Gene-ius at work: Hemophilia B treatment enters a new era.

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Insight into human Miro1/2 domain organization based on the structure of its N-terminal GTPase.

Dysfunction in mitochondrial dynamics is believed to contribute to a host of neurological disorders and has recently been implicated in cancer metastasis. The outer mitochondrial membrane adapter protein Miro functions in the regulation of mitochondrial mobility and degradation, however, the structural basis for its roles in mitochondrial regulation remain unknown. Here, we report a 1.

UbMES and UbFluor: Novel probes for ring-between-ring (RBR) E3 ubiquitin ligase PARKIN.

Ring-between-ring (RBR) E3 ligases have been implicated in autoimmune disorders and neurodegenerative diseases. The functions of many RBR E3s are poorly defined, and their regulation is complex, involving post-translational modifications and allosteric regulation with other protein partners. The functional complexity of RBRs, coupled with the complexity of the native ubiquitination reaction that requires ATP and E1 and E2 enzymes, makes it difficult to study these ligases for basic research and therapeutic purposes.

Src family kinase phosphorylation of the motor domain of the human kinesin-5, Eg5.

Spindle formation in mammalian cells requires precise spatial and temporal regulation of the kinesin-5, Eg5, which generates outward force to establish spindle bipolarity. Our results demonstrate that Eg5 is phosphorylated in cultured cells by Src family kinases (SFKs) at three sites in the motor head: Y125, Y211, and Y231. Mutation of these sites diminishes motor activity in vitro, and replacement of endogenous Eg5 with phosphomimetic Y211 in LLC-Pk1 cells results in monopolar spindles, consistent with loss of Eg5 activity.

Structural insights into Parkin substrate lysine targeting from minimal Miro substrates.

Hereditary Parkinson's disease is commonly caused by mutations in the protein kinase PINK1 or the E3 ubiquitin ligase Parkin, which function together to eliminate damaged mitochondria. PINK1 phosphorylates both Parkin and ubiquitin to stimulate ubiquitination of dozens of proteins on the surface of the outer mitochondrial membrane. However, the mechanisms by which Parkin recognizes specific proteins for modification remain largely unexplored.

UbFluor: A Mechanism-Based Probe for HECT E3 Ligases.

Homologous to E6AP Carboxyl Terminus E3 ubiquitin ligases (HECT, ~28 known) are genetically implicated in cancer, neurological, hypertensive, and autoimmune disorders, and are potential drug targets to treat these diseases. The major bottleneck in the field of HECT E3s is a lack of simple assays to quantify the enzymatic activity of these enzymes in the presence of small molecules. Typical assays require E1, E2, HECT E3, ubiquitin (Ub), ATP and additional reagents to detect the resulting free poly-ubiquitin chains.

Survey of phosphorylation near drug binding sites in the Protein Data Bank (PDB) and their effects.

While it is currently estimated that 40 to 50% of eukaryotic proteins are phosphorylated, little is known about the frequency and local effects of phosphorylation near pharmaceutical inhibitor binding sites. In this study, we investigated how frequently phosphorylation may affect the binding of drug inhibitors to target proteins. We examined the 453 non-redundant structures of soluble mammalian drug target proteins bound to inhibitors currently available in the Protein Data Bank (PDB).

Model-independent decomposition of two-state data.

Two-state models often provide a reasonable approximation of protein behaviors such as partner binding, folding, and conformational changes. Many different techniques have been developed to determine the population ratio between two states as a function of different experimental conditions. Data analysis is accomplished either by fitting individual measured spectra to a linear combination of known basis spectra or alternatively by decomposing the entire set of spectra into two components using a least-squares optimization of free parameters within an assumed population model.

Intrinsic Disorder in the Kinesin Superfamily.

Kinesin molecular motors perform a myriad of intracellular transport functions. While their mechanochemical mechanisms are well understood and well-conserved throughout the superfamily, the cargo-binding and regulatory mechanisms governing the activity of kinesins are highly diverse and in general, are incompletely characterized. Here we present evidence from bioinformatic predictions indicating that most kinesin superfamily members contain significant regions of intrinsically disordered (ID) residues.

Mechanism and regulation of kinesin-5, an essential motor for the mitotic spindle.

Mitotic cell division is the most fundamental task of all living cells. Cells have intricate and tightly regulated machinery to ensure that mitosis occurs with appropriate frequency and high fidelity. A core element of this machinery is the kinesin-5 motor protein, which plays essential roles in spindle formation and maintenance.

Structural coupling of the EF hand and C-terminal GTPase domains in the mitochondrial protein Miro.

Miro is a highly conserved calcium-binding GTPase at the regulatory nexus of mitochondrial transport and autophagy. Here we present crystal structures comprising the tandem EF hand and carboxy terminal GTPase (cGTPase) domains of Drosophila Miro. The structures reveal two previously unidentified 'hidden' EF hands, each paired with a canonical EF hand.

Plant Kinesin-Like Calmodulin Binding Protein Employs Its Regulatory Domain for Dimerization.

Kinesin-like calmodulin binding protein (KCBP), a Kinesin-14 family motor protein, is involved in the structural organization of microtubules during mitosis and trichome morphogenesis in plants. The molecular mechanism of microtubule bundling by KCBP remains unknown. KCBP binding to microtubules is regulated by Ca(2+)-binding proteins that recognize its C-terminal regulatory domain.

Kinesin tail domains are intrinsically disordered.

Kinesin motor proteins transport a wide variety of molecular cargoes in a spatially and temporally regulated manner. Kinesin motor domains, which hydrolyze ATP to produce a directed mechanical force along a microtubule, are well conserved throughout the entire superfamily. Outside of the motor domains, kinesin sequences diverge along with their transport functions.

The loop 5 element structurally and kinetically coordinates dimers of the human kinesin-5, Eg5.

Eg5 is a homotetrameric kinesin-5 motor protein that generates outward force on the overlapping, antiparallel microtubules (MTs) of the mitotic spindle. Upon binding an MT, an Eg5 dimer releases one ADP molecule, undergoes a slow (∼0.5 s(-1)) isomerization, and finally releases a second ADP, adopting a tightly MT-bound, nucleotide-free (APO) conformation.

The optical trapping dumbbell assay for nonprocessive motors or motors that turn around filaments.

In vitro motility assays enabled the analysis of coupling between ATP hydrolysis and movement of myosin along actin filaments or kinesin along microtubules. Single-molecule assays using laser trapping have been used to obtain more detailed information about kinesins, myosins, and processive DNA enzymes. The combination of in vitro motility assays with laser-trap measurements has revealed detailed dynamic structural changes associated with the ATPase cycle.

Attachment of anti-GFP antibodies to microspheres for optical trapping experiments.

In vitro motility assays enabled the analysis of coupling between ATP hydrolysis and movement of myosin along actin filaments or kinesin along microtubules. Single-molecule assays using laser trapping have been used to obtain more detailed information about kinesins, myosins, and processive DNA enzymes. The combination of in vitro motility assays with laser-trap measurements has revealed detailed dynamic structural changes associated with the ATPase cycle.

Optical traps to study properties of molecular motors.

In vitro motility assays enabled the analysis of coupling between ATP hydrolysis and movement of myosin along actin filaments or kinesin along microtubules. Single-molecule assays using laser trapping have been used to obtain more detailed information about kinesins, myosins, and processive DNA enzymes. The combination of in vitro motility assays with laser-trap measurements has revealed detailed dynamic structural changes associated with the ATPase cycle.

X-ray diffraction from intact tau aggregates in human brain tissue.

We describe an instrument to record x-ray diffraction patterns from diseased regions of human brain tissue by combining an in-line visible light fluorescence microscope with an x-ray diffraction microprobe. We use thiazine red fluorescence to specifically label and detect the filamentous tau protein pathology associated with Pick's disease, as several labs have done previously. We demonstrate that thiazine red-enhanced regions within the tissue show periodic structure in x-ray diffraction that is not observed in healthy tissue.

Analysis of the interaction of the Eg5 Loop5 with the nucleotide site.

Loop 5 (L5) is a conserved loop that projects from the α2-helix adjacent to the nucleotide site of all kinesin-family motors. L5 is critical to the function of the mitotic kinesin-5 family motors and is the binding site for several kinesin-5 inhibitors that are currently in clinical trials. Its conformational dynamics and its role in motor function are not fully understood.

Kinesin's light chains inhibit the head- and microtubule-binding activity of its tail.

Kinesin-1 is a microtubule-based motor comprising two heavy chains (KHCs) and two light chains (KLCs). Motor activity is precisely regulated to avoid futile ATP consumption and to ensure proper intracellular localization of kinesin-1 and its cargoes. The KHC tail inhibits ATPase activity by interacting with the enzymatic KHC heads, and the tail also binds microtubules.

The conserved L5 loop establishes the pre-powerstroke conformation of the Kinesin-5 motor, eg5.

Kinesin superfamily motor proteins contain a structurally conserved loop near the ATP binding site, termed L5. The function of L5 is unknown, although several drug inhibitors of the mitotic kinesin Eg5 bind to L5. We used electron paramagnetic resonance spectroscopy (EPR) to investigate the function of L5 in Eg5.

Microtubule-mediated transport of the tumor-suppressor protein Merlin and its mutants.

The neurofibromatosis type 2 (NF2) tumor-suppressor protein Merlin is a member of the ERM family of proteins that links the cytoskeleton to the plasma membrane. In humans, mutations in the NF2 gene cause neurofibromatosis type-2 (NF2), a cancer syndrome characterized by the development of tumors of the nervous system. Previous reports have suggested that the subcellular distribution of Merlin is critical to its function, and that several NF2 mutants that lack tumor-suppressor activity present improper localization.

Microtubule-associated protein-like binding of the kinesin-1 tail to microtubules.

The kinesin-1 molecular motor contains an ATP-dependent microtubule-binding site in its N-terminal head domain and an ATP-independent microtubule-binding site in its C-terminal tail domain. Here we demonstrate that a kinesin-1 tail fragment associates with microtubules with submicromolar affinity. Binding is largely electrostatic in nature, and is facilitated by a region of basic amino acids in the tail and the acidic E-hook at the C terminus of tubulin.

Opposite-polarity motors activate one another to trigger cargo transport in live cells.

Intracellular transport is typically bidirectional, consisting of a series of back and forth movements. Kinesin-1 and cytoplasmic dynein require each other for bidirectional transport of intracellular cargo along microtubules; i.e.