We have searched for a minimal interaction motif in protein that supports the aggregation into Alzheimer-like paired helical filaments. Digestion of the repeat domain with different proteases yields a GluC-induced fragment comprising 43 residues (termed PHF43), which represents the third repeat of plus some flanking residues. This fragment self assembles readily into thin filaments without a paired helical appearance, but these filaments are highly competent to nucleate bona fide PHFs from full-length . Probing the interactions of PHF43 with overlapping peptides derived from the full sequence yields a minimal hexapeptide interaction motif of 306 VQIVYK 311 at the beginning of the third internal repeat. This motif coincides with the highest predicted ␤-structure potential in . CD and Fourier transform infrared spectroscopy shows that PHF43 acquires pronounced ␤ structure in conditions of self assembly. Point mutations in the hexapeptide region by prolinescanning mutagenesis prevent the aggregation. The data indicate that PHF assembly is initiated by a short fragment containing the minimal interaction motif forming a local ␤ structure embedded in a largely random-coil protein.

Alzheimer's disease is characterized by the progressive deposition of two types of fibers in the affected brains, the amyloid fibers (consisting of the A peptide, generating the amyloid plaques) and paired helical filaments (PHFs, made up of tau protein, forming the neurofibrillary tangles). While the principles of amyloid aggregation are known in some detail, the investigation of PHF assembly has been hampered by the low efficiency of tau aggregation, the requirement of high protein concentrations, and the lack of suitable detection methods. Here we report a quantitative assay system that permits monitoring of the assembly of PHFs in real time by the fluorescence of dyes such as thioflavine S or T. Using this assay, we evaluated parameters that influence the efficiency of filament formation. Disulfide-linked dimers of tau constructs representing the repeat domain assemble into PHFs most efficiently, but other tau isoforms or constructs form bona fide PHFs as well. The rate of assembly is greatly enhanced by polyanions such as RNA, heparin, and notably polyglutamate which resembles the acidic tail of tubulin. The assembly is optimal at pH ∼6 and low ionic strengths (<50 mM) and increases steeply with temperatures above 30°C , indicating that it is an entropy-driven process.

The microtubule-associated protein tau is the main component of the paired helical filaments (PHFs) of Alzheimer's disease, the most common senile dementia. To understand the origin of tau's abnormal assembly we have studied the influence of other cytosolic components. Here we report that PHF assembly is strongly enhanced by RNA. The RNA-induced assembly of PHFs is dependent on the formation of intermolecular disulfide bridges involving Cys 322 in the third repeat of tau, and it includes the dimerization of tau as an early intermediate. Three-repeat constructs polymerize most efficiently, two repeat constructs are the minimum number required for assembly, and even all six full-length isoforms of tau can be induced to form PHFs by RNA.Key words: Paired helical filament; Alzheimer's disease; Microtubule-associated protein; RNA such factors in the cytoplasm. An initial hint was provided by the tubulin molecule, the natural partner of tau. Tubulin associates with tau, polymerizes into microtubules, and thus prevents tau's interaction with itself. Tubulin's C-terminus, to which tau binds [15], is unusually acidic, suggesting that tau might respond to other polyanionic molecules. Other prominent polyanions in the cytosol are the various RNA species. It turns out that these molecules have the capacity of promoting PHF assembly. In this respect, they are similar to polyanions of the extracellular matrix, such as heparin or heparan sulfate, whose effect on PHF assembly has been reported recently [8,19]. While it is conceptually difficult to imagine how components of the extracellular matrix might interact with cytosolic proteins, the potential role of cytosolic polyanions seems straightforward, making the RNA-PHF connection an attractive model for further investigation.

Alzheimer's disease is characterized by two types of fibrous aggregates in the affected brains, the amyloid fibers (consisting of the A␤-peptide, generating the amyloid plaques), and paired helical filaments (PHFs; made up of tau protein, forming the neurofibrillary tangles). Hence, tau protein, a highly soluble protein that normally stabilizes microtubules, becomes aggregated into insoluble fibers that obstruct the cytoplasm of neurons and cause a loss of microtubule stability. We have developed recently a rapid assay for monitoring PHF assembly and show here that PHFs arise from a nucleated assembly mechanism. The PHF nucleus comprises about 8-14 tau monomers. A prerequisite for nucleation is the dimerization of tau because tau dimers act as effective building blocks. PHF assembly can be seeded by preformed filaments (made either in vitro or isolated from Alzheimer brain tissue). These results suggest that dimerization and nucleation are the rate-limiting steps for PHF formation in vivo.Alzheimer's disease, the most common age-relate dementia, is characterized by two pathological protein deposits in the brain, the amyloid plaques, consisting largely of amyloid fibers assembled from the A␤-peptide [a derivative of the membrane protein APP (amyloid precursor protein); reviewed in ref. 1] and the neurofibrillary tangles (NFT), which are bundles of paired helical filaments (PHFs) whose main constituent is the microtubule-associated protein tau (for reviews, see refs. 2 and 3). The uncontrolled precipitation of these aggregates is believed to be largely responsible for the neuronal degeneration, and the disease has been classified into several stages on the basis of the spreading of neurofibrillary deposits (4). It is therefore important to understand the factors underlying the abnormal aggregation of A␤ and tau. Both form filaments of Ϸ10 nm in width; in the case of A␤ they are smooth, while most tau filaments from Alzheimer brains show a characteristic ''paired helical'' structure, resembling two strands wound around one another, with a crossover periodicity of Ϸ80 nm and width varying between 10 and 20 nm (for review, see ref.The membrane-derived A␤-peptide is partly hydrophobic so that its tendency to aggregate is intuitively understandable (6, 7). By contrast, the cytosolic tau has a very hydrophilic character and is highly soluble (8, 9). Thus, it shows hardly any tendency to aggregate in physiological buffer conditions, and the formation of aggregates is very slow (days or weeks; ref. 10). Soluble tau protein contains very little secondary structure (␣-helix or ␤-sheet content Ͻ 5%), and the same holds for Alzheimer PHFs, in spite of their long-range periodicity (11). It therefore is not obvious why tau should aggregate in a specific manner and which structural principle could be responsible for this. Progress in understanding the mechanism has been correspondingly slow. Several factors supporting assembly have emerged in recent years. (i) The repeat domain in the C-terminal half of tau forms PHFs mo...

Summary DNA mismatch repair corrects errors that have escaped polymerase proofreading, increasing replication fidelity 100- to 1000-fold in organisms ranging from bacteria to humans. The MutL protein plays a central role in mismatch repair by coordinating multiple protein-protein interactions that signal strand removal upon mismatch recognition by MutS. Here we report the crystal structure of the endonuclease domain of Bacillus subtilis MutL. The structure is organized in dimerization and regulatory subdomains connected by a helical lever spanning the conserved endonuclease motif. Additional conserved motifs cluster around the lever and define a Zn2+-binding site that is critical for MutL function in vivo. The structure unveils a powerful inhibitory mechanism to prevent undesired DNA nicking and allows us to propose a model describing how the interaction with MutS and the processivity clamp could license the endonuclease activity of MutL. The structure also provides a molecular framework to propose and test additional roles of MutL in mismatch repair.

To avoid mutations in the genome, DNA replication is generally followed by DNA mismatch repair (MMR). MMR starts when a MutS homolog recognizes a mismatch and undergoes an ATP-dependent transformation to an elusive sliding clamp state. How this transient state promotes MutL homolog recruitment and activation of repair is unclear. Here we present a crystal structure of the MutS/MutL complex using a site-specifically crosslinked complex and examine how large conformational changes lead to activation of MutL. The structure captures MutS in the sliding clamp conformation, where tilting of the MutS subunits across each other pushes DNA into a new channel, and reorientation of the connector domain creates an interface for MutL with both MutS subunits. Our work explains how the sliding clamp promotes loading of MutL onto DNA, to activate downstream effectors. We thus elucidate a crucial mechanism that ensures that MMR is initiated only after detection of a DNA mismatch.DOI: http://dx.doi.org/10.7554/eLife.06744.001

Based on crystal structure analysis of the Serratia nuclease and a sequence alignment of six related nucleases, conserved amino acid residues that are located in proximity to the previously identified catalytic site residue His89 were selected for a mutagenesis study. Five out of 12 amino acid residues analyzed turned out to be of particular importance for the catalytic activity of the enzyme: Arg57, Arg87, His89, Asn119 and Glu127. Their replacement by alanine, for example, resulted in mutant proteins of very low activity, < 1% of the activity of the wild-type enzyme. Steady-state kinetic analysis of the mutant proteins demonstrates that some of these mutants are predominantly affected in their kcat, others in their Km. These results and the determination of the pH and metal ion dependence of selected mutant proteins were used for a tentative assignment for the function of these amino acid residues in the mechanism of phosphodiester bond cleavage by the Serratia nuclease.

Over the past few years the systematic investigation of paired helical filament assembly from tau protein in vitro has become feasible. We review our current understanding of the structure and conformations of tau protein and how this affects tau's assembly into the pathological paired helical filaments in Alzheimer's disease.