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1.
Striated muscle contraction in most animals is regulated at least in part by the troponin-tropomyosin (Tn-Tm) switch on the thin (actin-containing) filaments. The only group that has been suggested to lack actin-linked regulation is the mollusks, where contraction is regulated through the myosin heads on the thick filaments. However, molluscan gene sequence data suggest the presence of troponin (Tn) components, consistent with actin-linked regulation, and some biochemical and immunological data also support this idea. The presence of actin-linked (in addition to myosin-linked) regulation in mollusks would simplify our general picture of muscle regulation by extending actin-linked regulation to this phylum as well. We have investigated this question structurally by determining the effect of Ca2+ on the position of Tm in native thin filaments from scallop striated adductor muscle. Three-dimensional reconstructions of negatively stained filaments were determined by electron microscopy and single-particle image analysis. At low Ca2+, Tm appeared to occupy the “blocking” position, on the outer domain of actin, identified in earlier studies of regulated thin filaments in the low-Ca2+ state. In this position, Tm would sterically block myosin binding, switching off filament activity. At high Ca2+, Tm appeared to move toward a position on the inner domain, similar to that induced by Ca2+ in regulated thin filaments. This Ca2+-induced movement of Tm is consistent with the hypothesis that scallop thin filaments are Ca2+ regulated. 相似文献
2.
S100A6 is a calcium binding protein that, like some other members of the S100 protein family, is able to bind p53. This interaction may be physiologically relevant considering the numerous connotations of S100 proteins and of S100A6, in particular, with cancer and metastasis. In this work, we show that the interaction with S100A6 is limited to unmodified or phosphorylated p53 and is inhibited by p53 acetylation. Using in vitro acetylation assay, we show that the presence of S100A6 attenuates p53 acetylation by p300. Furthermore, using ELISA, we show that S100A6 and the TAZ2 domain of p300 bind p53 with similar affinities and that S100A6 effectively competes with TAZ2 for binding to p53. Our results add another element to the complicated scheme of p53 activation. 相似文献
3.
Shchepkin DV Kopylova GV Nikitina LV 《Biochemical and biophysical research communications》2011,(1):104-108
Interaction of myosin with actin in striated muscle is controlled by Ca2+ via thin filament associated proteins: troponin and tropomyosin. In cardiac muscle there is a whole pattern of myosin and tropomyosin isoforms. The aim of the current work is to study regulatory effect of tropomyosin on sliding velocity of actin filaments in the in vitro motility assay over cardiac isomyosins. It was found that tropomyosins of different content of α- and β-chains being added to actin filament effects the sliding velocity of filaments in different ways. On the other hand the velocity of filaments with the same tropomyosins depends on both heavy and light chains isoforms of cardiac myosin. 相似文献
4.
Borovikov YS Avrova SV Karpicheva OE Robinson P Redwood CS 《Biochemical and biophysical research communications》2011,(3):496-500
Dilated cardiomyopathy (DCM), characterized by cardiac dilatation and contractile dysfunction, is a major cause of heart failure. DCM can result from mutations in the gene encoding cardiac α-tropomyosin (TM). In order to understand how the dilated cardiomyopathy-causing Glu40Lys mutation in TM affects actomyosin interactions, thin filaments have been reconstituted in muscle ghost fibers by incorporation of labeled Cys707 of myosin subfragment-1 and Cys374 of actin with fluorescent probe 1.5-IAEDANS and α-tropomyosin (wild-type or Glu40Lys mutant). For the first time, the effect of these α-tropomyosins on the mobility and rotation of subdomain-1 of actin and the SH1 helix of myosin subfragment-1 during the ATP hydrolysis cycle have been demonstrated directly by polarized fluorimetry. The Glu40Lys mutant TM inhibited these movements at the transition from AM∗∗·ADP·Pi to AM state, indicating a decrease of the proportion of the strong-binding sub-states in the actomyosin population. These structural changes are likely to underlie the contractile deficit observed in human dilated cardiomyopathy. 相似文献
5.
Regulation of muscle contraction via the myosin filaments occurs in vertebrate smooth and many invertebrate striated muscles. Studies of unphosphorylated vertebrate smooth muscle myosin suggest that activity is switched off through an intramolecular interaction between the actin-binding region of one head and the converter and essential light chains of the other, inhibiting ATPase activity and actin interaction. The same interaction (and additional interaction with the tail) is seen in three-dimensional reconstructions of relaxed, native myosin filaments from tarantula striated muscle, suggesting that such interactions are likely to underlie the off-state of myosin across a wide spectrum of the animal kingdom. We have tested this hypothesis by carrying out cryo-electron microscopy and three-dimensional image reconstruction of myosin filaments from horseshoe crab (Limulus) muscle. The same head-head and head-tail interactions seen in tarantula are also seen in Limulus, supporting the hypothesis. Other data suggest that this motif may underlie the relaxed state of myosin II in all species (including myosin II in nonmuscle cells), with the possible exception of insect flight muscle.The molecular organization of the myosin tails in the backbone of muscle thick filaments is unknown and may differ between species. X-ray diffraction data support a general model for crustaceans in which tails associate together to form 4-nm-diameter subfilaments, with these subfilaments assembling together to form the backbone. This model is supported by direct observation of 4-nm-diameter elongated strands in the tarantula reconstruction, suggesting that it might be a general structure across the arthropods. We observe a similar backbone organization in the Limulus reconstruction, supporting the general existence of such subfilaments. 相似文献
6.
Jung HS Billington N Thirumurugan K Salzameda B Cremo CR Chalovich JM Chantler PD Knight PJ 《Journal of molecular biology》2011,408(5):863-878
Myosin 2 from vertebrate smooth muscle or non-muscle sources is in equilibrium between compact, inactive monomers and thick filaments under physiological conditions. In the inactive monomer, the two heads pack compactly together, and the long tail is folded into three closely packed segments that are associated chiefly with one of the heads. The molecular basis of the folding of the tail remains unexplained. By using electron microscopy, we show that compact monomers of smooth muscle myosin 2 have the same structure in both the native state and following specific, intramolecular photo-cross-linking between Cys109 of the regulatory light chain (RLC) and segment 3 of the tail. Nonspecific cross-linking between lysine residues of the folded monomer by glutaraldehyde also does not perturb the compact conformation and stabilizes it against unfolding at high ionic strength. Sequence comparisons across phyla and myosin 2 isoforms suggest that the folding of the tail is stabilized by ionic interactions between the positively charged N-terminal sequence of the RLC and a negatively charged region near the start of tail segment 3 and that phosphorylation of the RLC could perturb these interactions. Our results support the view that interactions between the heads and the distal tail perform a critical role in regulating activity of myosin 2 molecules through stabilizing the compact monomer conformation. 相似文献
7.
Kritica Arora Lama Talje Ana B. Asenjo Parker Andersen Kaleem Atchia Monika Joshi Hernando Sosa John S. Allingham Benjamin H. Kwok 《Journal of molecular biology》2014
The mitotic kinesin motor protein KIF14 is essential for cytokinesis during cell division and has been implicated in cerebral development and a variety of human cancers. Here we show that the mouse KIF14 motor domain binds tightly to microtubules and does not display typical nucleotide-dependent changes in this affinity. It also has robust ATPase activity but very slow motility. A crystal structure of the ADP-bound form of the KIF14 motor domain reveals a dramatically opened ATP-binding pocket, as if ready to exchange its bound ADP for Mg·ATP. In this state, the central β-sheet is twisted ~ 10° beyond the maximal amount observed in other kinesins. This configuration has only been seen in the nucleotide-free states of myosins—known as the “rigor-like” state. Fitting of this atomic model to electron density maps from cryo-electron microscopy indicates a distinct binding configuration of the motor domain to microtubules. We postulate that these properties of KIF14 are well suited for stabilizing midbody microtubules during cytokinesis. 相似文献
8.
N Narayanan P Lee M Newland R L Khandelwal 《Biochemical and biophysical research communications》1982,108(3):1158-1164
A cytosolic protein fraction, termed CPF-I, derived by (NH4)2 SO4 fractionation of rabbit heart cytosol caused marked inhibition (up to 95%) of ATP-dependent Ca2+ uptake by cardiac sarcoplasmic reticulum. The inhibitory effect of CPF-I was concentration-dependent (50% inhibition with ~ 80–100 μg CPF-I) and heat labile. The inhibitor reduced the velocity of Ca2+ uptake without altering the apparent affinity of the transport system for Ca2+. Concomitant with the inhibition of Ca2+ uptake, Ca2+-sensitive ATP hydrolysis was also inhibited by CPF-I. The inhibitor did not cause release of Ca2+ from Ca2+-preloaded membrane vesicles. The inhibitor activity of CPF-I could be adsorbed to a DEAE cellulose column and could be eluted with a linear gradient of KCl. These results demonstrate the presence of a soluble protein inhibitor of sarcoplasmic reticulum calcium pump in cardiac muscle and raises the intriguing possibility of its participation in the regulation of calcium pump . 相似文献
9.
We have compared the physical properties of a 15.51-kb constitutive heterochromatin segment and a 16.17-kb facultative heterochromatin segment that form part of the chicken β-globin locus. These segments were excised from an avian erythroleukemia cell line by restriction enzyme digestion and released from the nucleus, thus allowing measurement of the sedimentation coefficients by use of calibrated sucrose gradients. A determination of the buoyant density of the cross-linked particle in CsCl led to the total mass of the particles and their frictional coefficients, f. Despite the slight differences in nucleosome density, the measured value of f for both fragments was consistent with a rodlike particle having a diameter of 33-45 nm and a length corresponding to approximately six to seven nucleosomes per 11-nm turn. At higher ionic strengths we found no evidence of any abrupt conformational change, demonstrating that these chromatin fragments released from the nucleus did not assume the more compact conformations recently described for some reconstituted structures. 相似文献
10.
Solubilized Ca2+, Mg2+-ATPase of sarcoplasmic reticulum was phosphorylated with ATP without added MgCl2. The phosphoenzyme formed was ADP-sensitive. Ca2+ in the medium was chelated after phosphorylation. This induced a slow transition of the phosphoenzyme from ADP-sensitive to ADP-insensitive forms. The ADP-sensitivity was restored by subsequent addition of CaCl2. These results showed that the transition was caused by dissociation of Ca2+ bound to the phosphoenzyme. Further observations indicated that, when Ca2+ in the medium was chelated, Ca2+ bound to the phosphoenzyme was dissociated much more slowly than Ca2+ bound to the dephosphoenzyme. This suggests a possible formation of the occluded form of the Ca2+-binding site in the phosphoenzyme. 相似文献
11.
Yasunobu Sugimoto Osamu Sato Shinya Watanabe Mitsuo Ikebe 《Journal of molecular biology》2009,392(2):420-2991
We used small-angle X-ray solution scattering (SAXS) technique to investigate the nucleotide-mediated conformational changes of the head domains [subfragment 1 (S1)] of myosin V and VI processive motors that govern their directional preference for motility on actin. Recombinant myosin V-S1 with two IQ motifs (MV-S1IQ2) and myosin VI-S1 (MVI-S1) were engineered from Sf9 cells using a baculovirus expression system. The radii of gyration (Rg) of nucleotide-free MV-S1IQ2 and MVI-S1 were 48.6 and 48.8 Å, respectively. In the presence of ATP, the Rg value of MV-S1IQ2 decreased to 46.7 Å, while that of MVI-S1 increased to 51.7 Å, and the maximum chord length of the molecule decreased by ca 9% for MV-S1IQ2 and increased by ca 6% for MVI-S1. These opposite directional changes were consistent with those occurring in S1s with ADP and Vi or AlF4− 2 bound (i.e., in states mimicking the ADP/Pi-bound state of ATP hydrolysis). Binding of AMPPNP induced Rg changes of both constructs similar to those in the presence of ATP, suggesting that the timing of the structural changes for their motion on actin is upon binding of ATP (the pre-hydrolysis state) during the ATPase cycle. Binding of ADP to MV-S1IQ2 and MVI-S1 caused their Rg values to drop below those in the nucleotide-free state. Thus, upon the release of Pi, the reverse conformational change could occur, coupling to drive the directional motion on actin. The amount of Rg change upon the release of Pi was ca 6.4 times greater in MVI-S1 than in MV-S1IQ2, relating to the production of the large stroke of the MVI motor during its translocation on actin. Atomic structural models for these S1s based upon the ab initio shape reconstruction from X-ray scattering data were constructed, showing that MVI-S1 has the light-chain-binding domain positioned in the opposite direction to MV-S1IQ2 in both the pre- and post-powerstroke transition. The angular change between the light chain-binding domains of MV-S1IQ2 in the pre- to post-powerstroke transition was ∼ 50°, comparable to that of MII-S1. On the other hand, that of MVI-S1 was ∼ 100° or ∼ 130° much less than the currently postulated changes to allow the maximal stroke size of myosin VI-S1 but still significantly larger than those of other myosins reported so far. The results suggest that some additional alterations or elements are required for MVI-S1 to take maximal working strokes along the actin filament. 相似文献
12.
Raman K. Roy James D. Potter Satyapriya Sarkar 《Biochemical and biophysical research communications》1976,70(1):28-36
The Ca2+-regulatory tropomyosin-troponin complex was purified from chick embryonic muscles by a combination of DEAE-cellulose chromatography and (NH4)2SO4 fractionation. The embryonic complex was very similar to that obtained from adult chicken muscles with respect to stoichiometry of components and biological activity. Tropomyosin of embryonic skeletal muscles contains both α and β subunits, the β form being the major species. In the adult stage the β form is decreased with a concomitant increase in the α form. These results indicate that i) the Ca2+-regulatory proteins are not deficient in early embryonic muscles as previously thought (Hitchcock, S.E., Develop. Biol. , 399, 1970), and ii) different structural genes coding for tropomyosin subunits are expressed differentially in embryonic and adult muscle fibers. 相似文献
13.
Dong H Li X Lou Z Xu X Su D Zhou X Zhou W Bartlam M Rao Z 《Journal of molecular biology》2008,383(3):455-464
Calcyphosine is an EF-hand protein involved in both Ca2 +-phosphatidylinositol and cyclic AMP signal cascades, as well as in other cellular functions. The crystal structure of Ca2 +-loaded calcyphosine was determined up to 2.65 Å resolution and reveals a protein containing two pairs of Ca2 +-binding EF-hand motifs. Calcyphosine shares a highly similar overall topology with calmodulin. However, there are striking differences between EF-hand 4, both N-terminal and C-terminal regions, and interdomain linkers. The C-terminal domain of calcyphosine possesses a large hydrophobic pocket in the presence of calcium ions that might be implicated in ligand binding, while its N-terminal hydrophobic pocket is almost shielded by an additional terminal helix. Calcyphosine is largely monomeric, regardless of the presence of Ca2 +. Differences in structure, oligomeric state in the presence and in the absence of Ca2 +, a highly conserved sequence with low similarity to other proteins, and phylogeny define a new EF-hand-containing family of calcyphosine proteins that extends from arthropods to humans. 相似文献
14.
Hiroyuki Iwamoto 《Journal of molecular biology》2009,390(1):99-192
Upon activation of living or skinned vertebrate skeletal muscle fibers, the sixth X-ray layer-line reflection from actin (6th ALL) is known to intensify, without a shift of its peak position along the layer line. Since myosin attachment to actin is expected to shift the peak towards the meridian, this intensification is considered to reflect the structural change of individual actin monomers in the thin filament. Here, we show that the 6th ALL of skinned insect flight muscles (IFMs) is rather weakened upon isometric calcium activation, and its peak shifts away from the meridian. This suggests that the actin monomers in the two types of muscles change their structures in substantially different manners. The changes that occurred in the 6th ALL of IFM were not diminished by lowering the temperature from 20 to 5 °C, while active force was greatly reduced. The inclusion of 100 μM blebbistatin (a myosin inhibitor) did not affect the changes either. This suggests that calcium binding to troponin C, rather than myosin binding to actin, causes the structural change of IFM actin. 相似文献
15.
Andrea E. Prota Franck Danel Felix Bachmann Katja Bargsten Rubén M. Buey Jens Pohlmann Stefan Reinelt Heidi Lane Michel O. Steinmetz 《Journal of molecular biology》2014
Microtubule-targeting agents are widely used for the treatment of cancer and as tool compounds to study the microtubule cytoskeleton. BAL27862 is a novel microtubule-destabilizing drug that is currently undergoing phase I clinical evaluation as the prodrug BAL101553. The drug is a potent inhibitor of tumor cell growth and shows a promising activity profile in a panel of human cancer models resistant to clinically relevant microtubule-targeting agents. Here, we evaluated the molecular mechanism of the tubulin–BAL27862 interaction using a combination of cell biology, biochemistry and structural biology methods. Tubulin-binding assays revealed that BAL27862 potently inhibited tubulin assembly at 37 °C with an IC50 of 1.4 μM and bound to unassembled tubulin with a stoichiometry of 1 mol/mol tubulin and a dissociation constant of 244 ± 30 nM. BAL27862 bound to tubulin independently of vinblastine, without the formation of tubulin oligomers. The kinetics of BAL27862 binding to tubulin were distinct from those of colchicine, with evidence of competition between BAL27862 and colchicine for binding. Determination of the tubulin–BAL27862 structure by X-ray crystallography demonstrated that BAL27862 binds to the same site as colchicine at the intradimer interface. Comparison of crystal structures of tubulin–BAL27862 and tubulin–colchicine complexes shows that the binding mode of BAL27862 to tubulin is similar to that of colchicine. However, comparative analyses of the effects of BAL27862 and colchicine on the microtubule mitotic spindle and in tubulin protease-protection experiments suggest different outcomes of tubulin binding. Taken together, our data define BAL27862 as a potent, colchicine site-binding, microtubule-destabilizing agent with distinct effects on microtubule organization. 相似文献
16.
Mohit C. Mathur P. Bryant Chase Joseph M. Chalovich 《Biochemical and biophysical research communications》2011,(1):4538
We examined the cardiomyopathy-causing tropomyosin mutations E180G, D175N, and V95A to determine their effects on actomyosin regulation. V95A reduced the ATPase rate when filaments were saturated with regulatory proteins both in the presence and absence of calcium, indicating either a stabilization of the inactive state or an inability to fully populate the active state. Effects of E180G and D175N were more complex. These two mutations increased ATPase rates at sub-saturating concentrations of troponin and tropomyosin as compared to wild type tropomyosin. At higher concentrations of regulatory proteins, ATPase rates became similar to wild type. Normal activation was achieved with the tight-binding myosin analog N-ethylmaleimide-S1, at saturating regulatory protein concentrations. These results suggest that the E180G and D175N mutations reduce the affinity of tropomyosin for actin and also destabilize troponin binding to the actin thin filaments. 相似文献
17.
In regulated myosin, motor and enzymatic activities are toggled between the on-state and off-state by a switch located on its lever arm domain, here called the regulatory domain (RD). This region consists of a long α-helical “heavy chain” stabilized by a “regulatory” light chain (RLC) and an “essential” light chain (ELC). The on-state is activated by phosphorylation of the RLC of vertebrate smooth muscle RD or by direct binding of Ca2+ to the ELC of molluscan RD. Crystal structures are available only for the molluscan RD. To understand in more detail the pathway between the on-state and the off-state, we have now also determined the crystal structure of a molluscan (scallop) RD in the absence of Ca2+. Our results indicate that loss of Ca2+ abolishes most of the interactions between the light chains and may increase the flexibility of the RD heavy chain. We propose that disruption of critical links with the C-lobe of the RLC is the key event initiating the off-state in both smooth muscle myosins and molluscan myosins. 相似文献
18.
Contraction of many muscles is activated in part by the binding of Ca2+ to, or phosphorylation of, the myosin heads on the surface of the thick filaments. In relaxed muscle, the myosin heads are helically ordered and undergo minimal interaction with actin. On Ca2+ binding or phosphorylation, the head array becomes disordered, reflecting breakage of the head-head and other interactions that underlie the ordered structure. Loosening of the heads from the filament surface enables them to interact with actin filaments, bringing about contraction. On relaxation, the heads return to their ordered positions on the filament backbone. In scallop striated adductor muscle, the disordering that takes place on Ca2+ binding occurs on the millisecond time scale, suggesting that it is a key element of muscle activation. Here we have studied the reverse process. Using time-resolved negative staining electron microscopy, we show that the rate of reordering on removal of Ca2+ also occurs on the same physiological time scale. Direct observation of images together with analysis of their Fourier transforms shows that activated heads regain their axial ordering within 20 ms and become ordered in their final helical positions within 50 ms. This rapid reordering suggests that reformation of the ordered structure, and the head-head and other interactions that underlie it, is a critical element of the relaxation process. 相似文献
19.
Prochniewicz E Pierre A McCullough BR Chin HF Cao W Saunders LP Thomas DD De La Cruz EM 《Journal of molecular biology》2011,413(3):584-592
The contractile and enzymatic activities of myosin VI are regulated by calcium binding to associated calmodulin (CaM) light chains. We have used transient phosphorescence anisotropy to monitor the microsecond rotational dynamics of erythrosin-iodoacetamide-labeled actin with strongly bound myosin VI (MVI) and to evaluate the effect of MVI-bound CaM light chain on actin filament dynamics. MVI binding lowers the amplitude but accelerates actin filament microsecond dynamics in a Ca2+- and CaM-dependent manner, as indicated from an increase in the final anisotropy and a decrease in the correlation time of transient phosphorescence anisotropy decays. MVI with bound apo-CaM or Ca2+-CaM weakly affects actin filament microsecond dynamics, relative to other myosins (e.g., muscle myosin II and myosin Va). CaM dissociation from bound MVI damps filament rotational dynamics (i.e., increases the torsional rigidity), such that the perturbation is comparable to that induced by other characterized myosins. Analysis of individual actin filament shape fluctuations imaged by fluorescence microscopy reveals a correlated effect on filament bending mechanics. These data support a model in which Ca2+-dependent CaM binding to the IQ domain of MVI is linked to an allosteric reorganization of the actin binding site(s), which alters the structural dynamics and the mechanical rigidity of actin filaments. Such modulation of filament dynamics may contribute to the Ca2+- and CaM-dependent regulation of myosin VI motility and ATP utilization. 相似文献
20.
Huang FC 《Cellular immunology》2011,(2):480-487
Our recent study demonstrated that a phosphatidylinositol-3 kinase (PI3K)/Akt-dependent anti-inflammatory pathway was activated by Salmonella in intestinal epithelial cells. Salmonella virulence is dependent on the ability of the bacterium to invade nonphagocytic host cells and then survive and replicate within modified Salmonella-containing vacuoles where cholesterol accumulates. In addition, cholesterol in membrane lipid rafts is frequently a platform for the activation of downstream signaling pathways, including the PI3K/Akt pathway. However, the role of plasma membrane cholesterol in the Salmonella-induced anti-inflammatory response in intestinal epithelial cells has not been elucidated. Here, we show that the effect of plasma membrane cholesterol depletion on the inhibition of Akt activation allows sustained ERK activation and the subsequent upregulation of IL-8 expression. These results demonstrate that plasma membrane cholesterol plays a critical role in the PI3K-dependent anti-inflammatory pathway activated by Salmonella in intestinal epithelial cells. 相似文献