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 1.7Å crystal structure of N-terminal GTPase domain (nGTPase) of human Miro1 bound unexpectedly to GTP, thereby revealing a non-catalytic configuration of the putative GTPase active site. We identify two conserved surfaces of the nGTPase, the "SELFYY" and "ITIP" motifs, that are potentially positioned to mediate dimerization or interaction with binding partners. Additionally, we report small angle X-ray scattering (SAXS) data obtained from the intact soluble HsMiro1 and its paralog HsMiro2. Taken together, the data allow modeling of a crescent-shaped assembly of the soluble domain of HsMiro1/2. PDB RSEFERENCE: Crystal structure of the human Miro1 N-terminal GTPase bound to GTP, 6D71.

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. To address this challenge, we developed novel chemical probes, ubiquitin C-terminal fluorescein thioesters UbMES and UbFluor, to qualitatively and quantitatively assess the activity of the RBR E3 ligase PARKIN in a simple experimental setup and in real time using fluorescence polarization. First, we confirmed that PARKIN does not require an E2 enzyme for substrate ubiquitination, lysine selection, and polyubiquitin chain formation. Second, we confirmed that UbFluor quantitatively detects naturally occurring activation states of PARKIN caused by Ser65 phosphorylation (pPARKIN) and phosphorylated ubiquitin (pUb). Third, we showed that both pUb and the ubiquitin-accepting substrate contribute to maximal pPARKIN ubiquitin conjugation turnover. pUb enhances the transthiolation step, whereas the substrate clears the pPARKIN∼Ub thioester intermediate. Finally, we established that UbFluor can quantify activation or inhibition of PARKIN by structural mutations. These results demonstrate the feasibility of using UbFluor for quantitative studies of the biochemistry of RBR E3s and for high-throughput screening of small-molecule activators or inhibitors of PARKIN and other RBR E3 ligases.

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. Cells treated with SFK inhibitors show defects in spindle formation, similar to those in cells expressing the nonphosphorylatable Y211 mutant, and distinct from inhibition of other mitotic kinases. We propose that this phosphoregulatory mechanism tunes Eg5 enzymatic activity for optimal spindle morphology.

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. Here, we show that the C-terminal GTPase (cGTPase) of the Parkin primary substrate human Miro is necessary and sufficient for efficient ubiquitination. We present several new X-ray crystal structures of both human Miro1 and Miro2 that reveal substrate recognition and ubiquitin transfer to be specific to particular protein domains and lysine residues. We also provide evidence that Parkin substrate recognition is functionally separate from substrate modification. Finally, we show that prioritization for modification of a specific lysine sidechain of the cGTPase (K572) within human Miro1 is dependent on both its location and chemical microenvironment. Activation of Parkin by phosphorylation or by binding of pUb is required for prioritization of K572 for modification, suggesting that Parkin activation and acquisition of substrate specificity are coupled.

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. To address this need, we developed UbFluor, a fluorescent thioester conjugate between the C-terminus of Ub and fluorescein-thiol (Fluor-SH). UbFluor is a mechanism-based probe that undergoes a direct transthiolation reaction with the catalytic cysteine of the model HECT E3 ligase Rsp5, producing the catalytically active Rsp5~Ub (~ indicates thioester) accompanied by release of Fluor-SH. The kinetics of this two-component reaction can be easily monitored with real-time fluorescence polarization (FP) assays. Importantly, UbFluor eliminates the need to use SDS-PAGE, ATP, E1, E2 enzymes, and extra poly-ubiquitin chain detection reagents. Although the developed system lacks ATP, E1 and E2 enzymes, we show that UbFluor can recapitulate the native ubiquitination reaction by detecting and quantifying defects in transthiolation and isopeptide ligation of Rsp5 HECT E3 alanine mutants. Based on our findings, we show that UbFluor can be utilized to conduct high-throughput screens (HTS) of small molecules against HECT ligases.

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). We cross-referenced these structures with phosphorylation data available from the PhosphoSitePlus database. Three hundred twenty-two of 453 (71%) of drug targets have evidence of phosphorylation that has been validated by multiple methods or labs. For 132 of 453 (29%) of those, the phosphorylation site is within 12 Å of the small molecule-binding site, where it would likely alter small molecule binding affinity. We propose a framework for distinguishing between drug-phosphorylation site interactions that are likely to alter the efficacy of drugs versus those that are not. In addition we highlight examples of well-established drug targets, such as estrogen receptor alpha, for which phosphorylation may affect drug affinity and clinical efficacy. Our data suggest that phosphorylation may affect drug binding and efficacy for a significant fraction of drug target proteins.

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. In this review, we discuss how the structural and mechanical properties of kinesin-5 motors uniquely suit them to their mitotic role. We describe some of the small molecule inhibitors and regulatory proteins that act on kinesin-5, and discuss how these regulators may influence the process of cell division. Finally, we touch on some more recently described functions of kinesin-5 motors in non-dividing cells. Throughout, we highlight a number of open questions that impede our understanding of both this motor's function and the potential utility of kinesin-5 inhibitors.

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. ID regions can bind to multiple partners with high specificity, and are highly labile to post-translational modification and degradation signals. In kinesins, the predicted ID regions are primarily found in areas outside the motor domains, where primary sequences diverge by family, suggesting that ID may be a critical structural element for determining the functional specificity of individual kinesins. To support this idea, we present a systematic analysis of the kinesin superfamily, family by family, for predicted regions of ID. We combine this analysis with a comprehensive review of kinesin binding partners and post-translational modifications. We find two key trends across the entire kinesin superfamily. First, ID residues tend to be in the tail regions of kinesins, opposite the superfamily-conserved motor domains. Second, predicted ID regions correlate to regions that are known to bind to cargoes and/or undergo post-translational modifications. We therefore propose that ID is a structural element utilized by the kinesin superfamily in order to impart functional specificity to individual kinesins.

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. Each EF hand pair is bound to a helix that structurally mimics an EF hand ligand. A key nucleotide-sensing element and a Pink1 phosphorylation site both lie within an extensive EF hand-cGTPase interface. Our results indicate structural mechanisms for calcium, nucleotide and phosphorylation-dependent regulation of mitochondrial function by Miro.

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. In this work, we have discovered a new function of the regulatory domain. We present a crystal structure of an Arabidopsis KCBP fragment showing that the C-terminal regulatory domain forms a dimerization interface for KCBP. This dimerization site is distinct from the dimerization interface within the N-terminal domain. Side chains of hydrophobic residues of the calmodulin binding helix of the regulatory domain form the C-terminal dimerization interface. Biochemical experiments show that another segment of the regulatory domain located beyond the dimerization interface, its negatively charged coil, is unexpectedly and absolutely required to stabilize the dimers. The strong microtubule bundling properties of KCBP are unaffected by deletion of the C-terminal regulatory domain. The slow minus-end directed motility of KCBP is also unchanged in vitro. Although the C-terminal domain is not essential for microtubule bundling, we suggest that KCBP may use its two independent dimerization interfaces to support different types of bundled microtubule structures in cells. Two distinct dimerization sites may provide a mechanism for microtubule rearrangement in response to Ca(2+) signaling since Ca(2+)- binding proteins can disengage KCBP dimers dependent on its C-terminal dimerization interface.

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. Here we demonstrate that it is possible to determine the population ratio in a two-state system directly from data without an a priori model for basis spectra or populations by applying physical constraints iteratively to a singular value decomposition of optical fluorescence, x-ray-scattering, and electron paramagnetic resonance data.

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 nonmotor regions, particularly the tails, respond to a wide variety of structural and molecular cues that enable kinesins to carry specific cargoes in response to particular cellular signals. Here, we demonstrate that intrinsic disorder is a common structural feature of kinesins. A bioinformatics survey of the full-length sequences of all 43 human kinesins predicts that significant regions of intrinsically disordered residues are present in all kinesins. These regions are concentrated in the nonmotor domains, particularly in the tails and near sites for ligand binding or post-translational modifications. In order to experimentally verify these predictions, we expressed and purified the tail domains of kinesins representing three different families (Kif5B, Kif10, and KifC3). Circular dichroism and NMR spectroscopy experiments demonstrate that the isolated tails are disordered in vitro, yet they retain their functional microtubule-binding activity. On the basis of these results, we propose that intrinsic disorder is a common structural feature that confers functional specificity to kinesins.

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. This article describes the use of optical traps to study processive and nonprocessive molecular motor proteins, focusing on the design of the instrument and the assays to characterize motility.

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. This protocol describes a method for attaching anti-GFP (green fluorescent protein) antibodies to microspheres. GFP-motor fusion proteins can then be adsorbed to the microspheres for use in single-molecule motility studies and optical trapping experiments.

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. This protocol describes the preparation of biotin-actin filaments and coverslips coated with polystyrene beads. These are then used in optical trapping dumbbell assays to study interactions between motors and filaments.

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. Our previous work using EPR spectroscopy suggested that L5 alters the nucleotide pocket conformation of the kinesin-5 motor Eg5 (Larson et al., 2010). EPR spectra of a spin-labeled nucleotide analog bound at the nucleotide site of Eg5 display a highly immobilized component that is absent if L5 is shortened or if the inhibitor STLC is added (Larson et al., 2010), which X-ray structures suggest stabilizes an L5 conformation pointing away from the nucleotide site. These data, coupled with the proximity of L5 to the nucleotide site suggest L5 could interact with a bound nucleotide, modulating function. Here we use molecular dynamics (MD) simulations of Eg5 to explore the interaction of L5 with the nucleotide site in greater detail. We performed MD simulations in which the L5-domain of the Eg5·ADP X-ray structure was manually deformed via backbone bond rotations. The L5-domain of Eg5 was sufficiently lengthy that portions of L5 could be located in proximity to bound ADP. The MD simulations evolved to thermodynamically stable structures at 300 K showing that L5 can interact directly with bound nucleotide with significant impingement on the ribose hydroxyls, consistent with the EPR spectroscopy results. Taken together, these data provide support for the hypothesis that L5 modulates Eg5 function via interaction with the nucleotide-binding site.

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. One observed periodicity (4.2 Å) is characteristic of cross-beta sheet structure, consistent with previous results from powder diffraction studies performed on purified, dried tau protein.

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. This conformation precedes ATP binding and stepping. Here, we use mutagenesis, steady-state and pre-steady-state kinetics, motility assays, and electron paramagnetic resonance spectroscopy to examine Eg5 monomers and dimers as they bind MTs and initiate stepping. We demonstrate that a critical element of Eg5, loop 5 (L5), accelerates ADP release during the initial MT-binding event. Furthermore, our electron paramagnetic resonance data show that L5 mediates the slow isomerization by preventing Eg5 dimer heads from binding the MT until they release ADP. Finally, we find that Eg5 having a seven-residue deletion within L5 can still hydrolyze ATP and move along MTs, suggesting that L5 is not required to accelerate subsequent steps of the motor along the MT. Taken together, these properties of L5 explain the kinetic effects of L5-directed inhibition on Eg5 activity and may direct further interventions targeting Eg5 activity.

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. We site-specifically attached EPR probes to ADP, L5, and the neck linker element that docks along the enzymatic head to drive forward motility on microtubules (MTs). Nucleotide-dependent spectral mobility shifts occurred in all of these structural elements, suggesting that they undergo coupled conformational changes. These spectral shifts were altered by deletion of L5 or addition of S-trityl-l-cysteine (STLC), an allosteric inhibitor that binds to L5. In particular, EPR probes attached to the neck linker of MT-bound Eg5 shifted to a more immobilized component in the nucleotide-free state relative to the ADP-bound state, consistent with the neck linker docking upon ADP release. In contrast, after L5 deletion or STLC addition, EPR spectra were highly immobilized in all nucleotide states. We conclude that L5 undergoes a conformational change that enables Eg5 to bind to MTs in a pre-powerstroke state. Deletion or inhibition of L5 with the small-molecule inhibitor STLC blocks this pre-powerstroke state, forcing the Eg5 neck linker to dock regardless of the nucleotide state.