Oligomer Formation of Tau Protein Hyperphosphorylated in Cells

Abnormal phosphorylation (“hyperphosphorylation”) and aggregation of Tau protein are hallmarks of Alzheimer disease and other tauopathies, but their causative connection is still a matter of debate. Tau with Alzheimer-like phosphorylation is also present in hibernating animals, mitosis, or during embryonic development, without leading to pathophysiology or neurodegeneration. Thus, the role of phosphorylation and the distinction between physiological and pathological phosphorylation needs to be further refined. So far, the systematic investigation of highly phosphorylated Tau was difficult because a reliable method of preparing reproducible quantities was not available. Here, we generated full-length Tau (2N4R) in Sf9 cells in a well defined phosphorylation state containing up to ∼20 phosphates as judged by mass spectrometry and Western blotting with phospho-specific antibodies. Despite the high concentration in living Sf9 cells (estimated ∼230 μm) and high phosphorylation, the protein was not aggregated. However, after purification, the highly phosphorylated protein readily formed oligomers, whereas fibrils were observed only rarely. Exposure of mature primary neuronal cultures to oligomeric phospho-Tau caused reduction of spine density on dendrites but did not change the overall cell viability.     

Alternative Conformations of the Tau Repeat Domain in Complex with an Engineered Binding Protein

The aggregation of Tau into paired helical filaments is involved in the pathogenesis of several neurodegenerative diseases, including Alzheimer disease. The aggregation reaction is characterized by conformational conversion of the repeat domain, which partially adopts a cross-β-structure in the resulting amyloid-like fibrils. Here, we report the selection and characterization of an engineered binding protein, β-wrapin TP4, targeting the Tau repeat domain. TP4 was obtained by phage display using the four-repeat Tau construct K18ΔK280 as a target. TP4 binds K18ΔK280 as well as the longest isoform of human Tau, hTau40, with nanomolar affinity. NMR spectroscopy identified two alternative TP4-binding sites in the four-repeat domain, with each including two hexapeptide motifs with high β-sheet propensity. Both binding sites contain the aggregation-determining PHF6 hexapeptide within repeat 3. In addition, one binding site includes the PHF6* hexapeptide within repeat 2, whereas the other includes the corresponding hexapeptide Tau(337–342) within repeat 4, denoted PHF6**. Comparison of TP4-binding with Tau aggregation reveals that the same regions of Tau are involved in both processes. TP4 inhibits Tau aggregation at substoichiometric concentration, demonstrating that it interferes with aggregation nucleation. This study provides residue-level insight into the interaction of Tau with an aggregation inhibitor and highlights the structural flexibility of Tau.     

Mass distribution and spatial organization of the linear bacterial motor of Spiroplasma citri R8A2

In the simple, helical, wall-less bacterial genus Spiroplasma, chemotaxis and motility are effected by a linear, contractile motor arranged as a flat cytoskeletal ribbon attached to the inner side of the membrane along the shortest helical line. With scanning transmission electron microscopy and diffraction analysis, we determined the hierarchical and spatial organization of the cytoskeleton of Spiroplasma citri R8A2. The structural unit appears to be a fibril, approximately 5 nm wide, composed of dimers of a 59-kDa protein; each ribbon is assembled from seven fibril pairs. The functional unit of the intact ribbon is a pair of aligned fibrils, along which pairs of dimers form tetrameric ring-like repeats. On average, isolated and purified ribbons contain 14 fibrils or seven well-aligned fibril pairs, which are the same structures observed in the intact cell. Scanning transmission electron microscopy mass analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified cytoskeletons indicate that the 59-kDa protein is the only constituent of the ribbons.     

Polymorphic C-terminal β-Sheet Interactions Determine the Formation of Fibril or Amyloid β-derived Diffusible Ligand-like Globulomer for the Alzheimer Aβ42 Dodecamer

The relationship between amyloid deposition and cellular toxicity is still controversial. In addition to fibril-forming oligomers, other soluble Aβ forms (amyloid β-derived diffusible ligands (ADDLs)) were also suggested to form and to present different morphologies and mechanisms of toxicity. One ADDL type, the “globulomer,” apparently forms independently of the fibril aggregation pathway. Even though many studies argue that such soluble Aβ oligomers are off fibril formation pathways, they may nonetheless share some structural similarity with protofibrils. NMR data of globulomer intermediates, “preglobulomers,” suggested parallel in-register C-terminal β-sheets, with different N-terminal conformations. Based on experimental data, we computationally investigate four classes of Aβ dodecamers: fibril, fibril oligomer, prefibril/preglobulomer cluster, and globulomer models. Our simulations of the solvent protection of double-layered fibril and globulomer models reproduce experimental observations. Using a single layer Aβ fibril oligomer β-sheet model, we found that the C-terminal β-sheet in the fibril oligomer is mostly curved, preventing it from quickly forming a fibril and leading to its breaking into shorter pieces. The simulations also indicate that β-sheets packed orthogonally could be the most stable species for Aβ dodecamers. The major difference between fibril-forming oligomers and ADDL-like oligomers (globulomers) could be the exposure of Met-35 patches. Although the Met-35 patches are necessarily exposed in fibril-forming oligomers to allow their maturation into fibrils, the Met-35 patches in the globulomer are covered by other residues in the orthogonally packed Aβ peptides. Our results call attention to the possible existence of certain “critical intermediates” that can lead to both seeds and other soluble ADDL-like oligomers.     

The Self-assembly of a Mini-fibril with Axial Periodicity from a Designed Collagen-mimetic Triple Helix

In this work we describe the self-assembly of a collagen-like periodic mini-fibril from a recombinant triple helix. The triple helix, designated Col108, is expressed in Escherichia coli using an artificial gene and consists of a 378-residue triple helix domain organized into three pseudo-repeating sequence units. The peptide forms a stable triple helix with a melting temperature of 41 °C. Upon increases of pH and temperature, Col108 self-assembles in solution into smooth mini-fibrils with the cross-striated banding pattern typical of fibrillar collagens. The banding pattern is characterized by an axially repeating feature of ∼35 nm as observed by transmission electron microscopy and atomic force microscopy. Both the negatively stained and the positively stained transmission electron microscopy patterns of the Col108 mini-fibrils are consistent with a staggered arrangement of triple helices having a staggering value of 123 residues, a value closely connected to the size of one repeat sequence unit. A mechanism is proposed for the mini-fibril formation of Col108 in which the axial periodicity is instigated by the built-in sequence periodicity and stabilized by the optimized interactions between the triple helices in a 1-unit staggered arrangement. Lacking hydroxyproline residues and telopeptides, two factors implicated in the fibrillogenesis of native collagen, the Col108 mini-fibrils demonstrate that sequence features of the triple helical domain alone are sufficient to “code” for axially repeating periodicity of fibrils. To our knowledge, Col108 is the first designed triple helix to self-assemble into periodic fibrils and offers a unique opportunity to unravel the specific molecular interactions of collagen fibrillogenesis.     

Atomic Force Microscopy Imaging and Nanomechanical Properties of Six Tau Isoform Assemblies

The amyloid fibrillar form of the protein Tau is involved in a number of neurodegenerative diseases, also known as tauopathies. In this work, six different fibrillar Tau isoforms were assembled in vitro. The morphological and nanomechanical properties of these isoforms were studied using atomic force microscopy at high resolution in air and buffer. Our results demonstrate that all Tau isoform fibrils exhibit paired-helical-filament-like structures consisting of two protofibrils separated by a shallow groove. Interestingly, whereas the N-terminal inserts do not contribute to any morphological or mechanical difference between the isoforms with the same carboxyl-terminal microtubule-binding domain repeats, isoforms with four microtubule repeats (4R) exhibited a persistence length ranging from 2.0 to 2.8 μm, almost twofold higher than those with three repeats (3R). In addition, the axial Young’s modulus values derived from the persistence lengths, as well as their radial ones determined via nanoindentation experiments, were very low compared to amyloid fibrils made of other proteins. This sheds light on the weak intermolecular interaction acting between the paired β-sheets within Tau fibrils. This may play an important role in their association into high molecular weight assemblies, their dynamics, their persistence, their clearance in cells, and their propagation.     

Ordered Regions of Channel Nucleoporins Nup62, Nup54, and Nup58 Form Dynamic Complexes in Solution

Three out of ∼30 nucleoporins, Nup62, Nup54, and Nup58, line the nuclear pore channel. These “channel” nucleoporins each contain an ordered region of ∼150–200 residues, which is predicted to be segmented into 3–4 α-helical regions of ∼40–80 residues. Notably, these segmentations are evolutionarily conserved between uni- and multicellular eukaryotes. Strikingly, the boundaries of these segments match our previously reported mapping and crystal data, which collectively identified two “cognate” segments of Nup54, each interacting with cognate segments, one in Nup58 and the other one in Nup62. Because Nup54 and Nup58 cognate segments form crystallographic hetero- or homo-oligomers, we proposed that these oligomers associate into inter-convertible “mid-plane” rings: a single large ring (40–50 nm diameter, consisting of eight hetero-dodecamers) or three small rings (10–20 nm diameter, each comprising eight homo-tetramers). Each “ring cycle” would recapitulate “dilation” and “constriction” of the nuclear pore complex''s central transport channel. As for the Nup54·Nup62 interactome, it forms a 1:2 triple helix (“finger”), multiples of which project alternately up and down from mid-plane ring(s). Collectively, our previous crystal data suggested a copy number of 128, 64, and 32 for Nup62, Nup54, and Nup58, respectively, that is, a 4:2:1 stoichiometry. Here, we carried out solution analysis utilizing the entire ordered regions of Nup62, Nup54, and Nup58, and demonstrate that they form a dynamic “triple complex” that is heterogeneously formed from our previously characterized Nup54·Nup58 and Nup54·Nup62 interactomes. These data are consistent both with our crystal structure-deduced copy numbers and stoichiometries and also with our ring cycle model for structure and dynamics of the nuclear pore channel.     

The dual fates of exogenous tau seeds: Lysosomal clearance versus cytoplasmic amplification

Tau assembly movement from the extracellular to intracellular space may underlie transcellular propagation of neurodegenerative tauopathies. This begins with tau binding to cell surface heparan sulfate proteoglycans, which triggers macropinocytosis. Pathological tau assemblies are proposed then to exit the vesicular compartment as “seeds” for replication in the cytoplasm. Tau uptake is highly efficient, but only ∼1 to 10% of cells that endocytose aggregates exhibit seeding. Consequently, we studied fluorescently tagged full-length (FL) tau fibrils added to native U2OS cells or “biosensor” cells expressing FL tau or repeat domain. FL tau fibrils bound tubulin. Seeds triggered its aggregation in multiple locations simultaneously in the cytoplasm, generally independent of visible exogenous aggregates. Most exogenous tau trafficked to the lysosome, but fluorescence imaging revealed a small percentage that steadily accumulated in the cytosol. Intracellular expression of Gal3-mRuby, which binds intravesicular galactosides and forms puncta upon vesicle rupture, revealed no evidence of vesicle damage following tau exposure, and most seeded cells had no evidence of endolysosome rupture. However, live-cell imaging indicated that cells with pre-existing Gal3-positive puncta were seeded at a slightly higher rate than the general population, suggesting a potential predisposing role for vesicle instability. Clearance of tau seeds occurred rapidly in both vesicular and cytosolic fractions. The lysosome/autophagy inhibitor bafilomycin inhibited vesicular clearance, whereas the proteasome inhibitor MG132 inhibited cytosolic clearance. Tau seeds that enter the cell thus have at least two fates: lysosomal clearance that degrades most tau, and entry into the cytosol, where seeds amplify, and are cleared by the proteasome.     

Acid-induced Dissociation of VacA, the Helicobacter pylori Vacuolating Cytotoxin, Reveals Its Pattern of Assembly

In this study, we describe the ultrastructural changes associated with acid activation of Helicobacter pylori vacuolating cytotoxin (VacA). Purified VacA molecules imaged by deep-etch electron microscopy form ~30-nm hexagonal “flowers,” each composed of an ~15-nm central ring surrounded by six ~6-nm globular “petals.” Upon exposure to acidic pH, these oligomeric flowers dissociate into collections of up to 12 teardrop-shaped subunits, each measuring ~6 × 14 nm. Correspondingly, glycerol density gradient centrifugation shows that at neutral pH VacA sediments at ~22 S, whereas at acidic pH it dissociates and sediments at ~5 S. Immunoblot and EM analysis of the 5-S material demonstrates that it represents ~90-kD monomers with 6 × 14–nm “teardrop” morphology. These data indicate that the intact VacA oligomer consists of 12 ~90-kD subunits assembled into two interlocked six-membered arrays, overlap of which gives rise to the flower-like appearance. Support for this interpretation comes from EM identification of small numbers of relatively “flat” oligomers composed of six teardrop-shaped subunits, interpreted to be halves of the complete flower. These flat forms adsorb to mica in two different orientations, corresponding to hexameric surfaces that are either exposed or sandwiched inside the dodecamer, respectively. This view of VacA structure differs from a previous model in which the flowers were interpreted to be single layers of six monomers and the flat forms were thought to be proteolysed flowers. Since acidification has been shown to potentiate the cytotoxic effects of VacA, the present results suggest that physical disassembly of the VacA oligomer is an important feature of its activation.     

In Sup35p filaments (the [PSI+] prion), the globular C-terminal domains are widely offset from the amyloid fibril backbone

In yeast cells infected with the [PSI+] prion, Sup35p forms aggregates and its activity in translation termination is downregulated. Transfection experiments have shown that Sup35p filaments assembled in vitro are infectious, suggesting that they reproduce or closely resemble the prion. We have used several EM techniques to study the molecular architecture of filaments, seeking clues as to the mechanism of downregulation. Sup35p has an N-terminal 'prion' domain; a highly charged middle (M-)domain; and a C-terminal domain with the translation termination activity. By negative staining, cryo-EM and scanning transmission EM (STEM), filaments of full-length Sup35p show a thin backbone fibril surrounded by a diffuse 65-nm-wide cloud of globular C-domains. In diameter (～8 nm) and appearance, the backbones resemble amyloid fibrils of N-domains alone. STEM mass-per-unit-length data yield ～1 subunit per 0.47 nm for N-fibrils, NM-filaments and Sup35p filaments, further supporting the fibril backbone model. The 30 nm radial span of decorating C-domains indicates that the M-domains assume highly extended conformations, offering an explanation for the residual Sup35p activity in infected cells, whereby the C-domains remain free enough to interact with ribosomes.     

Conformation Determines the Seeding Potencies of Native and Recombinant Tau Aggregates

Intracellular Tau inclusions are a pathological hallmark of several neurodegenerative diseases, collectively known as the tauopathies. They include Alzheimer disease, tangle-only dementia, Pick disease, argyrophilic grain disease, chronic traumatic encephalopathy, progressive supranuclear palsy, and corticobasal degeneration. Tau pathology appears to spread through intercellular propagation, requiring the formation of assembled “prion-like” species. Several cell and animal models have been described that recapitulate aspects of this phenomenon. However, the molecular characteristics of seed-competent Tau remain unclear. Here, we have used a cell model to understand the relationships between Tau structure/phosphorylation and seeding by aggregated Tau species from the brains of mice transgenic for human mutant P301S Tau and full-length aggregated recombinant P301S Tau. Deletion of motifs ²⁷⁵VQIINK²⁸⁰ and ³⁰⁶VQIVYK³¹¹ abolished the seeding activity of recombinant full-length Tau, suggesting that its aggregation was necessary for seeding. We describe conformational differences between native and synthetic Tau aggregates that may account for the higher seeding activity of native assembled Tau. When added to aggregated Tau seeds from the brains of mice transgenic for P301S Tau, soluble recombinant Tau aggregated and acquired the molecular properties of aggregated Tau from transgenic mouse brain. We show that seeding is conferred by aggregated Tau that enters cells through macropinocytosis and seeds the assembly of endogenous Tau into filaments.     

Co-immunoprecipitation with Tau Isoform-specific Antibodies Reveals Distinct Protein Interactions and Highlights a Putative Role for 2N Tau in Disease

Alternative splicing generates multiple isoforms of the microtubule-associated protein Tau, but little is known about their specific function. In the adult mouse brain, three Tau isoforms are expressed that contain either 0, 1, or 2 N-terminal inserts (0N, 1N, and 2N). We generated Tau isoform-specific antibodies and performed co-immunoprecipitations followed by tandem mass tag multiplexed quantitative mass spectrometry. We identified novel Tau-interacting proteins of which one-half comprised membrane-bound proteins, localized to the plasma membrane, mitochondria, and other organelles. Tau was also found to interact with proteins involved in presynaptic signal transduction. MetaCore analysis revealed one major Tau interaction cluster that contained 33 Tau pulldown proteins. To explore the pathways in which these proteins are involved, we conducted an ingenuity pathway analysis that revealed two significant overlapping pathways, “cell-to-cell signaling and interaction” and “neurological disease.” The functional enrichment tool DAVID showed that in particular the 2N Tau-interacting proteins were specifically associated with neurological disease. Finally, for a subset of Tau interactions (apolipoprotein A1 (apoA1), apoE, mitochondrial creatine kinase U-type, β-synuclein, synaptogyrin-3, synaptophysin, syntaxin 1B, synaptotagmin, and synapsin 1), we performed reverse co-immunoprecipitations, confirming the preferential interaction of specific isoforms. For example, apoA1 displayed a 5-fold preference for the interaction with 2N, whereas β-synuclein showed preference for 0N. Remarkably, a reverse immunoprecipitation with apoA1 detected only the 2N isoform. This highlights distinct protein interactions of the different Tau isoforms, suggesting that they execute different functions in brain tissue.     

Studying early stages of fibronectin fibrillogenesis in living cells by atomic force microscopy

Fibronectin (FN) is an extracellular matrix protein that can be assembled by cells into large fibrillar networks, but the dynamics of FN remodeling and the transition through intermediate fibrillar stages are incompletely understood. Here we used a combination of fluorescence microscopy and time-lapse atomic force microscopy (AFM) to visualize initial stages of FN fibrillogenesis in living fibroblasts at high resolution. Initial FN nanofibrils form within <5 min of cell–matrix contact and subsequently extend at a rate of 0.25 μm/min at sites of cell membrane retraction. FN nanofibrils display a complex linear array of globular features spaced at varying distances, indicating the coexistence of different conformational states within the fibril. In some cases, initial fibrils extended in discrete increments of ∼800 nm during a series of cyclical membrane retractions, indicating a stepwise fibrillar extension mechanism. In presence of Mn²⁺, a known activator of integrin adhesion to FN, fibrillogenesis was accelerated almost threefold to 0.68 μm/min and fibrillar dimensions were increased, underlining the importance of integrin activation for early FN fibrillogenesis. FN fibrillogenesis visualized by time-lapse AFM thus provides new structural and mechanistic insight into initial steps of cell-driven FN fibrillogenesis.     

Dual Role of an N-terminal Amyloidogenic Mutation in Apolipoprotein A-I: DESTABILIZATION OF HELIX BUNDLE AND ENHANCEMENT OF FIBRIL FORMATION*

A number of naturally occurring mutations of apolipoprotein (apo) A-I, the major protein of HDL, are known to be associated with hereditary amyloidosis and atherosclerosis. Here, we examined the effects of the G26R point mutation in apoA-I (apoA-I_Iowa) on the structure, stability, and aggregation propensity to form amyloid fibril of full-length apoA-I and the N-terminal fragment of apoA-I. Circular dichroism and fluorescence measurements demonstrated that the G26R mutation destabilizes the N-terminal helix bundle domain of full-length protein, leading to increased hydrophobic surface exposure, whereas it has no effect on the initial structure of the N-terminal 1–83 fragment, which is predominantly a random coil structure. Upon incubation for extended periods at neutral pH, the N-terminal 1–83 variants undergo a conformational change to β-sheet-rich structure with a great increase in thioflavin T fluorescence, whereas no structural change is observed in full-length proteins. Comparison of fibril-forming propensity among substituted mutants at Gly-26 position of 1–83 fragments demonstrated that the G26R mutation enhances the nucleation step of fibril formation, whereas G26K and G26E mutations have small or inhibiting effects on the formation of fibrils. These fibrils of the 1–83 variants have long and straight morphology as revealed by atomic force microscopy and exhibited significant toxicity with HEK293 cells. Our results indicate dual critical roles of the arginine residue at position 26 in apoA-I_Iowa: destabilization of the N-terminal helix bundle structure in full-length protein and enhancement of amyloid fibril formation by the N-terminal 1–83 fragment.     

Tau protein assembles into isoform- and disulfide-dependent polymorphic fibrils with distinct structural properties

Tauopathies are neurodegenerative diseases in which insoluble fibrillar aggregates of a microtubule-binding protein, Tau, are abnormally accumulated. Pathological Tau fibrils often exhibit structural polymorphisms that differ among phenotypically distinct tauopathies; however, a molecular mechanism to generate polymorphic Tau fibrils remains obscure. Here, we note the formation of a disulfide bond in isoforms of full-length Tau and show that the thiol-disulfide status as well as the isoform composition determines structural and morphological properties of Tau fibrils in vitro. Mainly two regions in a Tau primary sequence are found to act as structural blocks for building a protease-resistant core of Tau fibrils. Interactions among those two blocks for building a core structure depend upon the thiol-disulfide status in each isoform of Tau, which results in the formation of polymorphic fibrils with distinct structural properties. Furthermore, we have found that more diverse structures of Tau fibrils emerge through a cross-seeded fibrillation between heterologous pairs of Tau isoforms. We thus propose that isoform- and disulfide-dependent combinatorial interactions among multiple regions in a Tau sequence endow Tau fibrils with various structures, i.e. polymorphism.     

The Oxytricha trifallax Macronuclear Genome: A Complex Eukaryotic Genome with 16,000 Tiny Chromosomes

The macronuclear genome of the ciliate Oxytricha trifallax displays an extreme and unique eukaryotic genome architecture with extensive genomic variation. During sexual genome development, the expressed, somatic macronuclear genome is whittled down to the genic portion of a small fraction (∼5%) of its precursor “silent” germline micronuclear genome by a process of “unscrambling” and fragmentation. The tiny macronuclear “nanochromosomes” typically encode single, protein-coding genes (a small portion, 10%, encode 2–8 genes), have minimal noncoding regions, and are differentially amplified to an average of ∼2,000 copies. We report the high-quality genome assembly of ∼16,000 complete nanochromosomes (∼50 Mb haploid genome size) that vary from 469 bp to 66 kb long (mean ∼3.2 kb) and encode ∼18,500 genes. Alternative DNA fragmentation processes ∼10% of the nanochromosomes into multiple isoforms that usually encode complete genes. Nucleotide diversity in the macronucleus is very high (SNP heterozygosity is ∼4.0%), suggesting that Oxytricha trifallax may have one of the largest known effective population sizes of eukaryotes. Comparison to other ciliates with nonscrambled genomes and long macronuclear chromosomes (on the order of 100 kb) suggests several candidate proteins that could be involved in genome rearrangement, including domesticated MULE and IS1595-like DDE transposases. The assembly of the highly fragmented Oxytricha macronuclear genome is the first completed genome with such an unusual architecture. This genome sequence provides tantalizing glimpses into novel molecular biology and evolution. For example, Oxytricha maintains tens of millions of telomeres per cell and has also evolved an intriguing expansion of telomere end-binding proteins. In conjunction with the micronuclear genome in progress, the O. trifallax macronuclear genome will provide an invaluable resource for investigating programmed genome rearrangements, complementing studies of rearrangements arising during evolution and disease.     

Charge Neutralization of the Central Lysine Cluster in Prion Protein (PrP) Promotes PrPSc-like Folding of Recombinant PrP Amyloids

The structure of the infectious form of prion protein, PrP^Sc, remains unclear. Most pure recombinant prion protein (PrP) amyloids generated in vitro are not infectious and lack the extent of the protease-resistant core and solvent exclusion of infectious PrP^Sc, especially within residues ∼90–160. Polyanionic cofactors can enhance infectivity and PrP^Sc-like characteristics of such fibrils, but the mechanism of this enhancement is unknown. In considering structural models of PrP^Sc multimers, we identified an obstacle to tight packing that might be overcome with polyanionic cofactors, namely, electrostatic repulsion between four closely spaced cationic lysines within a central lysine cluster of residues 101–110. For example, in our parallel in-register intermolecular β-sheet model of PrP^Sc, not only would these lysines be clustered within the 101–110 region of the primary sequence, but they would have intermolecular spacings of only ∼4.8 Å between stacked β-strands. We have now performed molecular dynamics simulations predicting that neutralization of the charges on these lysine residues would allow more stable parallel in-register packing in this region. We also show empirically that substitution of these clustered lysine residues with alanines or asparagines results in recombinant PrP amyloid fibrils with extended proteinase-K resistant β-sheet cores and infrared spectra that are more reminiscent of bona fide PrP^Sc. These findings indicate that charge neutralization at the central lysine cluster is critical for the folding and tight packing of N-proximal residues within PrP amyloid fibrils. This charge neutralization may be a key aspect of the mechanism by which anionic cofactors promote PrP^Sc formation.     

A Residue-specific Shift in Stability and Amyloidogenicity of Antibody Variable Domains

Variable (V) domains of antibodies are essential for antigen recognition by our adaptive immune system. However, some variants of the light chain V domains (V_L) form pathogenic amyloid fibrils in patients. It is so far unclear which residues play a key role in governing these processes. Here, we show that the conserved residue 2 of V_L domains is crucial for controlling its thermodynamic stability and fibril formation. Hydrophobic side chains at position 2 stabilize the domain, whereas charged residues destabilize and lead to amyloid fibril formation. NMR experiments identified several segments within the core of the V_L domain to be affected by changes in residue 2. Furthermore, molecular dynamic simulations showed that hydrophobic side chains at position 2 remain buried in a hydrophobic pocket, and charged side chains show a high flexibility. This results in a predicted difference in the dissociation free energy of ∼10 kJ mol⁻¹, which is in excellent agreement with our experimental values. Interestingly, this switch point is found only in V_L domains of the κ family and not in V_Lλ or in V_H domains, despite a highly similar domain architecture. Our results reveal novel insight into the architecture of variable domains and the prerequisites for formation of amyloid fibrils. This might also contribute to the rational design of stable variable antibody domains.     

Double Strand Break Unwinding and Resection by the Mycobacterial Helicase-Nuclease AdnAB in the Presence of Single Strand DNA-binding Protein (SSB)

Mycobacterial AdnAB is a heterodimeric DNA helicase-nuclease and 3′ to 5′ DNA translocase implicated in the repair of double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Inclusion of mycobacterial single strand DNA-binding protein (SSB) in reactions containing linear plasmid dsDNA allowed us to study the AdnAB helicase under conditions in which the unwound single strands are coated by SSB and thereby prevented from reannealing or promoting ongoing ATP hydrolysis. We found that the AdnAB motor catalyzed processive unwinding of 2.7–11.2-kbp linear duplex DNAs at a rate of ∼250 bp s⁻¹, while hydrolyzing ∼5 ATPs per bp unwound. Crippling the AdnA phosphohydrolase active site did not affect the rate of unwinding but lowered energy consumption slightly, to ∼4.2 ATPs bp⁻¹. Mutation of the AdnB phosphohydrolase abolished duplex unwinding, consistent with a model in which the “leading” AdnB motor propagates a Y-fork by translocation along the 3′ DNA strand, ahead of the “lagging” AdnA motor domain. By tracking the resection of the 5′ and 3′ strands at the DSB ends, we illuminated a division of labor among the AdnA and AdnB nuclease modules during dsDNA unwinding, whereby the AdnA nuclease processes the unwound 5′ strand to liberate a short oligonucleotide product, and the AdnB nuclease incises the 3′ strand on which the motor translocates. These results extend our understanding of presynaptic DSB processing by AdnAB and engender instructive comparisons with the RecBCD and AddAB clades of bacterial helicase-nuclease machines.