Charge attraction and beta propensity are necessary for amyloid fibril formation from tetrapeptides

Amyloid fibrils in which specific proteins have polymerized into a cross-beta-sheet structure are found in about 20 diseases. In contrast to the close structural similarity of fibrils formed in different amyloid diseases, the structures of the corresponding native proteins differ widely. We show here that peptides as short as 4 residues with the sequences KFFE or KVVE can form amyloid fibrils that are practically identical to fibrils formed in association with disease, as judged by electron microscopy and Congo red staining. In contrast, KLLE or KAAE do not form fibrils. The fibril-forming KFFE and KVVE show partial beta-strand conformation in solution, whereas the non-fibril-forming KLLE and KAAE show random structure only, suggesting that inherent propensity for beta-strand conformation promotes fibril formation. The peptides KFFK or EFFE do not form fibrils on their own but do so in an equimolar mixture. Thus, intermolecular electrostatic interactions, either between charged dipolar peptides or between complementary charges of co-fibrillating peptides favor fibril formation.     

A short sequence responsible for both phosphoinositide binding and actin binding activities of cofilin.

Cofilin is a widely distributed actin-modulating protein that has abilities to bind along the side of F-actin and to depolymerize F-actin. Both abilities of cofilin can be inhibited by phosphoinositides such as phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate (PIP2). We have previously shown that the synthetic dodecapeptide corresponding to Trp104-Met115 of cofilin is a potent inhibitor of actin polymerization (Yonezawa, N., Nishida, E., Iida, K., Kumagai, H., Yahara, I., and Sakai, H. (1991) J. Biol. Chem. 266, 10485-10489). In this study, we have found that the inhibitory effect of the synthetic dodecapeptide on actin polymerization is canceled specifically by phosphatidylinositol, phosphatidylinositol 4-monophosphate and PIP2. We further show that the dodecapeptide as well as cofilin binds to PIP2 molecules and inhibits PIP2 hydrolysis by phospholipase C. Thus, the actin-binding dodecapeptide sequence of cofilin may constitute a multifunctional domain in cofilin.     

A structural motif of acetylcholinesterase that promotes amyloid beta-peptide fibril formation

Acetylcholinesterase (AChE) has been found to be associated with the core of senile plaques. We have shown that AChE interacts with the amyloid beta-peptide (Abeta) and promotes amyloid fibril formation by a hydrophobic environment close to the peripheral anionic binding site (PAS) of the enzyme. Here we present evidence for the structural motif of AChE involved in this interaction. First, we modeled the docking of Abeta onto the structure of Torpedo californica AChE, and identified four potential sites for AChE-Abeta complex formation. One of these, Site I, spans a major hydrophobic sequence exposed on the surface of AChE, which had been previously shown to interact with liposomes [Shin et al. (1996) Protein Sci. 5, 42-51]. Second, we examined several AChE-derived peptides and found that a synthetic 35-residue peptide corresponding to the above hydrophobic sequence was able to promote amyloid formation. We also studied the ability to promote amyloid formation of two synthetic 24-residue peptides derived from the sequence of a Omega-loop, which has been suggested as an AChE-Abeta interacting motif. Kinetic analyses indicate that only the 35-residue hydrophobic peptide mimics the effect of intact AChE on amyloid formation. Moreover, RP-HPLC analysis revealed that the 35-residue peptide was incorporated into the growing Abeta-fibrils. Finally, fluorescence binding studies showed that this peptide binds Abeta with a K(d) = 184 microM, independent of salt concentration, indicating that the interaction is primarily hydrophobic. Our results indicate that the homologous human AChE motif is capable of accelerating Abeta fibrillogenesis.     

Computational study of the fibril organization of polyglutamine repeats reveals a common motif identified in beta-helices

The formation of fibril aggregates by long polyglutamine sequences is assumed to play a major role in neurodegenerative diseases such as Huntington. Here, we model peptides rich in glutamine, through a series of molecular dynamics simulations. Starting from a rigid nanotube-like conformation, we have obtained a new conformational template that shares structural features of a tubular helix and of a beta-helix conformational organization. Our new model can be described as a super-helical arrangement of flat beta-sheet segments linked by planar turns or bends. Interestingly, our comprehensive analysis of the Protein Data Bank reveals that this is a common motif in beta-helices (termed beta-bend), although it has not been identified so far. The motif is based on the alternation of beta-sheet and helical conformation as the protein sequence is followed from the N to the C termini (beta-alpha(R)-beta-polyPro-beta). We further identify this motif in the ssNMR structure of the protofibril of the amyloidogenic peptide Abeta(1-40). The recurrence of the beta-bend suggests a general mode of connecting long parallel beta-sheet segments that would allow the growth of partially ordered fibril structures. The design allows the peptide backbone to change direction with a minimal loss of main chain hydrogen bonds. The identification of a coherent organization beyond that of the beta-sheet segments in different folds rich in parallel beta-sheets suggests a higher degree of ordered structure in protein fibrils, in agreement with their low solubility and dense molecular packing.     

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.     

Inhibition of actin polymerization by a synthetic dodecapeptide patterned on the sequence around the actin-binding site of cofilin

Cofilin is an F-actin side-binding and -depolymerizing protein with an apparent molecular mass of 21 kDa. By means of the end label fingerprinting method, the amino acid residue on cofilin sequence cross-linked to actin by zero length cross-linker, 1-ethyl-3-(3-dimethylamino propyl)carbodiimide, was identified as Lys112 and/or Lys114. A synthetic dodecapeptide patterned on the sequence around the actin-cross-linking site of cofilin (Trp104-Met115) inhibited the binding of cofilin to actin. Moreover, the dodecapeptide was found to be a potent inhibitor of actin polymerization. Thus, we conclude that the dodecapeptide sequence constitutes the region essential for the actin-binding and -depolymerizing activity of cofilin. A sequence similar to the dodecapeptide is found in other actin-depolymerizing proteins, destrin, actin-depolymerizing factor, and depactin. Therefore, the dodecapeptide sequence may be a consensus sequence essential for actin-binding and -depolymerizing activity in actin-depolymerizing proteins.     

Glucagon Fibril Polymorphism Reflects Differences in Protofilament Backbone Structure

Amyloid fibrils formed by the 29-residue peptide hormone glucagon at different concentrations have strikingly different morphologies when observed by transmission electron microscopy. Fibrils formed at low concentration (0.25 mg/mL) consist of two or more protofilaments with a regular twist, while fibrils at high concentration (8 mg/mL) consist of two straight protofilaments. Here, we explore the structural differences underlying glucagon polymorphism using proteolytic degradation, linear and circular dichroism, Fourier transform infrared spectroscopy (FTIR), and X-ray fiber diffraction. Morphological differences are perpetuated at all structural levels, indicating that the two fibril classes differ in terms of protofilament backbone regions, secondary structure, chromophore alignment along the fibril axis, and fibril superstructure. Straight fibrils show a conventional β-sheet-rich far-UV circular dichroism spectrum whereas that of twisted fibrils is dominated by contributions from β-turns. Fourier transform infrared spectroscopy confirms this and also indicates a more dense backbone with weaker hydrogen bonding for the twisted morphology. According to linear dichroism, the secondary structural elements and the aromatic side chains in the straight fibrils are more highly ordered with respect to the alignment axis than the twisted fibrils. A series of highly periodical reflections in the diffractogram of the straight fibrils can be fitted to the diffraction pattern expected from a cylinder. Thus, the highly integrated structural organization in the straight fibril leads to a compact and highly uniform fibril with a well-defined edge. Prolonged proteolytic digestion confirmed that the straight fibrils are very compact and stable, while parts of the twisted fibril backbone are much more readily degraded. Differences in the digest patterns of the two morphologies correlate with predictions from two algorithms, suggesting that the polymorphism is inherent in the glucagon sequence. Glucagon provides a striking illustration of how the same short sequence can be folded into two remarkably different fibrillar structures.     

The GB1 amyloid fibril: recruitment of the peripheral beta-strands of the domain swapped dimer into the polymeric interface

Three-dimensional domain swapping has been evoked as a mechanism for oligomerization of proteins. Here, we show for the immunoglobulin-binding domain B1 of streptococcal protein G (GB1) that fibril formation is observed readily for variants that exist as domain-swapped dimers. No fibril was formed by a revertant that exhibits the stable wild-type GB1 fold or a mutant comprising a highly destabilized, fluctuating ensemble of conformers. Structural features of the GB1 amyloid fibril were characterized by cysteine disulfide cross-linking. Residues in the outer edge beta-strands of the domain-swapped dimer readily form intermolecular disulfide bonds prior to and during fibril formation. On the basis of these data, a structural model for the assembly of domain-swapped dimers into a polymeric structure of the GB1 fibril is proposed.     

Analysis of the sequence and structural features of the left-handed beta-helical fold

The left-handed parallel beta-helix (LbetaH) is a structurally repetitive, highly regular, and symmetrical fold formed by coiling of elongated beta-sheets into helical "rungs." This canonical fold has recently received interest as a possible solution to the fibril structure of amyloid and as a building block of self-assembled nanotubular structures. In light of this interest, we aimed to understand the structural requirements of the LbetaH fold. We first sought to determine the sequence characteristics of the repeats by analyzing known structures to identify positional preferences of specific residues types. We then used molecular dynamics simulations to demonstrate the stabilizing effect of successive rungs and the hydrophobic core of the LbetaH. We show that a two-rung structure is the minimally stable LbetaH structure. In addition, we defined the structure-based sequence preference of the LbetaH and undertook a genome-wide sequence search to determine the prevalence of this unique protein fold. This profile-based LbetaH search algorithm predicted a large fraction of LbetaH proteins from microbial origins. However, the relative number of predicted LbetaH proteins per specie was approximately equal across the genomes from prokaryotes to eukaryotes.     

3MATRIX and 3MOTIF: a protein structure visualization system for conserved sequence motifs

Computational methods such as sequence alignment and motif construction are useful in grouping related proteins into families, as well as helping to annotate new proteins of unknown function. These methods identify conserved amino acids in protein sequences, but cannot determine the specific functional or structural roles of conserved amino acids without additional study. In this work, we present 3MATRIX (http://3matrix.stanford.edu) and 3MOTIF (http://3motif.stanford.edu), a web-based sequence motif visualization system that displays sequence motif information in its appropriate three-dimensional (3D) context. This system is flexible in that users can enter sequences, keywords, structures or sequence motifs to generate visualizations. In 3MOTIF, users can search using discrete sequence motifs such as PROSITE patterns, eMOTIFs, or any other regular expression-like motif. Similarly, 3MATRIX accepts an eMATRIX position-specific scoring matrix, or will convert a multiple sequence alignment block into an eMATRIX for visualization. Each query motif is used to search the protein structure database for matches, in which the motif is then visually highlighted in three dimensions. Important properties of motifs such as sequence conservation and solvent accessible surface area are also displayed in the visualizations, using carefully chosen color shading schemes.     

Structures of soluble amyloid oligomers from computer simulations

Alzheimer's, Parkinson's, and Creutzfeldt-Jakob's neurodegenerative diseases are all linked with the assembly of normally soluble proteins into amyloid fibrils. Because of experimental limitations, structural characterization of the soluble oligomers, which form early in the process of fibrillogenesis and are cytotoxic, remains to be determined. In this article, we study the aggregation paths of seven chains of the shortest amyloid-forming peptide, using an activitated method and a reduced atomic representation. Our simulations show that disordered KFFE monomers ultimately form three distinct topologies of similar energy: amorphous oligomers, incomplete rings with beta-barrel character, and cross-beta-sheet structures with the meridional but not the equatorial X-ray fiber reflections. The simulations also shed light on the pathways from misfolded aggregates to fibrillar-like structures. They also underline the multiplicity of building blocks that can lead to the formation of the critical nucleus from which rapid growth of the fibril occurs.     

Higher-order molecular packing in amyloid-like fibrils constructed with linear arrangements of hydrophobic and hydrogen-bonding side-chains

Various mutants of the protein fragment, barnase module-1 (1-24) were investigated in order to reveal the structural principle of amyloid-like fibrils. By means of circular dichroism spectroscopy, X-ray diffraction, electron microscopy, and thioflavin T binding assay, we found that the molecules containing two beta-strands and an intervening turn structure are assembled to form a cross-beta structure. Stabilization by both the hydrophobic interactions and hydrogen bonding between the respective paired side-chains on the coupled beta-strands was essential for fibril formation. These two types of interaction can also arrange the corresponding residues in lines on both sheet surfaces of protofilaments with a cross-beta structure. This leads to the most probable fibril structure constructed with the line-matching interactions between protofilaments. Consideration of the geometrical symmetry resulted in our finding that a limited number of essential models for molecular packing in fibril structure are stable, which would rationally explain the occurrence of two or three morphologies from an identical molecular species. The ribbon-like fibrils exhibited striped texture along the axis, which was assigned to a stacked two-sheet repeat as a structural unit. The comprehensively proposed structural model, that is, the sheet-sheet interaction between left-handed cross-beta structures, results in a slightly right-handed twist of beta-sheet stacking, which reasonably elucidates the intrinsic sizes of the fibril width and its helical period along the fibril axis, as the bias in the orientation of the hydrogen-bonded beta-strand pair at the lateral edge is larger than that at the central protofilament.     

The exception that confirms the rule: a higher-order telomeric G-quadruplex structure more stable in sodium than in potassium

DNA and RNA guanine-quadruplexes (G4s) are stabilized by several cations, in particular by potassium and sodium ions. Generally, potassium stabilizes guanine-quartet assemblies to a larger extent than sodium; in this article we report about a higher-order G4 structure more stable in sodium than in potassium. Repeats of the DNA GGGTTA telomeric motif fold into contiguous G4 units. Using three independent approaches (thermal denaturation experiments, isothermal molecular-beacon and protein-binding assays), we show that the (GGGTTA)₇GGG sequence, folding into two contiguous G4 units, exhibits an unusual feature among G4 motifs: despite a lower thermal stability, its sodium conformation is more stable than its potassium counterpart at physiological temperature. Using differential scanning calorimetry and mutated sequences, we show that this switch in the relative stability of the sodium and potassium conformations (occurring around 45°C in 100 mM cation concentration) is the result of a more favorable enthalpy change upon folding in sodium, generated by stabilizing interactions between the two G4 units in the sodium conformation. Our work demonstrates that interactions between G4 structural domains can make a higher-order structure more stable in sodium than in potassium, even though its G4 structural domains are individually more stable in potassium than in sodium.     

Kinetic analysis of beta-amyloid fibril elongation

We used surface plasmon resonance biosensors to evaluate the kinetics associated with the initial events of beta-amyloid (Abeta) fibril elongation. Fibrils were immobilized on the sensor chip surface and extended by exposure to soluble Abeta(1-40) peptide. The fibril surfaces bound Congo red, a marker for beta sheet structures, and exhibited a slow linear background decay that is consistent with fibril depolymerization. Sonicated fibrils supported elongation better than unsonicated fibrils, which is consistent with fibril extension reactions. The kinetic data revealed that peptide association and dissociation occurred in multiple steps. Kinetic rate constants for fibril extension were determined by globally fitting the response data with a three-step polymerization model. In the first step, the soluble peptide binds to the growing fibril tip in a readily reversible reaction. The subsequent steps likely allow bound peptide to be stabilized into the growing fiber through postbinding transitional events. Using a mutant peptide, F19P Abeta(1-40), we illustrate how the biosensor assay can be used to probe structure/function relationships of fibril elongation.     

The UA_handle: a versatile submotif in stable RNA architectures

Stable RNAs are modular and hierarchical 3D architectures taking advantage of recurrent structural motifs to form extensive non-covalent tertiary interactions. Sequence and atomic structure analysis has revealed a novel submotif involving a minimal set of five nucleotides, termed the UA_handle motif (5′XU/AN_nX3′). It consists of a U:A Watson–Crick: Hoogsteen trans base pair stacked over a classic Watson–Crick base pair, and a bulge of one or more nucleotides that can act as a handle for making different types of long-range interactions. This motif is one of the most versatile building blocks identified in stable RNAs. It enters into the composition of numerous recurrent motifs of greater structural complexity such as the T-loop, the 11-nt receptor, the UAA/GAN and the G-ribo motifs. Several structural principles pertaining to RNA motifs are derived from our analysis. A limited set of basic submotifs can account for the formation of most structural motifs uncovered in ribosomal and stable RNAs. Structural motifs can act as structural scaffoldings and be functionally and topologically equivalent despite sequence and structural differences. The sequence network resulting from the structural relationships shared by these RNA motifs can be used as a proto-language for assisting prediction and rational design of RNA tertiary structures.     

Automated classification of RNA 3D motifs and the RNA 3D Motif Atlas

The analysis of atomic-resolution RNA three-dimensional (3D) structures reveals that many internal and hairpin loops are modular, recurrent, and structured by conserved non-Watson–Crick base pairs. Structurally similar loops define RNA 3D motifs that are conserved in homologous RNA molecules, but can also occur at nonhomologous sites in diverse RNAs, and which often vary in sequence. To further our understanding of RNA motif structure and sequence variability and to provide a useful resource for structure modeling and prediction, we present a new method for automated classification of internal and hairpin loop RNA 3D motifs and a new online database called the RNA 3D Motif Atlas. To classify the motif instances, a representative set of internal and hairpin loops is automatically extracted from a nonredundant list of RNA-containing PDB files. Their structures are compared geometrically, all-against-all, using the FR3D program suite. The loops are clustered into motif groups, taking into account geometric similarity and structural annotations and making allowance for a variable number of bulged bases. The automated procedure that we have implemented identifies all hairpin and internal loop motifs previously described in the literature. All motif instances and motif groups are assigned unique and stable identifiers and are made available in the RNA 3D Motif Atlas (http://rna.bgsu.edu/motifs), which is automatically updated every four weeks. The RNA 3D Motif Atlas provides an interactive user interface for exploring motif diversity and tools for programmatic data access.     

A molecular model of Alzheimer amyloid beta-peptide fibril formation

Polymerization of the amyloid beta (Abeta) peptide into protease-resistant fibrils is a significant step in the pathogenesis of Alzheimer's disease. It has not been possible to obtain detailed structural information about this process with conventional techniques because the peptide has limited solubility and does not form crystals. In this work, we present experimental results leading to a molecular level model for fibril formation. Systematically selected Abeta-fragments containing the Abeta16-20 sequence, previously shown essential for Abeta-Abeta binding, were incubated in a physiological buffer. Electron microscopy revealed that the shortest fibril-forming sequence was Abeta14-23. Substitutions in this decapeptide impaired fibril formation and deletion of the decapeptide from Abeta1-42 inhibited fibril formation completely. All studied peptides that formed fibrils also formed stable dimers and/or tetramers. Molecular modeling of Abeta14-23 oligomers in an antiparallel beta-sheet conformation displayed favorable hydrophobic interactions stabilized by salt bridges between all charged residues. We propose that this decapeptide sequence forms the core of Abeta-fibrils, with the hydrophobic C terminus folding over this core. The identification of this fundamental sequence and the implied molecular model could facilitate the design of potential inhibitors of amyloidogenesis.     

Crystal structure and centromere binding of the plasmid segregation protein ParB from pCXC100

Plasmid pCXC100 from the Gram-positive bacterium Leifsonia xyli subsp. cynodontis uses a type Ib partition system that includes a centromere region, a Walker-type ATPase ParA and a centromere-binding protein ParB for stable segregation. However, ParB shows no detectable sequence homology to any DNA-binding motif. Here, we study the ParB centromere interaction by structural and biochemical approaches. The crystal structure of the C-terminal DNA-binding domain of ParB at 1.4 Å resolution reveals a dimeric ribbon–helix–helix (RHH) motif, supporting the prevalence of RHH motif in centromere binding. Using hydroxyl radical footprinting and quantitative binding assays, we show that the centromere core comprises nine uninterrupted 9-nt direct repeats that can be successively bound by ParB dimers in a cooperative manner. However, the interaction of ParB with a single subsite requires 18 base pairs covering one immediate repeat as well as two halves of flanking repeats. Through mutagenesis, sequence specificity was determined for each position of an 18-bp subsite. These data suggest an unique centromere recognition mechanism by which the repeat sequence is jointly specified by adjacent ParB dimers bound to an overlapped region.     

Partial denaturation of transthyretin is sufficient for amyloid fibril formation in vitro.

Amyloid diseases are caused by the self-assembly of a given protein into an insoluble cross-beta-sheet quaternary structural form which is pathogenic. An understanding of the biochemical mechanism of amyloid fibril formation should prove useful in understanding amyloid disease. Toward this end, a procedure for the conversion of the amyloidogenic protein transthyretin into amyloid fibrils under conditions which mimic the acidic environment of a lysosome has been developed. Association of a structured transthyretin denaturation intermediate is sufficient for amyloid fibril formation in vitro. The rate of fibril formation is pH dependent with significant rates being observed at pHs accessible within the lysosome (3.6-4.8). Far-UV CD spectroscopic studies suggest that transthyretin retains its secondary structural features at pHs where fibrils are formed. Near-UV CD studies demonstrate that transthyretin has retained the majority of its tertiary structure during fibril formation as well. Near-UV CD analysis in combination with glutaraldehyde cross-linking studies suggests that a pH-mediated tetramer to monomer transition is operative in the pH range where fibril formation occurs. The rate of fibril formation decreases markedly at pHs below pH 3.6, consistent with denaturation to a monomeric TTR intermediate which has lost its native tertiary structure and capability to form fibrils. It is difficult to specify with certainty which quaternary structural form of transthyretin is the amyloidogenic intermediate at this time. These difficulties arise because the maximal rate of fibril formation occurs at pH 3.6 where tetramer, traces of dimer, and significant amounts of monomer are observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of Protein Structural Motifs in Terms of Sets of Codes Representing Local Structures

An amino acid sequence pattern conserved among a family of proteins is called motif. It is usually related to the specific function of the family. On the other hand, functions of proteins are realized through their 3D structures. Specific local structures, called structural motifs, are considered as related to their functions. However, searching for common structural motifs in different proteins is much more difficult than for common sequence motifs. We are attempting in this study to convert the information about the structural motifs into a set of one-dimensional digital strings, i.e., a set of codes, to compare them more easily by computer and to investigate their relationship to functions more quantitatively. By applying the Delaunay tessellation to a 3D structure of a protein, we can assign each local structure to a unique code that is defined so as to reflect its structural feature. Since a structural motif is defined as a set of the local structures in this paper, the structural motif is represented by a set of the codes. In order to examine the ability of the set of the codes to distinguish differences among the sets of local structures with a given PROSITE pattern that contain both true and false positives, we clustered them by introducing a similarity measure among the set of the codes. The obtained clustering shows a good agreement with other results by direct structural comparison methods such as a superposition method. The structural motifs in homologous proteins are also properly clustered according to their sources. These results suggest that the structural motifs can be well characterized by these sets of the codes, and that the method can be utilized in comparing structural motifs and relating them with function.