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E Mandelkow

Publications and source records attributed to E Mandelkow.

At least 19 recordsLinked to original sources
PubMed2006

Spectroscopic approaches to the conformation of tau protein in solution and in paired helical filaments.

The abnormal aggregation of the microtubule-associated protein tau into paired helical filaments is one the hallmarks of Alzheimer's disease. This aggregation is based in the partial formation of beta-structure. In contrast, the soluble protein shows a mostly random coil structure, as judged by circular dichroism, Fourier transform infrared, X-ray scattering and biochemical assays. Here, we review the basis of the natively unstructured character of tau, as well as recent studies of residual structure and long-range interactions between different domains of the protein. Analysis of the primary structure reveals a very low content of hydrophobic amino acids and a high content of charged residues, both of which tend to counteract a well-folded globular state of proteins. In the case of tau, the low overall hydrophobicity is sufficient to explain the lack of folding. This is in contrast to other proteins which also carry an excess charge at physiological pH. By tryptophan scanning mutagenesis and fluorimetry we found that most of the sequence is solvent exposed. Analysis of the hydrodynamic radii confirms a mostly random coil structure of various tau isoforms and tau domains. The proteins can be further expanded by denaturation with GdHCl which indicates some global folding. This was substantiated by a FRET-based approach where the distances between different domains of tau were determined. The combined data show that tau is mostly disordered and flexible but tends to assume a hairpin-like overall fold which may be important in the transition to a pathological aggregate.

Animals↗
PubMed2005

Interaction of kinesin motors, microtubules, and MAPs.

Kinesins are a family of microtubule-dependent motor proteins that carry cargoes such as vesicles, organelles, or protein complexes along microtubules. Here we summarize structural studies of the "conventional" motor protein kinesin-1 and its interactions with microtubules, as determined by X-ray crystallography and cryo-electron microscopy. In particular, we consider the docking between the kinesin motor domain and tubulin subunits and summarize the evidence that kinesin binds mainly to beta tubulin with the switch-2 helix close to the intradimer interface between alpha and beta tubulin.

Animals↗
PubMed2005

The structure of microtubule motor proteins.

Microtubules are the intracellular tracks for two classes of motor proteins: kinesins and dyneins. During the past few years, the motor domain structures of several kinesins from different organisms have been determined by X-ray crystallography. Compared with kinesins, dyneins are much larger proteins and attempts to crystallize them have failed so far. Structural information about these proteins comes mostly from electron microscopy. In this chapter, we mainly focus on the crystal structures of kinesin motor domains.

Animals↗
PubMed2004

Surface-decoration of microtubules by human tau.

Tau is a neuronal, microtubule-associated protein that stabilizes microtubules and promotes neurite outgrowth. Tau is largely unfolded in solution and presumably forms mostly random coil. Because of its hydrophilic nature and flexible structure, tau complexed to microtubules is largely invisible by standard electron microscopy methods. We applied a combination of high-resolution metal-shadowing and cryo-electron microscopy to study the interactions between tau and microtubules. We used recombinant tau variants with different domain compositions, (1) full length tau, (2) the repeat domain that mediates microtubule binding (K19), and (3) two GFP-tau fusion proteins that contain a globular marker (GFP) attached to full-length tau at either end. All of these constructs bind exclusively to the outside of microtubules. Most of the tau-related mass appears randomly distributed, creating a "halo" of low-density mass spread across the microtubule surface. Only a small fraction of tau creates a periodic signal at an 8 nm interval, centered on alpha-tubulin subunits. Our data suggest that tau retains most of its disordered structure even when bound to the microtubule surface. Hence, it binds along, as well as across protofilaments. Nevertheless, even minute concentrations of tau have a strong stabilizing effect and effectively scavenge unpolymerized tubulin.

Cryoelectron Microscopy↗
PubMed2003

Clogging of axons by tau, inhibition of axonal traffic and starvation of synapses.

Loss of synapses and dying back of axons are considered early events in brain degeneration during Alzheimer's disease. This is accompanied by an aberrant behavior of the microtubule-associated protein tau (hyperphosphorylation, aggregation). Since microtubules are the tracks for axonal transport, we are testing the hypothesis that tau plays a role in the malfunctioning of transport. Experiments with various neuronal and non-neuronal cells show that tau is capable of reducing net anterograde transport of vesicles and cell organelles by blocking the microtubule tracks. Thus, a misregulation of tau could cause the starvation of synapses and enhanced oxidative stress, long before tau detaches from microtubules and aggregates into Alzheimer neurofibrillary tangles. In particular, the transport of amyloid precursor protein is retarded when tau is elevated, suggesting a possible link between the two key proteins that show abnormal behavior in Alzheimer's disease.

Alzheimer Disease↗
PubMed2002

Toward a unified scheme for the aggregation of tau into Alzheimer paired helical filaments.

Alzheimer's disease is characterized by aggregates of tau protein. Attempts to study the conditions for aggregation in vitro have led to different experimental systems, some of which appear mutually exclusive (e.g., oxidative vs reductive conditions, induction by polyanions vs fatty acids). We show here that different approaches and pathways can be viewed in a common framework, and that apparent differences can be explained by variations in the kinetics of subreactions. A unified view of PHF aggregation should help to analyze the causes of PHF aggregation and devise methods to prevent it.

Alzheimer Disease↗
PubMed2002

Tau blocks traffic of organelles, neurofilaments, and APP vesicles in neurons and enhances oxidative stress.

We studied the effect of microtubule-associated tau protein on trafficking of vesicles and organelles in primary cortical neurons, retinal ganglion cells, and neuroblastoma cells. Tau inhibits kinesin-dependent transport of peroxisomes, neurofilaments, and Golgi-derived vesicles into neurites. Loss of peroxisomes makes cells vulnerable to oxidative stress and leads to degeneration. In particular, tau inhibits transport of amyloid precursor protein (APP) into axons and dendrites, causing its accumulation in the cell body. APP tagged with yellow fluorescent protein and transfected by adenovirus associates with vesicles moving rapidly forward in the axon (approximately 80%) and slowly back (approximately 20%). Both movements are strongly inhibited by cotransfection with fluorescently tagged tau (cyan fluorescent protein-tau) as seen by two-color confocal microscopy. The data suggests a linkage between tau and APP trafficking, which may be significant in Alzheimer's disease.

Alzheimer Disease↗
PubMed2001

Structure of a fast kinesin: implications for ATPase mechanism and interactions with microtubules.

We determined the crystal structure of the motor domain of the fast fungal kinesin from Neurospora crassa (NcKin). The structure has several unique features. (i) Loop 11 in the switch 2 region is ordered and enables one to describe the complete nucleotide-binding pocket, including three inter-switch salt bridges between switch 1 and 2. (ii) Loop 9 in the switch 1 region bends outwards, making the nucleotide-binding pocket very wide. The displacement in switch 1 resembles that of the G-protein ras complexed with its guanosine nucleotide exchange factor. (iii) Loop 5 in the entrance to the nucleotide-binding pocket is remarkably long and interacts with the ribose of ATP. (iv) The linker and neck region is not well defined, indicating that it is mobile. (v) Image reconstructions of ice-embedded microtubules decorated with NcKin show that it interacts with several tubulin subunits, including a central beta-tubulin monomer and the two flanking alpha-tubulin monomers within the microtubule protofilament. Comparison of NcKin with other kinesins, myosin and G-proteins suggests that the rate-limiting step of ADP release is accelerated in the fungal kinesin and accounts for the unusually high velocity and ATPase activity.

Adenosine Triphosphatases↗
PubMed2001

Mutations of tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local beta-structure.

The microtubule-associated protein tau is a natively unfolded protein in solution, yet it is able to polymerize into the ordered paired helical filaments (PHF) of Alzheimer's disease. In the splice isoforms lacking exon 10, this process is facilitated by the formation of beta-structure around the hexapeptide motif PHF6 ((306)VQIVYK(311)) encoded by exon 11. We have investigated the structural requirements for PHF polymerization in the context of adult tau isoforms containing four repeats (including exon 10). In addition to the PHF6 motif there exists a related PHF6* motif ((275)VQIINK(280)) in the repeat encoded by the alternatively spliced exon 10. We show that this PHF6* motif also promotes aggregation by the formation of beta-structure and that there is a cross-talk between the two hexapeptide motifs during PHF aggregation. We also show that two of the tau mutations found in hereditary frontotemporal dementias, DeltaK280 and P301L, have a much stronger tendency for PHF aggregation which correlates with their high propensity for beta-structure around the hexapeptide motifs.

Amino Acid Motifs↗
PubMed2001

Dynamics and cooperativity of microtubule decoration by the motor protein kinesin.

We describe a theoretical and experimental analysis of the interaction between microtubules and dimeric motor proteins (kinesin, NCD), with special emphasis on the stoichiometry of the interaction, cooperative effects, and their consequences for the interpretation of biochemical and image reconstruction results. Monomeric motors can bind equivalently to microtubules without interference, at a stoichiometry of one motor head per tubulin subunit (alphabeta-heterodimer). By contrast, dimeric motors can interact with stoichiometries ranging between one and two heads per tubulin subunit, depending on binding constants of the first head and the subsequent binding of the second head, and the concentration of dimers in solution. Further, we show that an attractive interaction between the bound motor molecules can explain the higher periodicities observed in decorated microtubules (e.g. 16 nm periodicity), and the non-uniform decoration of a population of microtubules and give an estimate of the strength of this interaction.

Allosteric Site↗
PubMed2000

Conformational preferences of a synthetic 30mer peptide from the interface between the neck and stalk regions of kinesin.

The conformation of a synthetic peptide, consisting of 30 amino acids spanning the neck and hinge regions of rat brain kinesin, was investigated by NMR spectroscopy. The peptide extends from K357 to D386 and has the sequence KSVIQHLEVELNRWRNGEAVPEDEQISAKD. A total of 82 distance range constraints and 23 dihedral angle constraints could be obtained from NOESY and E.COSY spectra, respectively. These were used to calculate 500 structures by applying the REDAC algorithm of the software package DYANA. The first half of the peptide matched the helical structure of the neck determined from an X-ray crystal structure of kinesin. This part normally dimerizes into a coiled-coil by virtue of a leucine zipper interaction, but it is alpha-helical even in the monomeric state. The second half (not visible in the X-ray structure because of disorder) contains locally defined structure elements (extended chain, helical loop) connected by flexible joints. This is consistent with the "hinge" function postulated for this domain which is important for kinesin's motility and orientation.

Amino Acid Sequence↗
PubMed2000

Nonsaturable binding indicates clustering of tau on the microtubule surface in a paired helical filament-like conformation.

Tau protein modulates microtubule dynamics and forms insoluble aggregates in Alzheimer's disease. Because there is a discrepancy between reported affinities of Tau to microtubules, we determined the interaction over a wide concentration range using a sensitive enzyme-linked immunosorbent assay. We found that the interaction is biphasic and not monophasic as assumed earlier. The first binding phase is typical for identical and noninteracting binding sites, with dissociation constants around 0.1 micrometer and stoichiometries around 0.2 Tau/tubulin dimer. Surprisingly, the second phase is nonsaturable and shows a nearly linear increase in bound Tau versus free Tau for free Tau concentrations higher than 2 micrometer. The slope is proportional to the microtubule concentration. From this we define an overloading parameter with values around 50 micrometer. The influence of Tau isoform, phosphorylation, and dimerization on both phases was investigated. Remarkably the overloading of Tau on microtubules leads to a thioflavin S fluorescence increase reminiscent of that seen with Tau aggregated into Alzheimer paired helical filaments. Because polyanions stimulate Tau aggregation and because the C-terminal domain of tubulin is polyanionic, we suggest that an early conformational change in Tau leading to paired helical filament aggregation occurs right on the microtubule surface.

Alzheimer Disease↗
PubMed2000

Structure, microtubule interactions, and paired helical filament aggregation by tau mutants of frontotemporal dementias.

We have studied biochemical and structural parameters of several missense and deletion mutants of tau protein (G272V, N279K, DeltaK280, P301L, V337M, R406W) found in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). The mutant proteins were expressed on the basis of both full-length tau (htau40) and constructs derived from the repeat domain. They were analyzed with respect to the capacity to enhance microtubule assembly, binding of tau to microtubules, secondary structure content, and aggregation into Alzheimer-like paired helical or straight filaments. We find that the mutations cause a moderate decrease in microtubule interactions and stabilization, and they show no gross structural changes compared with the natively unfolded conformation of the wild-type protein, but the aggregation into PHFs is strongly enhanced, particularly for the mutants DeltaK280 and P301L. This gain of pathological aggregation would be consistent with the autosomal dominant nature of the disease.

Actin Cytoskeleton↗
PubMed2000

Structure of tau protein and assembly into paired helical filaments.

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.

Alzheimer Disease↗
PubMed2000

Assembly of tau protein into Alzheimer paired helical filaments depends on a local sequence motif ((306)VQIVYK(311)) forming beta structure.

We have searched for a minimal interaction motif in tau 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 tau 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 tau. Probing the interactions of PHF43 with overlapping peptides derived from the full tau 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 beta-structure potential in tau. CD and Fourier transform infrared spectroscopy shows that PHF43 acquires pronounced beta structure in conditions of self assembly. Point mutations in the hexapeptide region by proline-scanning mutagenesis prevent the aggregation. The data indicate that PHF assembly is initiated by a short fragment containing the minimal interaction motif forming a local beta structure embedded in a largely random-coil protein.

Alzheimer Disease↗
PubMed2000

A new look at the microtubule binding patterns of dimeric kinesins.

The interactions of monomeric and dimeric kinesin and ncd constructs with microtubules have been investigated using cryo-electron microscopy (cryo-EM) and several biochemical methods. There is a good consensus on the structure of dimeric ncd when bound to a tubulin dimer showing one head attached directly to tubulin, and the second head tethered to the first. However, the 3D maps of dimeric kinesin motor domains are still quite controversial and leave room for different interpretations. Here we reinvestigated the microtubule binding patterns of dimeric kinesins by cryo-EM and digital 3D reconstruction under different nucleotide conditions and different motor:tubulin ratios, and determined the molecular mass of motor-tubulin complexes by STEM. Both methods revealed complementary results. We found that the ratio of bound kinesin motor-heads to alphabeta-tubulin dimers was never reaching above 1.5 irrespective of the initial mixing ratios. It appears that each kinesin dimer occupies two microtubule-binding sites, provided that there is a free one nearby. Thus the appearances of different image reconstructions can be explained by non-specific excess binding of motor heads. Consequently, the use of different apparent density distributions for docking the X-ray structures onto the microtubule surface leads to different and mutually exclusive models. We propose that in conditions of stoichiometric binding the two heads of a kinesin dimer separate and bind to different tubulin subunits. This is in contrast to ncd where the two heads remain tightly attached on the microtubule surface. Using dimeric kinesin molecules crosslinked in their neck domain we also found that they stabilize protofilaments axially, but not laterally, which is a strong indication that the two heads of the dimers bind along one protofilament, rather than laterally bridging two protofilaments. A molecular walking model based on these results summarizes our conclusions and illustrates the implications of symmetry for such models.

Adenosine Triphosphate↗
PubMed2000

On the rigidity of the cytoskeleton: are MAPs crosslinkers or spacers of microtubules?

Microtubules are fibers of the cytoskeleton involved in mitosis, intracellular transport, motility and other functions. They contain microtubule-associated proteins (MAPs) bound to their surface which stabilize microtubules and promote their assembly. There has been a debate on additional functions of MAPs, e.g. whether MAPs crosslink microtubules and thus increase their rigidity, or whether they act as spacers between them. We have studied the packing of microtubules in the presence of MAPs by solution X-ray scattering using synchrotron radiation. Microtubules free in solution produce a scattering pattern typical of an isolated hollow cylinder, whereas tightly packed microtubules generate a pattern dominated by interparticle interference. The interference patterns are interpreted in terms of the Hosemann paracrystal concept, adapted for arrays of parallel fibers with hexagonal arrangement in the plane perpendicular to the fiber axes (Briki et al., 1998). Microtubules without MAPs can rapidly and efficiently be compressed by centrifugation, as judged by the transition from a "free microtubule" to a "packed microtubule" X-ray scattering pattern. MAPs make the microtubule array highly resistant to packing, even at high centrifugal forces. This emphasizes the role of MAPs as spacers of microtubules rather than crosslinkers. A possible function is to keep the microtubule tracks free for the approach of motor proteins carrying vesicle or organelle cargoes along microtubules.

Animals↗