Structure and Function of Palladin's Actin Binding Domain

Here, we report the NMR structure of the actin-binding domain contained in the cell adhesion protein palladin. Previously, we demonstrated that one of the immunoglobulin domains of palladin (Ig3) is both necessary and sufficient for direct filamentous actin binding in vitro. In this study, we identify two basic patches on opposite faces of Ig3 that are critical for actin binding and cross-linking. Sedimentation equilibrium assays indicate that the Ig3 domain of palladin does not self-associate. These combined data are consistent with an actin cross-linking mechanism that involves concurrent attachment of two actin filaments by a single palladin molecule by an electrostatic mechanism. Palladin mutations that disrupt actin binding show altered cellular distributions and morphology of actin in cells, revealing a functional requirement for the interaction between palladin and actin in vivo.     

Effects of ATP and Actin-Filament Binding on the Dynamics of the Myosin II S1 Domain

Actin and myosin interact with one another to perform a variety of cellular functions. Central to understanding the processive motion of myosin on actin is the characterization of the individual states along the mechanochemical cycle. We present an all-atom molecular dynamics simulation of the myosin II S1 domain in the rigor state interacting with an actin filament. We also study actin-free myosin in both rigor and post-rigor conformations. Using all-atom level and coarse-grained analysis methods, we investigate the effects of myosin binding on actin, and of actin binding on myosin. In particular, we determine the domains of actin and myosin that interact strongly with one another at the actomyosin interface using a highly coarse-grained level of resolution, and we identify a number of salt bridges and hydrogen bonds at the interface of myosin and actin. Applying coarse-grained analysis, we identify differences in myosin states dependent on actin-binding, or ATP binding. Our simulations also indicate that the actin propeller twist-angle and nucleotide cleft-angles are influenced by myosin at the actomyosin interface. The torsional rigidity of the myosin-bound filament is also calculated, and is found to be increased compared to previous simulations of the free filament.     

Effects of ATP and Actin-Filament Binding on the Dynamics of the Myosin II S1 Domain

Actin and myosin interact with one another to perform a variety of cellular functions. Central to understanding the processive motion of myosin on actin is the characterization of the individual states along the mechanochemical cycle. We present an all-atom molecular dynamics simulation of the myosin II S1 domain in the rigor state interacting with an actin filament. We also study actin-free myosin in both rigor and post-rigor conformations. Using all-atom level and coarse-grained analysis methods, we investigate the effects of myosin binding on actin, and of actin binding on myosin. In particular, we determine the domains of actin and myosin that interact strongly with one another at the actomyosin interface using a highly coarse-grained level of resolution, and we identify a number of salt bridges and hydrogen bonds at the interface of myosin and actin. Applying coarse-grained analysis, we identify differences in myosin states dependent on actin-binding, or ATP binding. Our simulations also indicate that the actin propeller twist-angle and nucleotide cleft-angles are influenced by myosin at the actomyosin interface. The torsional rigidity of the myosin-bound filament is also calculated, and is found to be increased compared to previous simulations of the free filament.     

Localization of Myosin 1b to Actin Protrusions Requires Phosphoinositide Binding

Myosin 1b (Myo1b), a class I myosin, is a widely expressed, single-headed, actin-associated molecular motor. Transient kinetic and single-molecule studies indicate that it is kinetically slow and responds to tension. Localization and subcellular fractionation studies indicate that Myo1b associates with the plasma membrane and certain subcellular organelles such as endosomes and lysosomes. Whether Myo1b directly associates with membranes is unknown. We demonstrate here that full-length rat Myo1b binds specifically and with high affinity to phosphatidylinositol 4,5-bisphosphate (PIP₂) and phosphatidylinositol 3,4,5-triphosphate (PIP₃), two phosphoinositides that play important roles in cell signaling. Binding is not Ca²⁺-dependent and does not involve the calmodulin-binding IQ region in the neck domain of Myo1b. Furthermore, the binding site is contained entirely within the C-terminal tail region, which contains a putative pleckstrin homology domain. Single mutations in the putative pleckstrin homology domain abolish binding of the tail domain of Myo1b to PIP₂ and PIP₃ in vitro. These same mutations alter the distribution of Myc-tagged Myo1b at membrane protrusions in HeLa cells where PIP₂ localizes. In addition, we found that motor activity is required for Myo1b localization in filopodia. These results suggest that binding of Myo1b to phosphoinositides plays an important role in vivo by regulating localization to actin-enriched membrane projections.     

Effects of Cardiac Myosin Binding Protein-C on Actin Motility Are Explained with a Drag-Activation-Competition Model

Although mutations in cardiac myosin binding protein-C (cMyBP-C) cause heart disease, its role in muscle contraction is not well understood. A mechanism remains elusive partly because the protein can have multiple effects, such as dual biphasic activation and inhibition observed in actin motility assays. Here we develop a mathematical model for the interaction of cMyBP-C with the contractile proteins actin and myosin and the regulatory protein tropomyosin. We use this model to show that a drag-activation-competition mechanism accurately describes actin motility measurements, while models lacking either drag or competition do not. These results suggest that complex effects can arise simply from cMyBP-C binding to actin.     

Transportation of Nanoscale Cargoes by Myosin Propelled Actin Filaments

Myosin II propelled actin filaments move ten times faster than kinesin driven microtubules and are thus attractive candidates as cargo-transporting shuttles in motor driven lab-on-a-chip devices. In addition, actomyosin-based transportation of nanoparticles is useful in various fundamental studies. However, it is poorly understood how actomyosin function is affected by different number of nanoscale cargoes, by cargo size, and by the mode of cargo-attachment to the actin filament. This is studied here using biotin/fluorophores, streptavidin, streptavidin-coated quantum dots, and liposomes as model cargoes attached to monomers along the actin filaments (“side-attached”) or to the trailing filament end via the plus end capping protein CapZ. Long-distance transportation (>100 µm) could be seen for all cargoes independently of attachment mode but the fraction of motile filaments decreased with increasing number of side-attached cargoes, a reduction that occurred within a range of 10–50 streptavidin molecules, 1–10 quantum dots or with just 1 liposome. However, as observed by monitoring these motile filaments with the attached cargo, the velocity was little affected. This also applied for end-attached cargoes where the attachment was mediated by CapZ. The results with side-attached cargoes argue against certain models for chemomechanical energy transduction in actomyosin and give important insights of relevance for effective exploitation of actomyosin-based cargo-transportation in molecular diagnostics and other nanotechnological applications. The attachment of quantum dots via CapZ, without appreciable modulation of actomyosin function, is useful in fundamental studies as exemplified here by tracking with nanometer accuracy.     

RNA结合域决定JDV Tat具有较强的激活能力

为分析JDV与BIV、HIV-1 LTR和Tat相互激活能力差异的原因,在氨基酸序列对比及HIV-1 Tat功能域划分的基础上构建了JH、HJ、JB、BJ几种嵌合Tat蛋白,并克隆到真核表达载体.将上述表达质粒与以JDV、BIV和HIV-1 LTR为启动子,以luc为报告基因的质粒共转染Hela细胞,证实了三种不同Tat激活能力的差异主要来自其结合域RNA结合能力的差异,排除了结构域不完整和细胞因子缺乏造成JH不激活HIV-1 LTR的可能性.     

RNA结合域决定JDV Tat具有较强的激活能力

为分析JDV与BIV、HIV-1LTR和Tat相互激活能力差异的原因,在氨基酸序列对比及HIV-1Tat功能域划分的基础上构建了JH、HJ、JB、BJ几种嵌合Tat蛋白,并克隆到真核表达载体。将上述表达质粒与以JDV、BIV和HIV-1LTR为启动子,以luc为报告基因的质粒共转染Hela细胞,证实了三种不同Tat激活能力的差异主要来自其结合域RNA结合能力的差异,排除了结构域不完整和细胞因子缺乏造成JH不激活HIV-1LTR的可能性。     

Actin Filaments Purified from Tobacco Cultured BY-2 Cells Can Be Translocated by Plant Myosin

Actin filaments (AFs) and microtubules (MTs) are essential constituentsof the cytoskeleton in plant cells. Sliding of motor proteinsalong these cytoskeletons is believed to be necessary in variouscellular functions. In our previous study [Yokota et al. (1995b)Plant Cell Physiol. 36: 1563], we succeeded in isolating tubulinfrom cultured tobacco BY-2 cells, which in its polymerized formcan be translocated by the MT-based motor protein, dynein, invitro. In the present study, the method was modified to purifyboth tubulin and actin. Purified actin could be polymerizedand decorated by subfragment-1 (S-1) of skeletal muscle myosin.In the motility assay in vitro, AFs, thus prepared, could betranslocated by plant myosin isolated from lily pollen tubes.The sliding velocity of those AFs was similar to that of animalAFs prepared from chicken breast muscle, and comparable withthe velocity of cytoplasmic streaming in living pollen tubesof lily. Using S-1, motility assay was carried out. The slidingvelocity of plant AFs and that of muscle AFs were also similar.As far as we know, this is the first report of the sliding ofisolated plant AFs with myosin. (Received April 30, 1999; Accepted September 7, 1999)     

Myosin Loop 2 Is Involved in the Formation of a Trimeric Complex of Twitchin, Actin, and Myosin

Molluscan smooth muscles exhibit a low energy cost contraction called catch. Catch is regulated by twitchin phosphorylation and dephosphorylation. Recently, we found that the D2 fragment of twitchin containing the D2 site (Ser-4316) and flanking immunoglobulin motifs (TWD2-S) formed a heterotrimeric complex with myosin and with actin in the region that interacts with myosin loop 2 (Funabara, D., Hamamoto, C., Yamamoto, K., Inoue, A., Ueda, M., Osawa, R., Kanoh, S., Hartshorne, D. J., Suzuki, S., and Watabe, S. (2007) J. Exp. Biol. 210, 4399–4410). Here, we show that TWD2-S interacts directly with myosin loop 2 in a phosphorylation-sensitive manner. A synthesized peptide, CAQNKEAETTGTHKKRKSSA, based on the myosin loop 2 sequence (loop 2 peptide), competitively inhibited the formation of the trimeric complex. Isothermal titration calorimetry showed that TWD2-S binds to the loop 2 peptide with a K_a of (2.44 ± 0.09) × 10⁵ m⁻¹ with two binding sites. The twitchin-binding peptide of actin, AGFAGDDAP, which also inhibited formation of the trimeric complex, bound to TWD2-S with a K_a of (5.83 ± 0.05) × 10⁴ m⁻¹ with two binding sites. The affinity of TWD2-S to actin and myosin was slightly decreased with an increase of pH, but this effect could not account for the marked pH dependence of catch in permeabilized fibers. The complex formation also showed a moderate Ca²⁺ sensitivity in that in the presence of Ca²⁺ complex formation was reduced.Molluscan smooth muscles, such as mussel anterior byssus retractor muscle (ABRM)² and adductor muscle, exhibit a low energy cost phase of tension maintenance termed catch. Catch muscle develops active tension following an increase of the intracellular [Ca²⁺] induced by secretion of acetylcholine. Myosin is activated by direct binding of Ca²⁺ to the regulatory myosin light chain and initiates a relative sliding between thick and thin filaments (1). After a decrease of intracellular [Ca²⁺] to resting levels, the catch state is formed where tension is maintained over long periods of time with little energy consumption (2, 3). Catch tension is abolished by secretion of serotonin and an increase of intracellular [cAMP] with the resulting activation of cAMP-dependent protein kinase and phosphorylation of twitchin (4, 5). Twitchin phosphorylation is required for relaxation of the muscle from catch. For this cycle to repeat, dephosphorylation of twitchin is necessary (6). Thus, in this scheme, twitchin is a major regulator of the catch state.Molluscan twitchin is known as a myosin-binding protein belonging to the titin/connectin superfamily. It is a single polypeptide of 530 kDa containing multiple repeats of immunoglobulin (Ig) and fibronectin type 3-like motifs in addition to a single kinase domain homologous to the catalytic domain of myosin light chain kinase of vertebrate smooth muscle (7). There are several possible phosphorylation sites in molluscan twitchin recognized by cAMP-dependent protein kinase, and two, D1 and D2, have been identified. The D1 phosphorylation site (Ser-1075) is in the linker region between the 7th and 8th Ig motifs (numbering from the N terminus). The D2 site (Ser-4316) is in the linker region between the 21st and 22nd Ig motifs. Additional sites are found close to D1, but are thought not to be vital for catch regulation.The molecular mechanisms underlying development and maintenance of the catch state have been controversial for several years. One theory proposes that catch reflected attached frozen or slowly cycling cross-bridges (8, 9). What distinguished the attached cross-bridge from the detached relaxed state is not clear. Also it was suggested that interactions between thick filaments, other than cross-bridges, or between thin and thick filaments are responsible for the catch contraction (10). In either of the latter cases, the cross-bridge (myosin head) was not involved.Recently we found that a twitchin fragment including the D2 phosphorylation site and its flanking Ig motifs (TWD2-S) interacted with myosin and actin in a phosphorylation-sensitive manner, and it was suggested that this trimeric complex contributed to tension maintenance in catch (11). TWD2-S bound to a region of the actin molecule known also to interact with loop 2 of myosin that is involved in the ATP-driven movement of myosin with actin (12). In the present study, we show that the myosin loop 2 binds to TWD2-S using competitive cosedimentation assays and isothermal titration calorimetry (ITC). These techniques were applied to also study in more detail the interactions of the twitchin-binding peptide of actin (identified in the previous study (11)). In addition, the effects of pH and Ca²⁺ on the binding of TWD2-S to myosin and actin were investigated.     

Phosphorylation of the Actin Binding Protein Drebrin at S647 Is Regulated by Neuronal Activity and PTEN

Defects in actin dynamics affect activity-dependent modulation of synaptic transmission and neuronal plasticity, and can cause cognitive impairment. A salient candidate actin-binding protein linking synaptic dysfunction to cognitive deficits is Drebrin (DBN). However, the specific mode of how DBN is regulated at the central synapse is largely unknown. In this study we identify and characterize the interaction of the PTEN tumor suppressor with DBN. Our results demonstrate that PTEN binds DBN and that this interaction results in the dephosphorylation of a site present in the DBN C-terminus - serine 647. PTEN and pS647-DBN segregate into distinct and complimentary compartments in neurons, supporting the idea that PTEN negatively regulates DBN phosphorylation at this site. We further demonstrate that neuronal activity increases phosphorylation of DBN at S647 in hippocampal neurons in vitro and in ex vivo hippocampus slices exhibiting seizure activity, potentially by inducing rapid dissociation of the PTEN:DBN complex. Our results identify a novel mechanism by which PTEN is required to maintain DBN phosphorylation at dynamic range and signifies an unusual regulation of an actin-binding protein linked to cognitive decline and degenerative conditions at the CNS synapse.     

The Actin Binding Domain of the Epidermal Growth Factor Receptor Is Required for EGF-Stimulated Tissue Invasion

NIH-3T3 fibroblasts expressing epidermal growth factor receptors (EGFRs) lacking the actin binding domain (ABD) were analyzed for their EGF-induced capacity to invade a bone marrow stromal cell (BMSC) monolayer. The fibroblasts display a reduction in the percentage of cytoskeleton-associated EGFRs. Furthermore, EGF-induced tyrosine kinase activity is unaffected by the mutation. Cells expressing the mutant EGFRs hardly invade a BMSC monolayer upon EGF stimulation in contrast to cells expressing wild-type EGFRs. Using the same cells no difference was observed in PDGF-induced invasion, which ligand was as potent in both cell types as EGF was in wild-type cells. Inhibition of both the phosphatidyl inositol-3-kinase (PI-3-K) and lipoxygenase pathways in wild-type cells mimicked the effect of the ABD deletion. Our results point to an important role for the ABD of the EGFR in EGF-induced tissue invasion.     

Strong Binding of Single-stranded DNA by Stem-loop Oligonucleotides

Abstract

We report that oligodeoxynucleotides which form stem-loop hairpin structures and which have pyrimidine-rich loops can form strong complexes with complementary single-stranded DNA sequences. Stem-loop oligonucleotides were constructed with a 25-nt T-rich loop and with variable Watson-Crick stems. The complexes of these oligomers with the sequence dA_gwere studied by thermal denaturation. Evidence is presented that the complexes are one-to-one, bimolecular complexes in which the pyrimidine loop bases comprise the outer strands in a pyr · pur · pyr triplex, in effect chelating the purine strand in the center of the loop. Melting temperatures for the loop complexes are shown to be up to 29 °C higher than Watson- Crick duplex of the same length. It is shown that the presence of a stem increases stability of the triplex relative to an analogous oligomer without a stem. The effect of stem length on the stability of such a complex is examined. Such hairpin oligomers represent a new approach to the sequence-specific binding of single-stranded RNA and DNA. In addition, the finding raises the possibility that such a complex may exist in natural RNA folded sequences.     

Longitudinal Distortions and Transversal Fluctuations of an Actin Filament Sliding on Myosin Molecules

An actin filament sliding on myosin moleculesdemonstrates both longitudinal distortions and transversal fluctuationswith the linear dimension far exceeding the diameter of an actinmonomer. Local swaying of a single actin filament was identified byreading speckled fluorescent markers attached on the filament. Theaccuracy of reading each speckled marker was about 10.4 nm (r.m.s.).Longitudinal distortions of an actin filament at a low ATP concentrationof 20 M were as much as 0.5 m for the average filament lengthof 5.4 m. The magnitude of transversal fluctuations was as much as60 nm, that was independent of the filament length. Both longitudinaldistortions and transversal fluctuations are suggested to play a pivotalrole for facilitating a smooth sliding movement of an actin filament.     

Alternative S2 Hinge Regions of the Myosin Rod Affect Myofibrillar Structure and Myosin Kinetics

The subfragment 2/light meromyosin “hinge” region has been proposed to significantly contribute to muscle contraction force and/or speed. Transgenic replacement of the endogenous fast muscle isovariant hinge A (exon 15a) in Drosophila melanogaster indirect flight muscle with the slow muscle hinge B (exon 15b) allows examination of the structural and functional changes when only this region of the myosin molecule is different. Hinge B was previously shown to increase myosin rod length, increase A-band and sarcomere length, and decrease flight performance compared to hinge A. We applied additional measures to these transgenic lines to further evaluate the consequences of modifying this hinge region. Structurally, the longer A-band and sarcomere lengths found in the hinge B myofibrils appear to be due to the longitudinal addition of myosin heads. Functionally, hinge B, although a significant distance from the myosin catalytic domain, alters myosin kinetics in a manner consistent with this region increasing myosin rod length. These structural and functional changes combine to decrease whole fly wing-beat frequency and flight performance. Our results indicate that this hinge region plays an important role in determining myosin kinetics and in regulating thick and thin filament lengths as well as sarcomere length.     

A Second Tubulin Binding Site on the Kinesin-13 Motor Head Domain Is Important during Mitosis

Kinesin-13s are microtubule (MT) depolymerases different from most other kinesins that move along MTs. Like other kinesins, they have a motor or head domain (HD) containing a tubulin and an ATP binding site. Interestingly, kinesin-13s have an additional binding site (Kin-Tub-2) on the opposite side of the HD that contains several family conserved positively charged residues. The role of this site in kinesin-13 function is not clear. To address this issue, we investigated the in-vitro and in-vivo effects of mutating Kin-Tub-2 family conserved residues on the Drosophila melanogaster kinesin-13, KLP10A. We show that the Kin-Tub-2 site enhances tubulin cross-linking and MT bundling properties of KLP10A in-vitro. Disruption of the Kin-Tub-2 site, despite not having a deleterious effect on MT depolymerization, results in abnormal mitotic spindles and lagging chromosomes during mitosis in Drosophila S2 cells. The results suggest that the additional Kin-Tub-2 tubulin biding site plays a direct MT attachment role in-vivo.     

UNC-45B Chaperone: The Role of its Domains in the Interaction with the Myosin Motor Domain

The proper folding of many proteins can only be achieved by interaction with molecular chaperones. The molecular chaperone UNC-45B is required for the folding of striated muscle myosin II. However, the precise mechanism by which it contributes to proper folding of the myosin head remains unclear. UNC-45B contains three domains: an N-terminal TPR domain known to bind Hsp90, a Central domain of unknown function, and a C-terminal UCS domain known to interact with the myosin head. Here we used fluorescence titrations methods, dynamic light scattering, and single-molecule atomic force microscopy (AFM) unfolding/refolding techniques to study the interactions of the UCS and Central domains with the myosin motor domain. We found that both the UCS and the Central domains bind to the myosin motor domain. Our data show that the domains bind to distinct subsites on the myosin head, suggesting distinct roles in forming the myosin−UNC-45B complex. To determine the chaperone activity of the UCS and Central domains, we used two different methods: 1), prevention of misfolding using single-molecule AFM, and 2), prevention of aggregation using dynamic light scattering. Using the first method, we found that the UCS domain is sufficient to prevent misfolding of a titin mechanical reporter. Application of the second method showed that the UCS domain but not the Central domain prevents the thermal aggregation of the myosin motor domain. We conclude that while both the UCS and the Central domains bind the myosin head with high affinity, only the UCS domain displays chaperone activity.     

苯丙氨酰-tRNA合成酶的进化与结构域丢失

基因的复制、融合以及基因的水平转移是许多蛋白质包括氨酰 tRNA合成酶 (aminoacyl tRNAsynthetase ,AARS)进化过程中的常见事件。然而作者研究的结果显示 ,苯丙氨酰 tRNA合成酶 (phenylalanyl tRNAsynthetase,PheRS)的进化主要表现为一些结构域的丢失 ;并且这种结构域的丢失不影响PheRS的功能或活性。通常在生物从细菌到真核生物的进化过程中 ,其基因组的大小和基因的数目都有所增加 ,然而有趣的是 ,真核生物中PheRS的结构域类型和数目都明显少于细菌的PheRS。PheRS通过结构域的丢失而进化的现象 ,似乎与某些AARS功能由多重专一性向单一专一性的演化有着“异曲同工”之妙。     

Metal Switch-controlled Myosin II from Dictyostelium discoideum Supports Closure of Nucleotide Pocket during ATP Binding Coupled to Detachment from Actin Filaments

G-proteins, kinesins, and myosins are hydrolases that utilize a common protein fold and divalent metal cofactor (typically Mg²⁺) to coordinate purine nucleotide hydrolysis. The nucleoside triphosphorylase activities of these enzymes are activated through allosteric communication between the nucleotide-binding site and the activator/effector/polymer interface to convert the free energy of nucleotide hydrolysis into molecular switching (G-proteins) or force generation (kinesins and myosin). We have investigated the ATPase mechanisms of wild-type and the S237C mutant of non-muscle myosin II motor from Dictyostelium discoideum. The S237C substitution occurs in the conserved metal-interacting switch-1, and we show that this substitution modulates the actomyosin interaction based on the divalent metal present in solution. Surprisingly, S237C shows rapid basal steady-state Mg²⁺- or Mn²⁺-ATPase kinetics, but upon binding actin, its MgATPase is inhibited. This actin inhibition is relieved by Mn²⁺, providing a direct and experimentally reversible linkage of switch-1 and the actin-binding cleft through the swapping of divalent metals in the reaction. Using pyrenyl-labeled F-actin, we demonstrate that acto·S237C undergoes slow and weak MgATP binding, which limits the rate of steady-state catalysis. Mn²⁺ rescues this effect to near wild-type activity. 2′(3′)-O-(N-Methylanthraniloyl)-ADP release experiments show the need for switch-1 interaction with the metal cofactor for tight ADP binding. Our results are consistent with strong reciprocal coupling of nucleoside triphosphate and F-actin binding and provide additional evidence for the allosteric communication pathway between the nucleotide-binding site and the filament-binding region.