RESUMO
BACKGROUND: The motor protein dynein is integral to retrograde transport along microtubules and interacts with numerous cargoes through the recruitment of cargo-specific adaptor proteins. This interaction is mediated by dynein light intermediate chain subunits LIC1 (DYNC1LI1) and LIC2 (DYNC1LI2), which govern the adaptor binding and are present in distinct dynein complexes with overlapping and unique functions. METHODS: Using bioinformatics, we analyzed the C-terminal domains (CTDs) of LIC1 and LIC2, revealing similar structural features but diverse post-translational modifications (PTMs). The methylation status of LIC2 and the proteins involved in this modification were examined through immunoprecipitation and immunoblotting analyses. The specific methylation sites on LIC2 were identified through a site-directed mutagenesis analysis, contributing to a deeper understanding of the regulatory mechanisms of the dynein complex. RESULTS: We found that LIC2 is specifically methylated at the arginine 397 residue, a reaction that is catalyzed by protein arginine methyltransferase 1 (PRMT1). CONCLUSIONS: The distinct PTMs of the LIC subunits offer a versatile mechanism for dynein to transport diverse cargoes efficiently. Understanding how these PTMs influence the functions of LIC2, and how they differ from LIC1, is crucial for elucidating the role of dynein-related transport pathways in a range of diseases. The discovery of the arginine 397 methylation site on LIC2 enhances our insight into the regulatory PTMs of dynein functions.
Assuntos
Arginina , Dineínas do Citoplasma , Proteína-Arginina N-Metiltransferases , Proteínas Repressoras , Metilação , Arginina/metabolismo , Arginina/química , Humanos , Dineínas do Citoplasma/metabolismo , Dineínas do Citoplasma/genética , Dineínas do Citoplasma/química , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Processamento de Proteína Pós-Traducional , Dineínas/metabolismo , Dineínas/genética , Dineínas/química , Sequência de AminoácidosRESUMO
Dynein is a ~1.2 MDa cytoskeletal motor protein that carries organelles via retrograde transport in eukaryotic cells. The motor protein belongs to the ATPase family of proteins associated with diverse cellular activities and plays a critical role in transporting cargoes to the minus end of the microtubules. The motor domain of dynein possesses a hexameric head, where ATP hydrolysis occurs. The presented work analyzes the structure-activity relationship (SAR) of dynapyrazole A and B, as well as ciliobrevin A and D, in their various protonated states and their 46 analogues for their binding in the AAA1 subunit, the leading ATP hydrolytic site of the motor domain. This study exploits in silico methods to look at the analogues' effects on the functionally essential subsites of the motor domain of dynein 1, since no similar experimental structural data are available. Ciliobrevin and its analogues bind to the ATP motifs of the AAA1, namely, the walker-A (W-A) or P-loop, the walker-B (W-B), and the sensor I and II. Ciliobrevin A shows a better binding affinity than its D analogue. Although the double bond in ciliobrevin A and D was expected to decrease the ligand potency, they show a better affinity to the AAA1 binding site than dynapyrazole A and B, lacking the bond. In addition, protonation of the nitrogen atom in ciliobrevin A and D, as well as dynapyrazole A and B, at the N9 site of ciliobrevin and the N7 of the latter increased their binding affinity. Exploring ciliobrevin A geometrical configuration suggests the E isomer has a superior binding profile over the Z due to binding at the critical ATP motifs. Utilizing the refined structure of the motor domain obtained through protein conformational search in this study exhibits that Arg1852 of the yeast cytoplasmic dynein could involve in the "glutamate switch" mechanism in cytoplasmic dynein 1 in lieu of the conserved Asn in AAA+ protein family.
Assuntos
Trifosfato de Adenosina/metabolismo , Dineínas/química , Quinazolinonas/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Sítios de Ligação , Transporte Biológico , Simulação por Computador , Citoplasma/metabolismo , Dineínas do Citoplasma/química , Dineínas do Citoplasma/metabolismo , Dineínas/antagonistas & inibidores , Dineínas/metabolismo , Hidrólise , Microtúbulos/metabolismo , Ligação Proteica , Conformação Proteica , Quinazolinonas/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-AtividadeRESUMO
Cytoplasmic dynein is the primary motor for microtubule minus-end-directed transport and is indispensable to eukaryotic cells. Although each motor domain of dynein contains three active AAA+ ATPases (AAA1, 3, and 4), only the functions of AAA1 and 3 are known. Here, we use single-molecule fluorescence and optical tweezers studies to elucidate the role of AAA4 in dynein's mechanochemical cycle. We demonstrate that AAA4 controls the priming stroke of the motion-generating linker, which connects the dimerizing tail of the motor to the AAA+ ring. Before ATP binds to AAA4, dynein remains incapable of generating motion. However, when AAA4 is bound to ATP, the gating of AAA1 by AAA3 prevails and dynein motion can occur. Thus, AAA1, 3, and 4 work together to regulate dynein function. Our work elucidates an essential role for AAA4 in dynein's stepping cycle and underscores the complexity and crosstalk among the motor's multiple AAA+ domains.
Assuntos
Dineínas do Citoplasma/química , Dineínas do Citoplasma/metabolismo , Domínio AAA , Trifosfato de Adenosina/metabolismo , Dineínas do Citoplasma/genética , Hidrólise , Microtúbulos/metabolismo , Movimento , Mutagênese , Pinças Ópticas , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismoRESUMO
Emerging evidence indicates that neurodegenerative diseases (NDs) result from a failure to clear toxic protein aggregates rather than from their generation. We previously showed N-acetylglucosamine kinase (NAGK) promotes dynein functionality and suggested this might promote aggregate removal and effectively address proteinopathies. Here, we report NAGK interacts with dynein light chain roadblock type 1 (DYNLRB1) and efficiently suppresses mutant huntingtin (mHtt) (Q74) and α-synuclein (α-syn) A53T aggregation in mouse brain cells. A kinase-inactive NAGKD107A also efficiently cleared Q74 aggregates. Yeast two-hybrid selection and in silico protein-protein docking analysis showed the small domain of NAGK (NAGK-DS) binds to the C-terminal of DYNLRB1. Furthermore, a small peptide derived from NAGK-DS interfered with Q74 clearance. We propose binding of NAGK-DS to DYNLRB1 'pushes up' the tail of dynein light chain and confers momentum for inactive phi- to active open-dynein transition.
Assuntos
Dineínas do Citoplasma/metabolismo , Dineínas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Agregados Proteicos , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Dineínas do Citoplasma/química , Células HEK293 , Humanos , Proteína Huntingtina/metabolismo , Camundongos , Mitocôndrias/metabolismo , Modelos Biológicos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/química , Ligação Proteica , Domínios Proteicos , RNA Interferente Pequeno/metabolismo , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Hub proteins participate in cellular regulation by dynamic binding of multiple proteins within interaction networks. The hub protein LC8 reversibly interacts with more than 100 partners through a flexible pocket at its dimer interface. To explore the diversity of the LC8 partner pool, we screened for LC8 binding partners using a proteomic phage display library composed of peptides from the human proteome, which had no bias toward a known LC8 motif. Of the identified hits, we validated binding of 29 peptides using isothermal titration calorimetry. Of the 29 peptides, 19 were entirely novel, and all had the canonical TQT motif anchor. A striking observation is that numerous peptides containing the TQT anchor do not bind LC8, indicating that residues outside of the anchor facilitate LC8 interactions. Using both LC8-binding and nonbinding peptides containing the motif anchor, we developed the "LC8Pred" algorithm that identifies critical residues flanking the anchor and parses random sequences to predict LC8-binding motifs with â¼78% accuracy. Our findings significantly expand the scope of the LC8 hub interactome.
Assuntos
Dineínas do Citoplasma/metabolismo , Peptídeos/química , Domínios e Motivos de Interação entre Proteínas , Algoritmos , Calorimetria , Proteínas de Ciclo Celular/metabolismo , Dineínas do Citoplasma/química , Dineínas do Citoplasma/genética , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Ligação Proteica , Proteômica , TermodinâmicaRESUMO
The tumor suppressor protein 53BP1 plays key roles in response to DNA double-strand breaks (DSBs) by serving as a master scaffold at the damaged chromatin. Current evidence indicates that 53BP1 assembles a cohort of DNA damage response (DDR) factors to distinctly execute its repertoire of DSB responses, including checkpoint activation and non-homologous end joining (NHEJ) repair. Here, we have uncovered LC8 (a.k.a. DYNLL1) as an important 53BP1 effector. We found that LC8 accumulates at laser-induced DNA damage tracks in a 53BP1-dependent manner and requires the canonical H2AX-MDC1-RNF8-RNF168 signal transduction cascade. Accordingly, genetic inactivation of LC8 or its interaction with 53BP1 resulted in checkpoint defects. Importantly, loss of LC8 alleviated the hypersensitivity of BRCA1-depleted cells to ionizing radiation and PARP inhibition, highlighting the 53BP1-LC8 module in counteracting BRCA1-dependent functions in the DDR. Together, these data establish LC8 as an important mediator of a subset of 53BP1-dependent DSB responses.
Assuntos
Dineínas do Citoplasma/fisiologia , Quebras de DNA de Cadeia Dupla , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Proteína BRCA1/genética , Linhagem Celular , Cromatina/metabolismo , Dineínas do Citoplasma/química , Dineínas do Citoplasma/metabolismo , Reparo do DNA , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases , Radiação IonizanteRESUMO
A power stroke of dynein is thought to be responsible for the stepping of dimeric dynein. However, the actual size of the displacement driven by a power stroke has not been directly measured. Here, the displacements of single-headed cytoplasmic dynein were measured by optical tweezers. The mean displacement of dynein interacting with microtubule was ~8 nm at 100 µM ATP, and decreased sigmoidally with a decrease in the ATP concentration. The ATP dependence of the mean displacement was explained by a model that some dynein molecules bind to microtubule in pre-stroke conformation and generate 8-nm displacement, while others bind in the post-stroke one and detach without producing a power stroke. Biochemical assays showed that the binding affinity of the post-stroke dynein to a microtubule was ~5 times higher than that of pre-stroke dynein, and the dissociation rate was ~4 times lower. Taking account of these rates, we conclude that the displacement driven by a power stroke is 8.3 nm. A working model of dimeric dynein driven by the 8-nm power stroke was proposed.
Assuntos
Dineínas do Citoplasma/química , Dineínas do Citoplasma/metabolismo , Dineínas/química , Dineínas/metabolismo , Pinças Ópticas , Trifosfato de Adenosina/metabolismo , Humanos , Cinética , Microtúbulos/metabolismo , Multimerização Proteica , Estrutura Quaternária de ProteínaRESUMO
Cytoplasmic dynein, whose motor domain belongs to the AAA+ family, walks on microtubules toward the minus end. Using the available structures in different nucleotide states, we performed simulations of a coarse-grained model to elucidate the dynamics of allosteric transitions. Binding of ATP closes the cleft between the AAA1 and AAA2 domains, triggering conformational changes in the rest of the motor domain, thus forming the pre-power stroke state. Interactions with the microtubule, modeled implicitly, enhance ADP release rate, and the formation of the post-power stroke state. The dynamics of the linker (LN), which reversibly changes from a straight to a bent state, is heterogeneous. Persistent interactions between the LN and the insert loops in the AAA2 domain prevent the formation of pre-power stroke state when ATP is bound to AAA3, thus locking dynein in a repressed non-functional state. Application of mechanical force to the LN restores motility in the repressed state.
Assuntos
Trifosfato de Adenosina/metabolismo , Dineínas do Citoplasma/química , Dineínas do Citoplasma/metabolismo , Regulação Alostérica , Animais , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , Domínios ProteicosRESUMO
Polarised mRNA transport is a prevalent mechanism for spatial control of protein synthesis. However, the composition of transported ribonucleoprotein particles (RNPs) and the regulation of their movement are poorly understood. We have reconstituted microtubule minus end-directed transport of mRNAs using purified components. A Bicaudal-D (BicD) adaptor protein and the RNA-binding protein Egalitarian (Egl) are sufficient for long-distance mRNA transport by the dynein motor and its accessory complex dynactin, thus defining a minimal transport-competent RNP. Unexpectedly, the RNA is required for robust activation of dynein motility. We show that a cis-acting RNA localisation signal promotes the interaction of Egl with BicD, which licenses the latter protein to recruit dynein and dynactin. Our data support a model for BicD activation based on RNA-induced occupancy of two Egl-binding sites on the BicD dimer. Scaffolding of adaptor protein assemblies by cargoes is an attractive mechanism for regulating intracellular transport.
Assuntos
Proteínas de Drosophila/genética , Complexo Dinactina/genética , Dineínas/genética , Animais , Sítios de Ligação , Dineínas do Citoplasma/química , Dineínas do Citoplasma/genética , Proteínas de Drosophila/química , Drosophila melanogaster/genética , Complexo Dinactina/química , Dineínas/química , Ligação Proteica/genética , Multimerização Proteica , Transporte Proteico/genética , Transporte de RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genéticaRESUMO
The transcription factor ASCIZ (ATMIN, ZNF822) has an unusually high number of recognition motifs for the product of its main target gene, the hub protein LC8 (DYNLL1). Using a combination of biophysical methods, structural analysis by NMR and electron microscopy, and cellular transcription assays, we developed a model that proposes a concerted role of intrinsic disorder and multiple LC8 binding events in regulating LC8 transcription. We demonstrate that the long intrinsically disordered C-terminal domain of ASCIZ binds LC8 to form a dynamic ensemble of complexes with a gradient of transcriptional activity that is inversely proportional to LC8 occupancy. The preference for low occupancy complexes at saturating LC8 concentrations with both human and Drosophila ASCIZ indicates that negative cooperativity is an important feature of ASCIZ-LC8 interactions. The prevalence of intrinsic disorder and multivalency among transcription factors suggests that formation of heterogeneous, dynamic complexes is a widespread mechanism for tuning transcriptional regulation.
Assuntos
Dineínas do Citoplasma/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Dineínas/metabolismo , Regulação da Expressão Gênica , Proteínas Intrinsicamente Desordenadas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Dineínas do Citoplasma/química , Dineínas do Citoplasma/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/fisiologia , Dineínas/química , Dineínas/genética , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Many motor proteins achieve high efficiency for chemomechanical conversion, and single-molecule force-resisting experiments are a major tool to detect the chemomechanical coupling of efficient motors. Here, we introduce several quantitative relations that involve only parameters extracted from force-resisting experiments and offer new benchmarks beyond mere efficiency to judge the chemomechanical optimality or deficit of evolutionary remote motors on the same footing. The relations are verified by the experimental data from F1-ATPase, kinesin-1, myosin V and cytoplasmic dynein, which are representative members of four motor protein families. A double-fitting procedure yields the chemomechanical parameters that can be cross-checked for consistency. Using the extracted parameters, two generic maps of chemomechanical optimality are constructed on which motors across families can be quantitatively compared. The maps reveal two chemomechanical coupling regimes, one conducive to high efficiency and high directionality, and the other advantageous to force generation. Surprisingly, an F1 rotor and a kinesin-1 walker belong to the first regime despite their obvious evolutionary gap, while myosin V and cytoplasmic dynein follow the second regime. This analysis also predicts the symmetries of directional biases and heat productions for the motors, which impose constraints on their chemomechanical coupling and are open to future experimental tests. The verified relations, six in total, present a unified fitting framework to analyze force-resisting experiments. The generic maps of optimality, to which many more motors can be added in future, provide a rigorous method for a systematic cross-family comparison of motors to expose their evolutionary connections and mechanistic similarities.
Assuntos
Dineínas do Citoplasma/química , Cinesinas/química , Cadeias Pesadas de Miosina/química , Miosina Tipo V/química , Miosinas/química , ATPases Translocadoras de Prótons/química , Trifosfato de Adenosina/química , Animais , Galinhas , Citoplasma/química , Drosophila , Camundongos , Microtúbulos/química , Ranidae , TermodinâmicaRESUMO
LC8 is a ubiquitous hub protein that binds intrinsically disordered proteins and promotes their assembly into higher-order complexes. A common feature among the more than 100 essential LC8 binding proteins is that in the 10-12-amino acid recognition sequence there is a conserved QT motif but variable amino acids N- and C-terminal to the QT pair. The sequence diversity among LC8 binding partners implies that structural factors also contribute to specificity. To investigate whether one such factor is the transient secondary structure favored by an LC8 binding sequence, we report here a molecular ensemble description of ICTL, a domain of the dynein intermediate chain that includes binding sites for light chains LC8 and Tctex1. Nuclear magnetic resonance secondary chemical shifts and residual dipolar coupling values combined with ensemble generation and selection algorithms indicate a deviation from statistical (random) coil behavior with an elevated population of polyproline II (PPII) conformations for the ICTL regions that bind LC8 and Tctex1. Independent measurements of one- and three-bond scalar couplings confirm the PPII transient secondary structure propensity. Given that in the IC/Tctex1/LC8 ternary complex ICTL forms a ß-strand at the interface of Tctex1 and LC8, we hypothesize that a PPII conformation may facilitate its initial docking and insertion into the binding cleft of the ß-sheet LC8 dimer interface. Molecular ensemble calculations for intrinsically disordered LC8 binding partners also reveal PPII conformational sampling within and proximate to the LC8 recognition motifs, suggesting that a preference for a PPII conformation is general for LC8 binding partners.
Assuntos
Dineínas do Citoplasma/química , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas Associadas aos Microtúbulos/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Conformação Proteica em Folha beta , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
The force-generating mechanism of dynein differs from the force-generating mechanisms of other cytoskeletal motors. To examine the structural dynamics of dynein's stepping mechanism in real time, we used polarized total internal reflection fluorescence microscopy with nanometer accuracy localization to track the orientation and position of single motors. By measuring the polarized emission of individual quantum nanorods coupled to the dynein ring, we determined the angular position of the ring and found that it rotates relative to the microtubule (MT) while walking. Surprisingly, the observed rotations were small, averaging only 8.3°, and were only weakly correlated with steps. Measurements at two independent labeling positions on opposite sides of the ring showed similar small rotations. Our results are inconsistent with a classic power-stroke mechanism, and instead support a flexible stalk model in which interhead strain rotates the rings through bending and hinging of the stalk. Mechanical compliances of the stalk and hinge determined based on a 3.3-µs molecular dynamics simulation account for the degree of ring rotation observed experimentally. Together, these observations demonstrate that the stepping mechanism of dynein is fundamentally different from the stepping mechanisms of other well-studied MT motors, because it is characterized by constant small-scale fluctuations of a large but flexible structure fully consistent with the variable stepping pattern observed as dynein moves along the MT.
Assuntos
Dineínas do Citoplasma/química , Trifosfato de Adenosina/metabolismo , Avidina , Fenômenos Biofísicos , Biotina , Dineínas do Citoplasma/metabolismo , Humanos , Microscopia de Fluorescência , Microtúbulos/metabolismo , Simulação de Dinâmica Molecular , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , Nanotubos , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , RotaçãoRESUMO
BRCA2 is responsible for familial breast and ovarian cancer and has been linked to DNA repair and centrosome duplication. Here we analyzed the mechanism by which the centrosomal localization signal (CLS) of BRCA2 interacts with cytoplasmic dynein 1 to localize BRCA2 to the centrosome. In vitro pull-down assays demonstrated that BRCA2 directly binds to the cytoplasmic dynein 1 light intermediate chain 2. A dominant-negative HA-CLS-DsRed fusion protein, the depletion of dynein by siRNA, and the inactivation of dynein by EHNA, inhibited the localization of BRCA2 at centrosomes and caused the separation of centrosome pairs during the S-phase. The double depletion of BRCA2 and C-Nap1 caused a larger dispersion of centrosome distances than the silencing of C-Nap1. These results suggest that cytoplasmic dynein 1 binds to BRCA2 through the latter's CLS and BRCA2 mediates the cohesion between centrosomes during the S phase, potentially serving as a cell-cycle checkpoint.
Assuntos
Proteína BRCA2/metabolismo , Centrossomo/metabolismo , Dineínas do Citoplasma/metabolismo , Sequência de Aminoácidos , Dineínas do Citoplasma/química , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Espectrometria de Massas , Modelos Biológicos , Proteínas Mutantes/metabolismo , Ligação Proteica , Fase SRESUMO
We report a new multidimensional magic angle spinning NMR methodology, which provides an accurate and detailed probe of molecular motions occurring on timescales of nano- to microseconds, in sidechains of proteins. The approach is based on a 3D CPVC-RFDR correlation experiment recorded under fast MAS conditions (ν(R) = 62 kHz), where (13)C-(1)H CPVC dipolar lineshapes are recorded in a chemical shift resolved manner. The power of the technique is demonstrated in model tripeptide Tyr-(d)Ala-Phe and two nanocrystalline proteins, GB1 and LC8. We demonstrate that, through numerical simulations of dipolar lineshapes of aromatic sidechains, their detailed dynamic profile, i.e., the motional modes, is obtained. In GB1 and LC8 the results unequivocally indicate that a number of aromatic residues are dynamic, and using quantum mechanical calculations, we correlate the molecular motions of aromatic groups to their local environment in the crystal lattice. The approach presented here is general and can be readily extended to other biological systems.
Assuntos
Dineínas do Citoplasma/química , Receptores de GABA-B/química , Isótopos de Carbono/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Teoria QuânticaRESUMO
It has been suggested that DYNLT, a dynein light chain known to bind to various cellular and viral proteins, can function as a microtubule-cargo adaptor. Recent data showed that DYNLT links the small GTPase Rab3D to microtubules and, for this to occur, the DYNLT homodimer needs to display a binding site for dynein intermediate chain together with a binding site for the small GTPase. We have analysed in detail how RagA, another small GTPase, associates to DYNLT. After narrowing down the binding site of RagA to DYNLT we could identify that a ß strand, part of the RagA G3 box involved in nucleotide binding, mediates this association. Interestingly, we show that both microtubule-associated DYNLT and cytoplasmic DYNLT are equally able to bind to the small GTPases Rab3D and RagA. Using NMR spectroscopy, we analysed the binding of dynein intermediate chain and RagA to mammalian DYNLT. Our experiments identify residues of DYNLT affected by dynein intermediate chain binding and residues affected by RagA binding, hence distinguishing the docking site for each of them. In summary, our results shed light on the mechanisms adopted by DYNLT when binding to protein cargoes that become transported alongside microtubules bound to the dynein motor.
Assuntos
Citoplasma/metabolismo , Dineínas do Citoplasma/metabolismo , Dineínas/metabolismo , Microtúbulos/metabolismo , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas rab3 de Ligação ao GTP/metabolismo , Substituição de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Citoplasma/enzimologia , Dineínas do Citoplasma/química , Dineínas do Citoplasma/genética , Dimerização , Dineínas/química , Dineínas/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Microtúbulos/enzimologia , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas rab3 de Ligação ao GTP/química , Proteínas rab3 de Ligação ao GTP/genéticaRESUMO
Members of the dynein family, consisting of cytoplasmic and axonemal isoforms, are motors that move towards the minus ends of microtubules. Cytoplasmic dynein-1 (dynein-1) plays roles in mitosis and cellular cargo transport, and is implicated in viral infections and neurodegenerative diseases. Cytoplasmic dynein-2 (dynein-2) performs intraflagellar transport and is associated with human skeletal ciliopathies. Dyneins share a conserved motor domain that couples cycles of ATP hydrolysis with conformational changes to produce movement. Here we present the crystal structure of the human cytoplasmic dynein-2 motor bound to the ATP-hydrolysis transition state analogue ADP.vanadate. The structure reveals a closure of the motor's ring of six AAA+ domains (ATPases associated with various cellular activites: AAA1-AAA6). This induces a steric clash with the linker, the key element for the generation of movement, driving it into a conformation that is primed to produce force. Ring closure also changes the interface between the stalk and buttress coiled-coil extensions of the motor domain. This drives helix sliding in the stalk which causes the microtubule binding domain at its tip to release from the microtubule. Our structure answers the key questions of how ATP hydrolysis leads to linker remodelling and microtubule affinity regulation.
Assuntos
Citoplasma , Dineínas do Citoplasma/química , Dineínas do Citoplasma/metabolismo , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Hidrólise , Modelos Moleculares , Movimento , Conformação ProteicaRESUMO
Cytoplasmic dynein play an important role in transporting various intracellular cargos by coupling their ATP hydrolysis cycle with their conformational changes. Recent experimental results showed that the cytoplasmic dynein had a highly variable stepping pattern including "hand-over-hand", "inchworm" and "nonalternating-inchworm". Here, we developed a model to describe the coordinated stepping patterns of cytoplasmic dynein, based on its working cycle, construction and the interaction between its leading head and tailing head. The kinetic model showed how change in the distance between the two heads influences the rate of cytoplasmic dynein under different stepping patterns. Numerical simulations of the distribution of step size and striding rate are in good quantitative agreement with experimental observations. Hence, our coordinated stepping model for cytoplasmic dynein successfully explained its diverse stepping patterns as a molecular motor. The cooperative mechanism carried out by the two heads of cytoplasmic dynein shed light on the strategies adopted by the cytoplasmic dynein in executing various functions.
Assuntos
Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Dineínas do Citoplasma/química , Dineínas do Citoplasma/metabolismo , Mecanotransdução Celular/fisiologia , Modelos Biológicos , Modelos Químicos , Animais , Sítios de Ligação , Transporte Biológico Ativo/fisiologia , Simulação por Computador , Dineínas do Citoplasma/ultraestrutura , Transferência de Energia/fisiologia , Humanos , Hidrólise , Movimento (Física) , Ligação ProteicaRESUMO
LC8 dynein light chains (DYNLL) are conserved homodimeric eukaryotic hub proteins that participate in diverse cellular processes. Among the binding partners of DYNLL2, myosin 5a (myo5a) is a motor protein involved in cargo transport. Here we provide a profound characterization of the DYNLL2 binding motif of myo5a in free and DYNLL2-bound form by using nuclear magnetic resonance spectroscopy, X-ray crystallography, and molecular dynamics simulations. In the free form, the DYNLL2 binding region, located in an intrinsically disordered domain of the myo5a tail, has a nascent helical character. The motif becomes structured and folds into a ß-strand upon binding to DYNLL2. Despite differences of the myo5a sequence from the consensus binding motif, one peptide is accommodated in each of the parallel DYNLL2 binding grooves, as for all other known partners. Interestingly, while the core motif shows a similar interaction pattern in the binding groove as seen in other complexes, the flanking residues make several additional contacts, thereby lengthening the binding motif. The N-terminal extension folds back and partially blocks the free edge of the ß-sheet formed by the binding motif itself. The C-terminal extension contacts the dimer interface and interacts with symmetry-related residues of the second myo5a peptide. The involvement of flanking residues of the core binding site of myo5a could modify the quaternary structure of the full-length myo5a and affect its biological functions. Our results deepen the knowledge of the diverse partner recognition of DYNLL proteins and provide an example of a Janus-faced linear motif.
Assuntos
Dineínas do Citoplasma/química , Dineínas do Citoplasma/metabolismo , Miosinas/química , Miosinas/metabolismo , Motivos de Aminoácidos/fisiologia , Sequência de Aminoácidos , Cristalografia por Raios X , Dineínas do Citoplasma/genética , Humanos , Dados de Sequência Molecular , Miosinas/genética , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Dyneins are large protein complexes that act as microtubule based molecular motors. The dynein heavy chain contains a motor domain which is a member of the AAA+ protein family (ATPases Associated with diverse cellular Activities). Proteins of the AAA+ family show a diverse range of functionalities, but share a related core AAA+ domain, which often assembles into hexameric rings. Dynein is unusual because it has all six AAA+ domains linked together, in one long polypeptide. The dynein motor domain generates movement by coupling ATP driven conformational changes in the AAA+ ring to the swing of a motile element called the linker. Dynein binds to its microtubule track via a long antiparallel coiled-coil stalk that emanates from the AAA+ ring. Recently the first high resolution structures of the dynein motor domain were published. Here we provide a detailed structural analysis of the six AAA+ domains using our Saccharomycescerevisiae crystal structure. We describe how structural similarities in the dynein AAA+ domains suggest they share a common evolutionary origin. We analyse how the different AAA+ domains have diverged from each other. We discuss how this is related to the function of dynein as a motor protein and how the AAA+ domains of dynein compare to those of other AAA+ proteins.