44, Phe53-Gln54, and Asn56-Asn57. All these peptide bonds are located at level of the beta-subdomain of the protein (chain region 34-57). Similar sites of preferential cleavage have been observed with the TFE- and oleic acid-induced molten globule of apo-alpha-LA. A protein species given by the N-terminal fragment 1-34 linked via the four disulfide bridges to the C-terminal fragment 54-123 or 57-123 can be isolated from the proteolytic mixture. The results of this study indicate that the same molten globule state of apo-alpha-LA can be obtained at neutral pH under mildly denaturing conditions, as indicated by using a classical spectroscopic technique such as CD and a simple biochemical approach as limited proteolysis. We conclude that the molten globule of alpha-LA maintains a native-like tertiary fold characterized by a rather well-structured alpha-domain and a disordered chain region encompassing the beta-subdomain 34-57 of the protein.     

Inhibition of transthyretin amyloid fibril formation by 2,4-dinitrophenol through tetramer stabilization

Transthyretin (TTR), a homotetrameric thyroxine transport protein found in the plasma and cerebrospinal fluid, circulates normally as a innocuous soluble protein. In some individuals, TTR polymerizes to form insoluble amyloid fibrils. TTR amyloid fibril formation and deposition have been associated with several diseases like familial amyloid polyneuropathy and senile systemic amyloidosis. Inhibition of the fibril formation is considered a potential strategy for the therapeutic intervention. The effect of small water-soluble, hydrophobic ligand 2,4-dinitrophenol (2,4-DNP) on TTR amyloid formation has been tested. 2,4-DNP binds to TTR both at acidic and physiological pH, as shown by the quenching of TTR intrinsic fluorescence. Interestingly, 2,4-DNP not only binds to TTR at acidic pH but also inhibits amyloid fibril formation as shown by the light scattering and Congo red-binding assay. Inhibition of fibril formation by 2,4-DNP appears to be through the stabilization of TTR tetramer upon binding to the protein, which includes active site. These findings may have implications for the development of mechanism based small molecular weight compounds as therapeutic agents for the prevention/inhibition of the amyloid diseases.     

Domain structure and molecular flexibility of streptococcal M proteinIn Situ probed by limited proteolysis

Serologically distinct group A streptococcal M proteins, the antiphagocytic determinants of the bacteria, have a highly repetitive sequence and exhibit a heptad periodicity characteristic of alpha-helical coiled-coil proteins. Based on the differences in the pattern of heptad periodicity, the coiled-coil region of the complete M molecule has been divided into three distinct domains: I, II, and III. Domains I and II together constitute the variable part of M protein, whereas domain III is conserved among serotypes. Pepsin treatment of the M5, M6, and M24 streptococci results in a preferential cleavage of their M molecules between the predicted domains II and III, releasing biologically active fragments of the respective M proteins. Thus, a pepsin cleavage site at the junction of their variable and conserved regions is conserved in the M5, M6, and M24 proteins. In contrast, in the case of the M49 streptococci, the primary site of pepsin cleavage was observed to be within the conserved region of the M49 molecule, rather than at the junction of its variable and conserved regions. Despite containing part of the conserved region, the PepM49 protein is significantly smaller than the pepsin fragments of the M5, M6, and M24 proteins, which contain only the variable regions. However, in addition to the major PepM49 species, the pepsin digest of the type-49 streptococci also contained a smaller fragment, PepM49/a, as a minor component. Its formation was extremely sensitive to thepH of pepsin digestion. PepM49/a, which retains both the propensity to attain an alpha-helical conformation and the opsonic antibody epitope of the M49 molecule, contains only domains I and II like the other PepM proteins. Thus, as in the M5, M6, and M24 proteins, a pepsin cleavage site at the junction of the variable and conserved regions is indeed present in the M49 molecule, but is much less accessible relative to the other serotypes. Thus, the pepsin cleavage sites in the M protein correlate quite well with the boundaries of structurally distinct domains reflected by the predictive analysis. These sites apparently represent the flexible/hinge regions of the molecule. PepM49/a is the least repetitive and the shortest of the M protein pepsin fragments isolated so far. These results suggest that the flexibility of the interdomain regions in M protein may be dependent on the molecular size of their variable domains. The placement of a more accessible hinge within the conserved part of the M49 molecule, rather than at the junction of the variable and conserved domains, suggests that a critical molecular size may be essential for the efficient functioning of the M molecule.