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1.
Chem Commun (Camb) ; 56(8): 1187-1190, 2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31922177

RESUMO

Trimethylamine N-oxide is found to be effective in regulating the interaction between microtubules and kinesins over a wide temperature range. The lifetime of the motility of microtubules on kinesins at high temperatures is prolonged using trimethylamine N-oxide. The activation energy of microtubule motility is increased by trimethylamine N-oxide. Prolonged operation at high temperatures decreased the activation energy of MT motility despite the increase in concentration of trimethylamine N-oxide.


Assuntos
Cinesina/metabolismo , Metilaminas/farmacologia , Microtúbulos/metabolismo , Temperatura Ambiente , Cinesina/química , Cinética , Metilaminas/química , Microtúbulos/química
2.
Int J Mol Sci ; 20(19)2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31623357

RESUMO

A general kinetic model is presented for the chemomechanical coupling of dimeric kinesin molecular motors with and without extension of their neck linkers (NLs). A peculiar feature of the model is that the rate constants of ATPase activity of a kinesin head are independent of the strain on its NL, implying that the heads of the wild-type kinesin dimer and the mutant with extension of its NLs have the same force-independent rate constants of the ATPase activity. Based on the model, an analytical theory is presented on the force dependence of the dynamics of kinesin dimers with and without extension of their NLs at saturating ATP. With only a few adjustable parameters, diverse available single molecule data on the dynamics of various kinesin dimers, such as wild-type kinesin-1, kinesin-1 with mutated residues in the NLs, kinesin-1 with extension of the NLs and wild-type kinesin-2, under varying force and ATP concentration, can be reproduced very well. Additionally, we compare the power production among different kinesin dimers, showing that the mutation in the NLs reduces the power production and the extension of the NLs further reduces the power production.


Assuntos
Trifosfato de Adenosina/metabolismo , Cinesina/metabolismo , Modelos Biológicos , Proteínas Motores Moleculares/metabolismo , Trifosfato de Adenosina/química , Algoritmos , Fenômenos Químicos , Ativação Enzimática , Hidrólise , Cinesina/química , Fenômenos Mecânicos , Proteínas Motores Moleculares/química
3.
Dev Genes Evol ; 229(5-6): 161-181, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31486889

RESUMO

KIF3A and KIF3B are homologous motor subunits of the Kinesin II protein family. KIF3A, KIF3B, and KAP3 form a heterotrimeric complex and play a significant role in spermatogenesis. Here, we first cloned full-length kif3a/3b cDNAs from Larimichthys polyactis. Lp-kif3a/3b are highly related to their homologs in other animals. The proteins are composed of three domains, an N-terminal head domain, a central stalk domain, and a C-terminus tail domain. Lp-kif3a/3b mRNAs were found to be ubiquitously expressed in the examined tissues, with high expression in the testis. Fluorescence in situ hybridization (FISH) was used to analyze the expression of Lp-kif3a/3b mRNAs during spermiogenesis. The results showed that Lp-kif3a/3b mRNAs had similar expression pattern and were continuously expressed during spermiogenesis. From middle spermatid to mature sperm, Lp-kif3a/3b mRNAs gradually localized to the side of the spermatid where the midpiece and tail form. In addition, we used immunofluorescence (IF) to observe that Lp-KIF3A protein co-localizes with tubulin during spermiogenesis. In early spermatid, Lp-KIF3A protein and microtubule signals were randomly distributed in the cytoplasm. In middle spermatid, however, the protein was detected primarily around the nucleus. In late spermatid, the protein migrated primarily to one side of the nucleus where the tail forms. In mature sperm, Lp-KIF3A and microtubules accumulated in the midpiece. Moreover, Lp-KIF3A co-localized with the mitochondria. In mature sperm, Lp-KIF3A and mitochondria were present in the midpiece. Therefore, Lp-KIF3A/KIF3B may be involved in spermiogenesis in L. polyactis, particularly during nuclear reshaping and tail formation.


Assuntos
Proteínas de Peixes/metabolismo , Peixes/fisiologia , Cinesina/metabolismo , Espermatogênese , Espermatozoides/citologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Núcleo Celular/metabolismo , Clonagem Molecular , Proteínas de Peixes/química , Proteínas de Peixes/genética , Humanos , Cinesina/química , Cinesina/genética , Masculino , Filogenia , Alinhamento de Sequência , Espermatozoides/metabolismo
4.
Molecules ; 24(18)2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31510043

RESUMO

S-trityl-l-cysteine (STLC) is a well-recognized lead compound known for its anticancer activity owing to its potent inhibitory effect on human mitotic kinesin Eg5. STLC contains two free terminal amino and carboxyl groups that play pivotal roles in binding to the Eg5 pocket. On the other hand, such a zwitterion structure complicates the clinical development of STLC because of the solubility issues. Masking either of these radicals reduces or abolishes STLC activity against Eg5. We recently identified and characterized a new class of nicotinamide adenine dinucleotide-dependent deacetylase isoform 2 of sirtuin protein (SIRT2) inhibitors that can be utilized as cytotoxic agents based on an S-trityl-l-histidine scaffold. Herein, we propose new STLC-derived compounds that possess pronounced SIRT2 inhibition effects. These derivatives contain modified amino and carboxyl groups, which conferred STLC with SIRT2 bioactivity, representing an explicit repurposing approach. Compounds STC4 and STC11 exhibited half maximal inhibitory concentration values of 10.8 ± 1.9 and 9.5 ± 1.2 µM, respectively, against SIRT2. Additionally, introduction of the derivatizations in this study addressed the solubility limitations of free STLC, presumably due to interruption of the zwitterion structure. Therefore, we could obtain drug-like STLC derivatives that work by a new mechanism of action. The new derivatives were designed, synthesized, and their structure was confirmed using different spectroscopic approaches. In vitro and cellular bioassays with various cancer cell lines and in silico molecular docking and solubility calculations of the synthesized compounds demonstrated that they warrant attention for further refinement of their bioactivity.


Assuntos
Neoplasias/tratamento farmacológico , Sirtuína 2/antagonistas & inibidores , Compostos de Tritil/farmacologia , Linhagem Celular Tumoral , Simulação por Computador , Cisteína/química , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Cinesina/química , Cinesina/genética , Neoplasias/genética , Neoplasias/patologia , Sirtuína 2/genética , Solubilidade , Compostos de Tritil/química
5.
Nature ; 572(7768): 224-229, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31391558

RESUMO

Living systems are capable of locomotion, reconfiguration and replication. To perform these tasks, cells spatiotemporally coordinate the interactions of force-generating, 'active' molecules that create and manipulate non-equilibrium structures and force fields of up to millimetre length scales1-3. Experimental active-matter systems of biological or synthetic molecules are capable of spontaneously organizing into structures4,5 and generating global flows6-9. However, these experimental systems lack the spatiotemporal control found in cells, limiting their utility for studying non-equilibrium phenomena and bioinspired engineering. Here we uncover non-equilibrium phenomena and principles of boundary-mediated control by optically modulating structures and fluid flow in an engineered system of active biomolecules. Our system consists of purified microtubules and light-activatable motor proteins that crosslink and organize the microtubules into distinct structures upon illumination. We develop basic operations-defined as sets of light patterns-to create, move and merge the microtubule structures. By combining these operations, we create microtubule networks that span several hundred micrometres in length and contract at speeds up to an order of magnitude higher than the speed of an individual motor protein. We manipulate these contractile networks to generate and sculpt persistent fluid flows. The principles of boundary-mediated control that we uncover may be used to study emergent cellular structures and forces and to develop programmable active-matter devices.


Assuntos
Bioengenharia/métodos , Cinesina/metabolismo , Cinesina/efeitos da radiação , Luz , Microtúbulos/química , Microtúbulos/efeitos da radiação , Cinesina/química , Microtúbulos/metabolismo
6.
Biophys Chem ; 253: 106216, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31288174

RESUMO

Kinesin is a biological molecular motor that can move continuously on microtubule until it unbinds. Here, we studied computationally the force dependence of the unbinding rate of the motor. Our results showed that while the unbinding rate under the forward load has the expected characteristic of "slip bond", with the unbinding rate increasing monotonically with the increase of the forward load, the unbinding rate under the backward load shows counterintuitive characteristic of "slip-catch-slip bond": as the backward load increases, the unbinding rate increases exponentially firstly, then drops rapidly and then increases again. Our calculated data are in agreement with the available single-molecule data from different research groups. The mechanism of the slip-catch-slip bond was revealed.


Assuntos
Cinesina/química , Microtúbulos/química , Cinesina/metabolismo , Microtúbulos/metabolismo
7.
Nanoscale ; 11(24): 11562-11568, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31168545

RESUMO

Structural defects can determine and influence various properties of materials, and many technologies rely on the manipulation of defects (e.g., semiconductor industries). In biological systems, management of defects/errors (e.g. DNA repair) is critical to an organism's survival, which has inspired the design of artificial nanomachines that mimic nature's ability to detect defects and repair damage. Biological motors have captured considerable attention in developing such capabilities due to their ability to convert energy into directed motion in response to environmental stimuli, which maximizes their ability for detection and repair. The objective of the present study was to develop an understanding of how the presence of non-bonding domains, here considered as a "defect", in microtubule (MT) building blocks affect the kinesin-driven, active assembly of MT spools. The assembly/joining of micron-scale bonding (i.e., biotin-containing) and non-bonding (i.e., no biotin) MTs resulted in segmented MT building blocks consisting of alternating bonding and non-bonding domains. Here, the introduction of these MT building blocks into a kinesin gliding motility assay along with streptavidin-coated quantum dots resulted in the active assembly of spools with altered morphology but retained functionality. Moreover, it was noted that non-bonding domains were autonomously and preferentially released from the spools over time, representing a mechanism by which defects may be removed from these structures. Overall, our findings demonstrate that this active assembly system has an intrinsic ability for quality control, which can be potentially expanded to a wide range of applications such as self-regulation and healing of active materials.


Assuntos
Proteínas de Drosophila/química , Cinesina/química , Microtúbulos/química , Pontos Quânticos/química , Animais , Drosophila melanogaster , Domínios Proteicos
8.
Elife ; 82019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31084716

RESUMO

Kinesin force generation involves ATP-induced docking of the neck linker (NL) along the motor core. However, the roles of the proposed steps of NL docking, cover-neck bundle (CNB) and asparagine latch (N-latch) formation, during force generation are unclear. Furthermore, the necessity of NL docking for transport of membrane-bound cargo in cells has not been tested. We generated kinesin-1 motors impaired in CNB and/or N-latch formation based on molecular dynamics simulations. The mutant motors displayed reduced force output and inability to stall in optical trap assays but exhibited increased speeds, run lengths, and landing rates under unloaded conditions. NL docking thus enhances force production but at a cost to speed and processivity. In cells, teams of mutant motors were hindered in their ability to drive transport of Golgi elements (high-load cargo) but not peroxisomes (low-load cargo). These results demonstrate that the NL serves as a mechanical element for kinesin-1 transport under physiological conditions.


Assuntos
Trifosfato de Adenosina/metabolismo , Cinesina/química , Cinesina/metabolismo , Animais , Células COS , Cinesina/genética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Conformação Proteica
9.
Nat Cell Biol ; 21(6): 768-777, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061466

RESUMO

Controlling cellular processes with light can help elucidate their underlying mechanisms. Here we present zapalog, a small-molecule dimerizer that undergoes photolysis when exposed to blue light. Zapalog dimerizes any two proteins tagged with the FKBP and DHFR domains until exposure to light causes its photolysis. Dimerization can be repeatedly restored with uncleaved zapalog. We implement this method to investigate mitochondrial motility and positioning in cultured neurons. Using zapalog, we tether mitochondria to constitutively active kinesin motors, forcing them down the axon towards microtubule (+) ends until their instantaneous release via blue light, which results in full restoration of their endogenous motility. We find that one-third of stationary mitochondria cannot be pulled away from their position and that these firmly anchored mitochondria preferentially localize to VGLUT1-positive presynapses. Furthermore, inhibition of actin polymerization with latrunculin A reduces this firmly anchored pool. On release from exogenous motors, mitochondria are preferentially recaptured at presynapses.


Assuntos
Axônios/metabolismo , Mitocôndrias/genética , Fotólise , Multimerização Proteica/efeitos da radiação , Actinas/antagonistas & inibidores , Animais , Axônios/química , Axônios/efeitos da radiação , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Células COS , Cinesina/química , Luz , Microtúbulos/genética , Microtúbulos/efeitos da radiação , Mitocôndrias/química , Mitocôndrias/efeitos da radiação , Neurônios/química , Neurônios/efeitos da radiação , Polimerização/efeitos dos fármacos , Domínios Proteicos/genética , Domínios Proteicos/efeitos da radiação , Multimerização Proteica/genética , Sinapses/química , Sinapses/genética , Sinapses/efeitos da radiação , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/genética , Tiazolidinas/farmacologia , Proteína Vesicular 1 de Transporte de Glutamato/genética
10.
Nano Lett ; 19(6): 3933-3938, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31037942

RESUMO

DNA has been well-known for its applications in programmable self-assembly of materials. Nonetheless, utility of DNA origami, which offers more opportunity to realize complicated operations, has been very limited. Here we report self-assembly of a biomolecular motor system, microtubule-kinesin mediated by DNA origami nanostructures. We demonstrate that a rodlike DNA origami motif facilitates self-assembly of microtubules into asters. A smooth-muscle like molecular contraction system has also been realized using the DNA origami in which self-assembled microtubules exhibited fast and dynamic contraction in the presence of kinesins through an energy dissipative process. This work provides potential nanotechnological applications of DNA and biomolecular motor proteins.


Assuntos
DNA/química , Cinesina/química , Microtúbulos/química , Nanoestruturas/química , Microtúbulos/ultraestrutura , Músculo Liso/química , Músculo Liso/ultraestrutura , Nanoestruturas/ultraestrutura , Nanotecnologia , Conformação de Ácido Nucleico
11.
Dev Cell ; 49(5): 711-730.e8, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31031197

RESUMO

The correct localization of Hedgehog effectors to the tip of primary cilia is critical for proper signal transduction. The conserved non-motile kinesin Kif7 defines a "cilium-tip compartment" by localizing to the distal ends of axonemal microtubules. How Kif7 recognizes microtubule ends remains unknown. We find that Kif7 preferentially binds GTP-tubulin at microtubule ends over GDP-tubulin in the mature microtubule lattice, and ATP hydrolysis by Kif7 enhances this discrimination. Cryo-electron microscopy (cryo-EM) structures suggest that a rotated microtubule footprint and conformational changes in the ATP-binding pocket underlie Kif7's atypical microtubule-binding properties. Finally, Kif7 not only recognizes but also stabilizes a GTP-form of tubulin to promote its own microtubule-end localization. Thus, unlike the characteristic microtubule-regulated ATPase activity of kinesins, Kif7 modulates the tubulin mechanochemical cycle. We propose that the ubiquitous kinesin fold has been repurposed in Kif7 to facilitate organization of a spatially restricted platform for localization of Hedgehog effectors at the cilium tip.


Assuntos
Cílios/fisiologia , Guanosina Trifosfato/metabolismo , Cinesina/metabolismo , Mecanotransdução Celular , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Humanos , Cinesina/química , Cinesina/genética , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Transdução de Sinais , Tubulina (Proteína)/genética
12.
Biochemistry ; 58(18): 2326-2338, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30973712

RESUMO

Chromokinesins NOD and KID have similar DNA binding domains and functions during cell division, while their motor domain sequences show significant variations. It has been unclear whether these motors have the similar structure, chemistry, and microtubule interactions necessary to follow a similar mechanism of force generation. We used biochemical rate measurements, cosedimentation, and structural analysis to investigate the ATPase mechanisms of the NOD and KID core domains. These studies revealed that NOD and KID have different ATPase mechanisms, microtubule interactions, and catalytic domain structures. The ATPase cycles of NOD and KID have different rate-limiting steps. The ATPase rate of NOD was robustly stimulated by microtubules, and its microtubule affinity was weakened in all nucleotide-bound states. KID bound microtubules tightly in all nucleotide states and remained associated with the microtubule for more than 100 cycles of ATP hydrolysis before dissociating. The structure of KID was most like that of conventional kinesin (KIF5). Key differences in the microtubule binding region and allosteric communication pathway between KID and NOD are consistent with our biochemical data. Our results support the model in which NOD and KID utilize distinct mechanistic pathways to achieve the same function during cell division.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Cinesina/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação/genética , Domínio Catalítico , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Humanos , Cinesina/química , Cinesina/genética , Cinética , Microtúbulos/química , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação Proteica , Domínios Proteicos
13.
Nanoscale ; 11(11): 4987-4998, 2019 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-30839012

RESUMO

This research proposed to create the next generation of versatile electrochemical-based biosensors capable of monitoring target capture and release as dictated by molecular binding or unbinding. The biosensor integrates cellular machines (i.e., microtubules, structural elements of cells and kinesin molecular motors involved in cellular transport) as functional units; its assembly is based on molecular self-assembly and self-recognition. Our results demonstrate that the designed biosensor was capable of allowing detection of binding and unbinding events based on redox reactions at user-controlled electrode interfaces. The analysis also showed that the sensitivity of the designed biosensor or its ability to record such events could be user-controlled at any given time by adjusting the energy source that "fuels" the system.


Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Trifosfato de Adenosina , Técnicas Eletroquímicas , Eletrodos , Cinesina/química , Cinesina/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Nanotecnologia , Oxirredução , Ligação Proteica
14.
Nanoscale ; 11(20): 9879-9887, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-30888373

RESUMO

Motor proteins function in in vivo ensembles to achieve cargo transport, flagellum motion, and mitotic cell division. Although the cooperativity of multiple motors is indispensable for physiological function, reconstituting the arrangement of motors in vitro is challenging, so detailed analysis of the functions of motor ensembles has not yet been achieved. Here, we developed an assay platform to study the motility of microtubules driven by a defined number of kinesin motors spaced in a definite manner. Gold (Au) nano-pillar arrays were fabricated on a silicon/silicon dioxide (Si/SiO2) substrate with spacings of 100 nm to 500 nm. The thiol-polyethylene glycol (PEG)-biotin self-assembled monolayer (SAM) and silane-PEG-CH3 SAM were then selectively formed on the pillars and SiO2 surface, respectively. This allowed for both immobilization of kinesin molecules on Au nano-pillars in a precise manner and repulsion of kinesins from the SiO2 surface. Using arrayed kinesin motors, we report that motor number and spacing do not influence the motility of microtubules driven by kinesin-1 motors. This assay platform is applicable to all kinds of biotinylated motors, allows the study of the effects of motor number and spacing, and is expected to reveal novel behaviors of motor proteins.


Assuntos
Ouro/química , Cinesina/química , Biotina/química , Proteínas Imobilizadas/química , Polietilenoglicóis/química , Dióxido de Silício/química , Compostos de Sulfidrila/química , Propriedades de Superfície
15.
Biol Cell ; 111(6): 143-160, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30784092

RESUMO

Chromosome congression is essential for faithful chromosome segregation and genomic stability in cell division. Centromere-associated protein E (CENP-E), a plus-end-directed kinesin motor, is required for congression of pole-proximal chromosomes in metaphase. CENP-E accumulates at the outer plate of kinetochores and mediates the kinetochore-microtubule capture. CENP-E also transports the chromosomes along spindle microtubules towards the equatorial plate. CENP-E interacts with Bub1-related kinase, Aurora B and core kinetochore components during kinetochore-microtubule attachment. In this review, we introduce the structures and mechanochemistry of kinesin-7 CENP-E. We highlight the complicated interactions between CENP-E and partner proteins during chromosome congression. We summarise the detailed roles and mechanisms of CENP-E in mitosis and meiosis, including the kinetochore-microtubule capture, chromosome congression/alignment in metaphase and the regulation of spindle assembly checkpoint. We also shed a light on the roles of CENP-E in tumourigenesis and CENP-E's specific inhibitors.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/fisiologia , Cinesina/metabolismo , Cinetocoros/metabolismo , Metáfase/fisiologia , Microtúbulos/metabolismo , Animais , Aurora Quinase B/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Células HeLa , Humanos , Cinesina/química , Camundongos , Proteínas Nucleares/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis
16.
Molecules ; 24(2)2019 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-30646587

RESUMO

Kinesin-1, kinesin-2 and kinesin-5 are three families of a superfamily of motor proteins; which can walk processively on microtubule filaments by hydrolyzing ATP. It was experimentally shown that while the three kinesin dimers show similar feature on the force dependence of velocity, they show rather different features on the force dependence of run length. However, why the three families of kinesins show these rather different features is unclear. Here, we computationally studied the movement dynamics of the three dimers based on our proposed model. The simulated results reproduce well the available experimental data on the force dependence of velocity and run length. Moreover, the simulated results on the velocity and run length for the three dimers with altered neck linker lengths are also in quantitative agreement with the available experimental data. The studies indicate that the three families of kinesins show much similar movement mechanism and the rather different features on the force dependence of run length arise mainly from the difference in rate constants of the ATPase activity and neck linker docking. Additionally, the asymmetric (limping) movement dynamics of the three families of homodimers with and without altered neck linker lengths are studied, providing predicted results.


Assuntos
Cinesina/química , Modelos Teóricos , Proteínas Motores Moleculares/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Cinesina/genética , Proteínas Motores Moleculares/genética , Família Multigênica , Multimerização Proteica
17.
Gene ; 683: 169-183, 2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-30316921

RESUMO

Kinesin-14 KIFC1 plays an important role in vesicular transport, microtubule organization, and spermiogenesis. In this study, we first investigated the microtubule distribution and expression pattern of KIFC1 during spermiogenesis of P. esculenta. Microtubules are abundant during spermiogenesis of P. esculenta and may be related to the generation and maintenance of pseudopodia-like cytoplasmic protrusions and nuclear reshaping. The Pe-KIFC1 protein is conserved with a motor domain where microtubule and ATP binding sites are predicted, a coiled-coil domain and a divergent tail domain. The Pe-kifc1 gene was extensively expressed and showed the highest expression in coelomic fluid where spermiogenesis occurs. We further observed the expression of kifc1 mRNA and protein and found that Pe-KIFC1 protein primarily co-localized with microtubules during spermiogenesis, indicating that KIFC1 might play several roles during this process via its cargo transport and/or microtubule organization function. In addition, co-localization of mitochondria and KIFC1 was also detected during spermiogenesis, which were located in the midpiece in mature sperm, suggesting that mitochondria might be a cargo of Pe-KIFC1 that participates in the intracellular distribution of mitochondria and formation of the midpiece. Based on our detailed observations of the dynamic distribution of microtubules, KIFC1, and mitochondria during spermiogenesis and the conserved function of KIFC1 in cargo transport and microtubule organization, functional models of Pe-KIFC1 during spermiogenesis are proposed, including the participation of KIFC1 in nuclear reshaping and midpiece formation.


Assuntos
Cinesina/genética , Cinesina/metabolismo , Poliquetos/fisiologia , Espermatogênese , Animais , Sítios de Ligação , Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Cinesina/química , Masculino , Microtúbulos/metabolismo , Modelos Moleculares , Filogenia , Poliquetos/genética , Conformação Proteica , Domínios Proteicos , Transporte Proteico
18.
Proteins ; 87(1): 12-22, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30370948

RESUMO

Accurate protein-protein complex prediction, to atomic detail, is a challenging problem. For flexible docking cases, current state-of-the-art docking methods are limited in their ability to exhaustively search the high dimensionality of the problem space. In this study, to obtain more accurate models, an investigation into the local optimization of initial docked solutions is presented with respect to a reference crystal structure. We show how physics-based refinement of protein-protein complexes in contact map space (CMS), within a metadynamics protocol, can be performed. The method uses 5 times replicated 10 ns simulations for sampling and ranks the generated conformational snapshots with ZRANK to identify an ensemble of n snapshots for final model building. Furthermore, we investigated whether the reconstructed free energy surface (FES), or a combination of both FES and ZRANK, referred to as CSα , can help to reduce snapshot ranking error.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Simulação por Computador , Cinesina/química , Cinesina/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas
19.
J Chem Theory Comput ; 15(1): 424-435, 2019 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-30537823

RESUMO

Accurate prediction of ligand binding affinities is of key importance in small molecule lead optimization and a central task in computational medicinal chemistry. Over the years, advances in both computer hardware and computational methodologies have established free energy perturbation (FEP) methods as among the most reliable and rigorous approaches to compute protein-ligand binding free energies. However, accurate description of ionization and tautomerism of ligands is still a major challenge in structure-based prediction of binding affinities. Druglike molecules are often weak acid or bases with multiple accessible protonation and tautomeric states that can contribute significantly to the binding process. To address this issue, we introduce in this work the p Ka and tautomeric state correction approach. This approach is based on free energy perturbation formalism and provides a rigorous treatment of the ionization and tautomeric equilibria of ligands in solution and in the protein complexes. A series of Kinesin Spindle Protein (KSP) and Factor Xa inhibitor molecules were used as test cases. Our results demonstrate that the p Ka and tautomeric state correction approach is able to rigorously and accurately incorporate multiple protonation and tautomeric states in the binding affinity calculations.


Assuntos
Inibidores Enzimáticos/química , Proteínas/química , Inibidores do Fator Xa/química , Cinesina/química , Ligantes , Prótons , Estereoisomerismo , Termodinâmica
20.
J Cell Sci ; 132(4)2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30578316

RESUMO

Kinesin-13 motors regulate precise microtubule dynamics and limit microtubule length throughout metazoans by depolymerizing microtubule ends. Recently, the kinesin-13 motor family member MCAK (also known Kif2C) has been proposed to undergo large conformational changes during its catalytic cycle, as it switches from being in solution to being bound to microtubules. Here, we reveal that MCAK has a compact conformation in solution through crosslinking and electron microscopy experiments. When MCAK is bound to the microtubule ends, it adopts an extended conformation with the N-terminus and neck region of MCAK interacting with the microtubule. Interestingly, the region of MCAK that interacts with the microtubule is the region phosphorylated by Aurora B and contains an end binding (EB) protein-binding motif. The level of phosphorylation of the N-terminus results in a graded microtubule depolymerase activity. Here, we show that the N-terminus of MCAK forms a platform to integrate Aurora B kinase downstream signals and in response fine-tunes its depolymerase activity during mitosis. We propose that this allosteric control mechanism allows decoupling of the N-terminus from the motor domain of MCAK to allow MCAK depolymerase activity at kinetochores.


Assuntos
Aurora Quinase B/química , Cinesina/química , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Animais , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinesina/genética , Cinesina/metabolismo , Cinetocoros/ultraestrutura , Microtúbulos/ultraestrutura , Mitose , Modelos Moleculares , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera
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