Intermediates control domain swapping during folding of p13suc1

The 13-kDa protein p13(suc1) has two folded states, a monomer and a structurally similar domain-swapped dimer formed by exchange of a beta-strand. The refolding reaction of p13(suc1) is multiphasic, and in this paper we analyze the kinetics as a function of denaturant and protein concentration and compare the behavior of wild type and a set of mutants previously designed with dimerization propensities that span 9 orders of magnitude. We show that the folding reactions of wild type and all mutants produce the monomer predominantly despite their very different equilibrium behavior. However, the addition of low concentrations of denaturant in the refolding buffer leads to thermodynamic control of the folding reaction with products that correspond to the wild type and mutant equilibrium dimerization propensities. We present evidence that the kinetic control in the absence of urea arises because of the population of the folding intermediates. Intermediates are usually considered to be detrimental to folding because they slow down the reaction; however, our work shows that intermediates buffer the monomeric folding pathway against the effect of mutations that favor the nonfunctional, dimeric state at equilibrium.     

Amyloid fibrils formed by the programmed cell death regulator Bcl-xL

The accumulation of amyloid fibers due to protein misfolding is associated with numerous human diseases. For example, the formation of amyloid deposits in neurodegenerative pathologies is correlated with abnormal apoptosis. We report here the in vitro formation of various types of aggregates by Bcl-xL, a protein of the Bcl-2 family involved in the regulation of apoptosis. Bcl-xL forms aggregates in three states, micelles, native-like fibrils, and amyloid fibers, and their biophysical characterization has been performed in detail. Bcl-xL remains in its native state within micelles and native-like fibrils, and our results suggest that native-like fibrils are formed by the association of micelles. Formation of amyloid structures, that is, nonnative intermolecular β-sheets, is favored by the proximity of proteins within fibrils at the expense of the Bcl-xL native structure. Finally, we provide evidence of a direct relationship between the amyloid character of the fibers and the tertiary-structure stability of the native Bcl-xL. The potential causality between the accumulation of Bcl-xL into amyloid deposits and abnormal apoptosis during neurodegenerative diseases is discussed.     

Folding and association of the human cell cycle regulatory proteins ckshs1 and ckshs2.

The two human proteins ckshs1 and ckshs2 are each 79 amino acids in length and consist of a four-stranded beta-sheet capped at one end by two alpha-helices. They are members of the cks family of essential cell cycle regulatory proteins that can adopt two native states, a monomer and a domain-swapped dimer formed by exchange of a C-terminal beta-strand. ckshs1 and ckshs2 both have marginal thermodynamic stability (the free energies of unfolding at 25 degrees C are 3.0 and 2.5 kcal/mol, respectively) and low kinetic stability (the rates of unfolding in water are approximately 1 s(-1)). Refolding of their denatured states to the monomeric forms of the proteins is slowed by transient oligomerization that is likely to occur via domain swapping. The folding behavior of ckshs1 and ckshs2 is markedly different from that of suc1, the cks protein from Schizosaccharomyces pombe, but the domain swapping propensities are similar. The greater thermodynamic and kinetic stability of suc1 and the population of a folding intermediate are most likely a consequence of its larger size (113 residues). The similarity in the domain swapping propensities, despite the contrast in other biophysical properties, may be attributable to the common double-proline motif in the hinge loop that connects the swapped domain to the rest of the protein. The motif was shown previously for suc1 to control the equilibrium between the monomer and the domain-swapped dimer. Finally, according to our model, the kinetic barrier separating the monomer and the domain-swapped dimer arises because the protein must unfold for beta-strand exchange to occur. Consistent with this, interconversion between the two states is much faster in the human proteins than it is for suc1, reflecting the faster unfolding rates of the former.     

A model for the aggregation of the acylphosphatase from Sulfolobus solfataricus in its native-like state

Evidence is accumulating that normally folded proteins retain a significant tendency to form amyloid fibrils through a direct assembly of monomers in their native-like conformation. However, the factors promoting such processes are not yet well understood. The acylphosphatase from Sulfolobus solfataricus (Sso AcP) aggregates under conditions in which a native-like state is initially populated and forms, as a first step, aggregates in which the monomers maintain their native-like topology. An unstructured N-terminal segment and an edge beta-strand were previously shown to play a major role in the process. Using kinetic experiments on a set of Sso AcP variants we shall show that the major event of the first step is the establishment of an inter-molecular interaction between the unstructured segment of one Sso AcP molecule and the globular unit of another molecule. This interaction is determined by the primary sequence of the unstructured segment and not by its physico-chemical properties. Moreover, we shall show that the conversion of these initial aggregates into amyloid-like protofibrils is an intra-molecular process in which the Sso AcP molecules undergo conformational modifications. The obtained results allow the formulation of a model for the assembly of Sso AcP into amyloid-like aggregates at a molecular level.     

The yeast prion Ure2p native-like assemblies are toxic to mammalian cells regardless of their aggregation state

The yeast prion Ure2p assembles in vitro into oligomers and fibrils retaining the alpha-helix content and binding properties of the soluble protein. Here we show that the different forms of Ure2p native-like assemblies (dimers, oligomers, and fibrils) are similarly toxic to murine H-END cells when added to the culture medium. Interestingly, the amyloid fibrils obtained by heat treatment of the toxic native-like fibrils appear harmless. Moreover, the Ure2p C-terminal domain, lacking the N-terminal segment necessary for aggregation but containing the glutathione binding site, is not cytotoxic. This finding strongly supports the idea that Ure2p toxicity depends on the structural properties of the flexible N-terminal prion domain and can therefore be considered as an inherent feature of the protein, unrelated to its aggregation state but rather associated with a basic toxic fold shared by all of the Ure2p native-like assemblies. Indeed, the latter are able to interact with the cell surface, leading to alteration of calcium homeostasis, membrane permeabilization, and oxidative stress, whereas the heat-treated amyloid fibrils do not. Our results support the idea of a general mechanism of toxicity of any protein/peptide aggregate endowed with structural features, making it able to interact with cell membranes and to destabilize them. This evidence extends the widely accepted view that the toxicity by protein aggregates is restricted to amyloid prefibrillar aggregates and provides new insights into the mechanism by which native-like oligomers compromise cell viability.     

Evidence for a mechanism of amyloid formation involving molecular reorganisation within native-like precursor aggregates

The aggregation of the alpha/beta protein acylphosphatase from Sulfolobus solfataricus has been studied under conditions in which the protein maintains a native-like, although destabilised, conformation and that therefore bear resemblance to a physiological medium. Static and dynamic light-scattering measurements indicate that under these conditions the protein aggregates rapidly, within two minutes. The initial aggregates are enzymatically active and have a secondary structure that is not yet characterized by the high content of cross-beta structure typical of amyloid, as inferred from Fourier transform infra-red and circular dichroism measurements. These species then convert slowly into enzymatically inactive aggregates that bind thioflavin T and Congo red, characteristic of amyloid structures, and contain extensive beta-sheet structure. Transmission electron microscopy reveals the presence in the latter aggregates of spherical species and thin, elongated protofibrils, both with diameters of 3-5 nm. Kinetic tests reveal that this process occurs without the need for dissolution and re-nucleation of the aggregates. Formation of thioflavin T-binding and beta-structured aggregates is substantially more rapid than unfolding of the native state, indicating that the initial aggregation process promotes formation of amyloid structures. Taken together, these findings suggest a mechanism of amyloid formation that may have physiological relevance and in which the amyloid structures result from reorganisation of the molecular interactions within the initially formed non-amyloid aggregates.     

Synthetic Lipid Vesicles Recruit Native-Like Aggregates and Affect the Aggregation Process of the Prion Ure2p: Insights on Vesicle Permeabilization and Charge Selectivity

The yeast prion Ure2p polymerizes into native-like fibrils, retaining the overall structure and binding properties of the soluble protein. Recently we have shown that, similar to amyloid oligomers, the native-like Ure2p fibrils and their precursor oligomers are highly toxic to cultured mammalian cells when added to the culture medium, whereas Ure2p amyloid fibrils generated by heating the native-like fibrils are substantially harmless. We show here that, contrary to the nontoxic amyloid fibrils, the toxic, native-like Ure2p assemblies induce a significant calcein release from negatively charged phosphatidylserine vesicles. A minor and less-specific effect was observed with zwitterionic phosphatidylcholine vesicles, suggesting that the toxic aggregates preferentially bind to negatively charged sites on lipid membranes. We also found that cholesterol-enriched phospholipid membranes are protected against permeabilization by native-like Ure2p assemblies. Moreover, vesicle permeabilization appears charge-selective, allowing calcium, but not chloride, influx to be monitored. Finally, we found that the interaction with phosphatidylserine membranes speeds up Ure2p polymerization into oligomers and fibrils structurally and morphologically similar to the native-like Ure2p assemblies arising in free solution, although less cytotoxic. These data suggest that soluble Ure2p oligomers and native-like fibrils, but not amyloid fibrils, interact intimately with negatively charged lipid membranes, where they allow selective cation influx.     

Observation of signal transduction in three-dimensional domain swapping

p13suc1 (suc1) has two native states, a monomer and a domain-swapped dimer. The structure of each subunit in the dimer is identical to that of the monomer, except for the hinge loop that connects the exchanging domains. Here we find that single point mutations at sites throughout the protein and ligand binding both shift the position of the equilibrium between monomer and dimer. The hinge loop was shown previously to act as a loaded molecular spring that releases tension present in the monomer by adopting an alternative conformation in the dimer. The results here indicate that the release of strain propagates throughout the entire protein and alters the energetics of regions remote from the hinge. Our data illustrate how the signal conferred by the conformational change of a protein loop, elicited by domain swapping, ligand binding or mutation, can be sensed by a distant active site. This work highlights the potential role of strained loops in proteins: the energy they store can be used for both signal transduction and allostery, and they could steer the evolution of protein function. Finally, a structural mechanism for the role of suc1 as an adapter molecule is proposed.     

The structure of the transition state for folding of domain-swapped dimeric p13suc1

suc1 has two native states, a monomer and a domain-swapped dimer, in which one molecule exchanges a beta strand with an identical partner. Thus, monomer and dimer have the same structures but are topologically distinct. Importantly, residues that exchange are part of the folding nucleus of the monomer and therefore forming these interactions in the dimer would be expected to incur a large entropic cost. Here we present the transition state for folding/unfolding of domain-swapped dimeric suc1 and compare it with its monomeric counterpart. The same overall structure is observed in the two transition states but the phi values are consistently higher for the domain-swapped dimer. Thus, a greater entropic penalty for bringing together the key interactions in the dimer is overcome by mobilizing more contacts in the transition state, thereby achieving a greater enthalpic gain.     

Stability and Inter-domain Interactions Modulate Amyloid Binding Activity of a General Amyloid Interaction Motif

The M13 tip protein, g3p, binds the C-terminal domain of the bacterial membrane protein TolA via β-sheet augmentation, facilitating viral entry into Escherichia coli. G3p binding leads to rearrangement of the β strands and partial unfolding of TolA. G3p also binds multiple amyloid assemblies with high affinity, and it can remodel them into amorphous aggregates. We previously showed that amyloid binding activity is defined by the two g3p N-terminal domains, which we call the general amyloid interaction motif (GAIM). GAIM–hIgG1Fc fusions, which add immune effector function to amyloid targeting of GAIM, mediate reduction of two CNS amyloid deposits, Aβ plaques and tau tangles, in transgenic animal models of neurodegenerative disease. We carried out site-directed mutagenesis of GAIM to identify variants with altered amyloid binding and remodeling activity. A small set of residues along the inner strands of the two domains regulates both activities. The specificity of amyloid binding is governed by individual domain stability and inter-domain interactions. Our studies reveal several lines of similarity between GAIM binding to amyloids and g3p binding to its E. coli membrane target, TolA. Based on these studies, we designed new GAIM fusions that show enhanced binding potency towards multiple amyloid aggregates.     

The crystal structure of p13suc1, a p34cdc2-interacting cell cycle control protein.

p13suc1 binds to p34cdc2 kinase and is essential for cell cycle progression in eukaryotic cells. The crystal structure of S.pombe p13suc1 has been solved to 2.7 A resolution using data collected at the ESRF source, Grenoble, from both native crystals and crystals of a seleno-methionine derivative. The starting point for structure solution was the determination of the six selenium sites by direct methods. The structure is dominated by a four-stranded beta-sheet, with four further alpha-helical regions. p13suc1 crystallizes as a dimer in the asymmetric unit stabilized by the binding of two zinc ions. A third zinc site stabilizes the higher-order crystal packing. The sites are consistent with a requirement for zinc during crystal growth. A likely site for p13suc1-protein interaction is immediately evident on one face of the p13suc1 surface. This region comprises a group of conserved, exposed aromatic and hydrophobic residues below a flexible negatively charged loop. A conserved positively charged area would also present a notable surface feature in the monomer, but is buried at the dimer interface. p13suc1 is larger than its recently solved human homologue p9CKS2, with the extra polypeptide forming a helical N-terminal extension and a surface loop between alpha-helices 3 and 4. Notably, p13suc1 does not show the unusual beta-strand exchange that creates an intimate p9CKS2 dimer. p13suc1 cannot oligomerize to form a stable hexamer as has been proposed for p9CKS2.     

Oligomeric assembly of native-like precursors precedes amyloid formation by beta-2 microglobulin

The deposition of beta-2-microglobulin (beta2m) as amyloid fibers results in debilitating complications for renal failure patients who are treated by hemodialysis. In vitro, wild-type beta2m can be converted to amyloid under physiological conditions by exposure to biomedically relevant concentrations of Cu(2+). In this work, we have made comparative measurements of the structural and oligomeric changes in beta2m at time points preceding fibrillogenesis. Our results show Cu(2+) mediates the formation of a monomeric, activated state followed by the formation of a discrete dimeric intermediate. The dimeric intermediates then assemble into tetra- and hexameric forms which display little additional oligomerization on the time scales of their own formation (<1 h). Amyloid fiber formation progresses from these intermediate states but on much longer time scales (>1 week). Although Cu(2+) is necessary for the generation and stabilization of these intermediates, it is not required for the stability of mature amyloid fibers. This suggests that Cu(2+) acts as an initiating factor of amyloidosis by inducing oligomer formation. (1)H NMR and near-UV circular dichroism are used to establish that oligomeric intermediates are native-like in structure. The native-like structure and discrete oligomeric size of beta2m amyloid intermediates suggest that this protein forms fibrils by structural domain swapping.     

Human pancreatitis-associated protein forms fibrillar aggregates with a native-like conformation

Human pancreatitis-associated protein was identified in pathognomonic lesions of Alzheimer disease, a disease characterized by the presence of filamentous protein aggregates. Here, we showed that at physiological pH, human pancreatitis-associated protein forms non-Congo Red-binding, proteinase K-resistant fibrillar aggregates with diameters from 6 up to as large as 68 nm. Interestingly, circular dichroism and Fourier transform infrared spectra showed that, unlike typical amyloid fibrils, which have a cross-beta-sheet structure, these aggregates have a very similar secondary structure to that of the native protein, which is composed of two alpha-helices and eight beta-strands, as determined by NMR techniques. Surface structure analysis showed that the positively charged and negatively charged residues were clustered on opposite sides, and strong electrostatic interactions between molecules were therefore very likely, which was confirmed by cross-linking experiments. In addition, several hydrophobic residues were found to constitute a continuous hydrophobic surface. These results and protein aggregation prediction using the TANGO algorithm led us to synthesize peptide Thr(84) to Ser(116), which, very interestingly, was found to form amyloid-like fibrils with a cross-beta structure. Thus, our data suggested that human pancreatitis-associated protein fibrillization is initiated by protein aggregation primarily because of electrostatic interactions, and the loop from residues 84 to 116 may play an important role in the formation of fibrillar aggregates with a native-like conformation.     

A Bacteriophage Capsid Protein Provides a General Amyloid Interaction Motif (GAIM) That Binds and Remodels Misfolded Protein Assemblies

Misfolded protein aggregates, characterized by a canonical amyloid fold, play a central role in the pathobiology of neurodegenerative diseases. Agents that bind and sequester neurotoxic intermediates of amyloid assembly, inhibit the assembly or promote the destabilization of such protein aggregates are in clinical testing. Here, we show that the gene 3 protein (g3p) of filamentous bacteriophage mediates potent generic binding to the amyloid fold. We have characterized the amyloid binding and conformational remodeling activities using an array of techniques, including X-ray fiber diffraction and NMR. The mechanism for g3p binding with amyloid appears to reflect its physiological role during infection of Escherichia coli, which is dependent on temperature-sensitive interdomain unfolding and cis–trans prolyl isomerization of g3p. In addition, a natural receptor for g3p, TolA-C, competitively interferes with Aβ binding to g3p. NMR studies show that g3p binding to Aβ fibers is predominantly through middle and C-terminal residues of the Aβ subunit, indicating β strand–g3p interactions. A recombinant bivalent g3p molecule, an immunoglobulin Fc (Ig) fusion of the two N-terminal g3p domains, (1) potently binds Aβ fibers (fAβ) (K_D = 9.4 nM); (2); blocks fAβ assembly (IC₅₀ ~ 50 nM) and (3) dissociates fAβ (EC₅₀ = 40–100 nM). The binding of g3p to misfolded protein assemblies is generic, and amyloid-targeted activities can be demonstrated using other misfolded protein systems. Taken together, our studies show that g3p(N1N2) acts as a general amyloid interaction motif.     

p13suc1 suppresses the catalytic function of p34cdc2 kinase for intermediate filament proteins, in vitro.

The regulation of p34cdc2 kinase activity controls the entry into and exit from mitosis. Although genetic and biochemical evidence suggested close interactions between cyclins, p13suc1 and p34cdc2 kinase, the roles of p13suc1 on p34cdc2 kinase functions remain unclear. To examine the effects of p13suc1 on p34cdc2 kinase function we developed a simple purification procedure for p34cdc2 kinase, unassociated with p13suc1. The key to the purification procedures we used was buffer containing 0.5 M NaCl and 50% ethylene glycol, as a specific elutant of p34cdc2 kinase from p13suc1-Sepharose. This purified p34cdc2 kinase stoichiometrically phosphorylated vimentin and desmin. Exogenous p13suc1 suppressed the phosphorylation of these filament proteins by the kinase and prevented disassembly, although histone H1 phosphorylation was not affected. Peptide mapping analysis showed a similar extent of inhibition by p13suc1 for all five phosphorylation sites by p34cdc2 kinase of vimentin and desmin, hence these p13suc1-induced inhibitions are probably not site-specific. It thus appears that p13suc1 has a selective effect on the catalytic activity of p34cdc2 kinase for these filament proteins.     

Dimorphic aggregation behavior of a fusion polypeptide incorporating a stable protein domain (EGFP) with an amyloidogenic sequence (retroCspA)

We describe the behavior of a polypeptide consisting of the genetic fusion of a structurally stable single-domain protein, EGFP (an analog of the green fluorescent protein) with an amyloidogenic sequence, retroCspA (known to readily form amyloid fibrils). Refolding of the fusion protein through single-step removal of denaturant and salt results in precipitation into amyloid aggregates displaying fibrillar morphology, thioflavin T binding as well as green fluorescence. Refolding through step-wise reduction of denaturant concentration in the presence of salt yields a soluble aggregate containing a folded, thermally-stable, non-fluorescent EGFP domain. Together, these results indicate that retroCspA forces the fusion protein to aggregate; however, the EGFP domain remains folded in a native-like structural format in both soluble aggregates and precipitates.     

Insights into the molecular mechanism of protein native-like aggregation upon glycation

Several human neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Familial Amyloidotic Polyneuropathy, have long been associated with, structural and functional changes in disease related proteins leading to aggregation into amyloid fibrils. Such changes can be triggered by post-translational modifications. Methylglyoxal modifications have been shown to induce the formation of small and stable native-like aggregates in the case of the amyloidogenic proteins insulin and α-synuclein. However, the fundamental biophysical mechanism underlying such methylglyoxal-induced protein aggregation is not yet fully understood. In this work cytochrome c (Cyt c) was used as a model protein for the characterization of specific glycation targets and to study their impact on protein structure, stability, and ability to form native-like aggregates. Our results show that methylglyoxal covalently modifies Cyt c at a single residue and induces early conformational changes that lead to the formation of native-like aggregates. Furthermore, partially unfolded species are formed, but do not seem to be implicated in the aggregation process. This shows a clear difference from the amyloid fibril mechanisms which involve partially or totally unfolded intermediates. Equilibrium-unfolding experiments show that glycation strongly decreases Cyt c conformational stability, which is balanced with an increase of conformational stability upon aggregation. Data collected from analytical and spectroscopic techniques, along with kinetic analysis based on least-squares parameter fitting and statistical model discrimination are used to help to understand the driving force underlying glycation-induced native-like aggregation, and enable the proposal of a comprehensive thermodynamic and kinetic model for native-like aggregation of methylglyoxal glycated Cyt c.     

p13suc1 acts in the fission yeast cell division cycle as a component of the p34cdc2 protein kinase.

cdc2+ encodes a protein kinase that is required during both G1 and G2 phases of the cell division cycle in fission yeast. suc1+ is an essential gene that was originally identified as a plasmid-borne sequence that could rescue certain temperature-sensitive cdc2 mutants. To investigate the role of the suc1+ gene product in the cell cycle p13suc1 has been expressed in Escherichia coli and purified. An immunoaffinity purified anti-p13suc1 polyclonal serum has been prepared and used to identify p13suc1 in fission yeast. The abundance of this protein did not alter either during the cell cycle or during entry into stationary phase. p13suc1 was found in yeast lysates in a complex with the cdc2+ gene product. Approximately 5% of cellular p34cdc2 was associated with p13suc1, and this fraction of p34cdc2 was active as a protein kinase. The stability of the complex was disrupted in yeast strains carrying temperature-sensitive alleles of cdc2 that are suppressible by overexpression of suc1+. The level of association between p13suc1 and p34cdc2 was not affected by cell cycle arrest in adverse nutritional conditions. p13suc1 is not a substrate of the p34cdc2 protein kinase. We propose instead that it acts as a regulatory component of p34cdc2 that facilitates interaction with other proteins.     

Structural basis for the protective role of sulfite against transthyretin amyloid formation

Transthyretin (TTR) is a plasma protein, which under conditions not yet completely understood, aggregates forming amyloid deposits that occur extracellularly. It is a protein composed of four identical subunits. Each monomer has a single cysteine residue (Cys10), which in the plasma is reduced (Cys-SH), oxidized (Cys-SO3-), sulfonated (Cys-S-SO3-) or bound to various sulfhydryls. There is evidence that these chemical modifications of the SH group alter the stability and the amyloidogenic potential of the protein. The sulfonated form was found to enhance the stability of the native conformation of TTR, avoiding misassembly of the protein leading to amyloid. Consequently, the potential treatment of TTR-type amyloidosis by sulfite has been suggested. The structure of TTR pre-incubated with sulfite at physiological pH, was determined by X-ray crystallography to provide structural insight for the stabilizing effect of sulfite. Each subunit has a beta-sandwich conformation, with two four stranded beta-pleated sheets (DAGH and CBEF) and a small alpha-helix between strands. The sulfonated cysteines have two sulfite oxygens involved in intramonomer hydrogen bonds that bridge Cys10, the amino acid immediately before beta-strand A, to the amino acids immediately after the edge beta-strand D. Implications of the newly observed interactions in the inhibition of fibril formation are discussed in light of the recent structural models of TTR amyloid fibrils.