Stable remodeling of tailless nucleosomes by the human SWI-SNF complex

The histone N-terminal tails have been shown previously to be important for chromatin assembly, remodeling, and stability. We have tested the ability of human SWI-SNF (hSWI-SNF) to remodel nucleosomes whose tails have been cleaved through a limited trypsin digestion. We show that hSWI-SNF is able to remodel tailless mononucleosomes and nucleosomal arrays, although hSWI-SNF remodeling of tailless nucleosomes is less effective than remodeling of nucleosomes with tails. Analogous to previous observations with tailed nucleosomal templates, we show both (i) that hSWI-SNF-remodeled trypsinized mononucleosomes and arrays are stable for 30 min in the remodeled conformation after removal of ATP and (ii) that the remodeled tailless mononucleosome can be isolated on a nondenaturing acrylamide gel as a novel species. Thus, nucleosome remodeling by hSWI-SNF can occur via interactions with a tailless nucleosome core.     

SWI-SNF-mediated nucleosome remodeling: role of histone octamer mobility in the persistence of the remodeled state

SWI-SNF is an ATP-dependent chromatin remodeling complex that disrupts DNA-histone interactions. Several studies of SWI-SNF activity on mononucleosome substrates have suggested that remodeling leads to novel, accessible nucleosomes which persist in the absence of continuous ATP hydrolysis. In contrast, we have reported that SWI-SNF-dependent remodeling of nucleosomal arrays is rapidly reversed after removal of ATP. One possibility is that these contrasting results are due to the different assays used; alternatively, the lability of the SWI-SNF-remodeled state might be different on mononucleosomes versus nucleosomal arrays. To investigate these possibilities, we use a coupled SWI-SNF remodeling-restriction enzyme assay to directly compare the remodeling of mononucleosome and nucleosomal array substrates. We find that SWI-SNF action causes a mobilization of histone octamers for both the mononucleosome and nucleosomal array substrates, and these changes in nucleosome positioning persist in the absence of continued ATP hydrolysis or SWI-SNF binding. In the case of mononucleosomes, the histone octamers accumulate at the DNA ends even in the presence of continued ATP hydrolysis. On nucleosomal arrays, SWI-SNF and ATP lead to a more dynamic state where nucleosomes appear to be constantly redistributed and restriction enzyme sites throughout the array have increased accessibility. This random positioning of nucleosomes within the array persists after removal of ATP, but inactivation of SWI-SNF is accompanied by an increased occlusion of many restriction enzyme sites. Our results also indicate that remodeling of mononucleosomes or nucleosomal arrays does not lead to an accumulation of novel nucleosomes that maintain an accessible state in the absence of continuous ATP hydrolysis.     

Chromatin remodelling at the PHO8 promoter requires SWI-SNF and SAGA at a step subsequent to activator binding

The SWI-SNF and SAGA complexes possess ATP-dependent nucleosome remodelling activity and histone acetyltransferase (HAT) activity, respectively. Mutations that eliminate the ATPase activity of the SWI-SNF complex, or the HAT activity of SAGA, abolish proper chromatin remodelling at the PHO8 promoter in vivo. These effects are mechanistically distinct, since the absence of SWI-SNF freezes chromatin in the repressed state, while the absence of Gcn5 permits a localized perturbation of chromatin structure immediately adjacent to the upstream transactivator binding site. However, this remodelling is not propagated to the proximal promoter, and no activation is observed under all conditions. Furthermore, Pho4 is bound to the PHO8 promoter in the absence of Snf2 or Gcn5, confirming a role for SWI-SNF and SAGA in chromatin remodelling independent of activator binding. These data provide new insights into the roles of the SWI-SNF and SAGA complexes in chromatin remodelling in vivo.     

Molecular Dynamics Simulation of the Allosteric Regulation of eIF4A Protein from the Open to Closed State,Induced by ATP and RNA Substrates

Background

Eukaryotic initiation factor 4A (eIF4A) plays a key role in the process of protein translation initiation by facilitating the melting of the 5′ proximal secondary structure of eukaryotic mRNA for ribosomal subunit attachment. It was experimentally postulated that the closed conformation of the eIF4A protein bound by the ATP and RNA substrates is coupled to RNA duplex unwinding to promote protein translation initiation, rather than an open conformation in the absence of ATP and RNA substrates. However, the allosteric process of eIF4A from the open to closed state induced by the ATP and RNA substrates are not yet fully understood.

Methodology

In the present work, we constructed a series of diplex and ternary models of the eIF4A protein bound by the ATP and RNA substrates to carry out molecular dynamics simulations, free energy calculations and conformation analysis and explore the allosteric properties of eIF4A.

Results

The results showed that the eIF4A protein completes the conformational transition from the open to closed state via two allosteric processes of ATP binding followed by RNA and vice versa. Based on cooperative allosteric network analysis, the ATP binding to the eIF4A protein mainly caused the relative rotation of two domains, while the RNA binding caused the proximity of two domains via the migration of RNA bases in the presence of ATP. The cooperative binding of ATP and RNA for the eIF4A protein plays a key role in the allosteric transition.     

The kinetic mechanism of kinesin

The chemical kinetic mechanism of kinesin (K) is considered by using a consensus scheme incorporating biochemically defined open, closed and trapped states. In the absence of microtubules, the dominant species is a trapped K*ADP state, which is defined by its ultra-slow release of ADP (off rate, k(off) approximately 0.002 s(-1)) and weak microtubule binding (dissociation constant, K(d) approximately 10-20 microM). Once bound, this trapped state equilibrates with a strongly binding open state that rapidly releases ADP (k(off) approximately 300 s(-1)). After ADP release, Mg*ATP binds (on rate, k(on) approximately 2 microM(-1)s(-1)) driving formation of a closed state that is defined by hydrolysis competence and by strong binding to microtubules. Hydrolysis (k(hyd) approximately 100-300 s(-1)) and phosphate release (k(off)>100 s(-1)) both occur in this microtubule-bound closed state. Phosphate release acts as a gate that controls reversion to the trapped K*ADP state, which detaches from the microtubule, completing the cycle.     

ATP dependent histone phosphorylation and nucleosome assembly in a human cell free extract.

Physiologically spaced nucleosome formation in HeLa cell extracts is ATP dependent. ATP hydrolysis is required for chromatin assembly on both linear and covalently closed circular DNA. The link between the phosphorylation state of histones and nucleosome formation has been examined and we demonstrate that in the absence of histone phosphorylation no stable and regularly spaced nucleosomes are formed. Phosphorylated H3 stabilizes the nucleosome core; while phosphorylation of histone H2a is necessary to increase the linker length between nucleosomes from 0 to approximately 45 bp. Histone H1 alone, whether phosphorylated or unphosphorylated, does not increase the nucleosome repeat length in the absence of core histone phosphorylation. Phosphorylations of H1 and H3 correlate with condensation of chromatin. Maximum ATP hydrolysis which is necessary to increase the periodicity of nucleosomes from approximately 150 to approximately 185 bp, not only inhibits H1 and H3 phosphorylation but facilitates their dephosphorylation.     

Circular closed DNA. Topology, energy, and stress at different equilibrium configurations and deformation conditions

The topology of circular closed DNA in different equilibrium configurations and deformation conditions has been investigated. The distribution of twisting and bending energy and stress has been assessed. It is shown that the twisting/bending stiffness ratio qualitatively influences the deformation of a circular DNA molecule.     

SWI/SNF chromatin remodeling requires changes in DNA topology

ySWI/SNF complex belongs to a family of enzymes that use the energy of ATP hydrolysis to remodel chromatin structure. Here we examine the role of DNA topology in the mechanism of ySWI/SNF remodeling. We find that the ability of ySWI/SNF to enhance accessibility of nucleosomal DNA is nearly eliminated when DNA topology is constrained in small circular nucleosomal arrays and that this inhibition can be alleviated by topoisomerases. Furthermore, we demonstrate that remodeling of these substrates does not require dramatic histone octamer movements or displacement. Our results suggest a model in which ySWI/SNF remodels nucleosomes by using the energy of ATP hydrolysis to drive local changes in DNA twist.     

Behavior of the ATP grasp domain of biotin carboxylase monomers and dimers studied using molecular dynamics simulations

The enzyme biotin carboxylase (BC) uses adenosine triphosphate (ATP) to carboxylate biotin and is involved in fatty acid synthesis. Structural evidence suggests that the B domain of BC undergoes a large hinge motion of ～45° when binding and releasing substrates. Escherichia coli BC can function as a natural homodimer and as a mutant monomer. Using molecular dynamics simulations, we evaluate the free energy profile along a closure angle of the B domain of E. coli BC for three cases: a monomer without bound Mg(2)ATP, a monomer with bound Mg(2)ATP, and a homodimer with bound Mg(2)ATP in one subunit. The simulation results show that a closed state is the most probable for the monomer with or without bound Mg(2)ATP. For the dimer with Mg(2)ATP in one of its subunits, communication between the two subunits was observed. Specifically, in the dimer, the opening of the subunit without Mg(2)ATP caused the other subunit to open, and hysteresis was observed upon reclosing it. The most stable state of the dimer is one in which the B domain of both subunits is closed; however, the open state for the B domain without Mg(2)ATP is only approximately 2k(B)T higher in free energy than the closed state. A simple diffusion model indicates that the mean times for opening and closing of the B domain in the monomer with and without Mg(2)ATP are much smaller than the overall reaction time, which is on the order of seconds.     

Allosteric regulation of Hsp70 chaperones by a proline switch

Crucial to the function of Hsp70 chaperones is the nucleotide-regulated transition between two conformational states, the ATP bound state with high association and dissociation rates for substrates and the ADP bound state with two and three orders of magnitude lower association and dissociation rates. The spontaneous transition between the two states is extremely slow, indicating a high energy barrier for the switch that regulates the transition. Here we provide evidence that a universally conserved proline in the ATPase domain constitutes the switch that assumes alternate conformations in response to ATP binding and hydrolysis. The conformation of the proline, acting through an invariant arginine as relay, determines and stabilizes the opened and closed conformation of the substrate binding domain and thereby regulates the chaperone activity of Hsp70.     

Coupling between ATP hydrolysis and protein conformational change in maltose transporter

As the intracellular part of maltose transporter, MalK dimer utilizes the energy of ATP hydrolysis to drive protein conformational change, which then facilitates substrate transport. Free energy evaluation of the complete conformational change before and after ATP hydrolysis is helpful to elucidate the mechanism of chemical‐to‐mechanical energy conversion in MalK dimer, but is lacking in previous studies. In this work, we used molecular dynamics simulations to investigate the structural transition of MalK dimer among closed, semi‐open and open states. We observed spontaneous structural transition from closed to open state in the ADP‐bound system and partial closure of MalK dimer from the semi‐open state in the ATP‐bound system. Subsequently, we calculated the reaction pathways connecting the closed and open states for the ATP‐ and ADP‐bound systems and evaluated the free energy profiles along the paths. Our results suggested that the closed state is stable in the presence of ATP but is markedly destabilized when ATP is hydrolyzed to ADP, which thus explains the coupling between ATP hydrolysis and protein conformational change of MalK dimer in thermodynamics. Proteins 2017; 85:207–220. © 2016 Wiley Periodicals, Inc.     

Halobacterial megaplasmids are negatively supercoiled

Several covalently closed circular halobacterial megaplasmids (up to more than 500 kb) from different strains of Halolerax mediterranei, have been resolved by orthogonal-field alternating gel electro-phoresis (OFAGE). These molecules seem to be negatively supercoiled in vivo, as deduced from the effect of intercalating agents affecting their topology and, therefore, their electrophoretic mobility. It has also been demonstrated that the topolsomerase II Inhibitor novobiocin affects the native topological state of halobacterial megaplasmids impeding their migration in OFAGE under standard conditions for resolution of large supercoiled molecules.     

An adenosine triphosphate-dependent deoxyribonuclease from Bacillus laterosporus. Improved purification, subunit structure and substrate specificity

The ATP-dependent deoxyribonuclease from Bacillus laterosporus has been purified to near homogeneity by a procedure involving ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-150 gel filtration, DEAE-Sephadex A-25 chromatography and DNA-cellulose affinity chromatography. The purified enzyme has a molecular weight of 210,000 +/- 8,000 as determined by sucrose gradient sedimentation. It is composed of two nonidentical polypeptide chains with close molecular weights of around 110,000. The substrate preference of the pure enzyme is essentially identical with the previous result obtained with the partially purified enzyme preparation (Anai, M., Mihara, T., Yamanaka, M., Shibata, T., & Takagi, Y. (1975) J. Biochem. 78, 105-114). Thus, the enzyme degrades double-stranded DNA about 100 times faster than heat-denatured DNA in the presence of ATP. Double-stranded DNA is not degraded to any measurable extent in the absence of ATP, but the enzyme exhibits activity toward denatured DNA in the absence of ATP. Furthermore, no endonuclease activity is observed on covalently closed circular duplex DNA and open circular duplex DNA.     

Adventures in membrane protein topology. A study of the membrane-bound state of colicin E1.

The molecular aggregate size of the closed state of the colicin E1 channel was determined by fluorescence resonance energy transfer experiments involving a fluorescence donor (three tryptophans, wild-type protein) and a fluorescence acceptor (5-(((acetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid (AEDANS), Trp-deficient protein). There was no evidence of energy transfer between the donor and acceptor species when bound to membrane large unilamellar vesicles. These experiments led to the conclusion that the colicin E1 channel is monomeric in the membrane-bound closed channel state. Experiments were also conducted to study the membrane topology of the closed colicin channel in membrane large unilamellar vesicles using acrylamide as the membrane-impermeant, nonionic quencher of tryptophan fluorescence in a battery of single tryptophan mutant proteins. Furthermore, additional fluorescence parameters, including fluorescence emission maximum, fluorescence quantum yield, and fluorescence decay times, were used to assist in mapping the topology of the closed channel. Results suggest that the closed channel comprises most of the polypeptide of the channel domain and that the hydrophobic anchor domain does not transverse the membrane bilayer but nonetheless is deeply embedded within the hydrocarbon core of the membrane. Finally, a model is proposed which features at least two states that are in rapid equilibrium with each other and in which one state is more heavily populated than the other.     

The kinetic and physical basis of K(ATP) channel gating: toward a unified molecular understanding

K(ATP) channels can be formed from Kir6.2 subunits with or without SUR1. The open-state stability of K(ATP) channels can be increased or reduced by mutations throughout the Kir6.2 subunit, and is increased by application of PIP(2) to the cytoplasmic membrane. Increase of open-state stability is manifested as an increase in the channel open probability in the absence of ATP (Po(zero)) and a correlated decrease in sensitivity to inhibition by ATP. Single channel lifetime analyses were performed on wild-type and I154C mutant channels expressed with, and without, SUR1. Channel kinetics include a single, invariant, open duration; an invariant, brief, closed duration; and longer closed events consisting of a "mixture of exponentials," which are prolonged in ATP and shortened after PIP(2) treatment. The steady-state and kinetic data cannot be accounted for by assuming that ATP binds to the channel and causes a gate to close. Rather, we show that they can be explained by models that assume the following regarding the gating behavior: 1) the channel undergoes ATP-insensitive transitions from the open state to a short closed state (C(f)) and to a longer-lived closed state (C(0)); 2) the C(0) state is destabilized in the presence of SUR1; and 3) ATP can access this C(0) state, stabilizing it and thereby inhibiting macroscopic currents. The effect of PIP(2) and mutations that stabilize the open state is then to shift the equilibrium of the "critical transition" from the open state to the ATP-accessible C(0) state toward the O state, reducing accessibility of the C(0) state, and hence reducing ATP sensitivity.     

High positive supercoiling in vitro catalyzed by an ATP and polyethylene glycol-stimulated topoisomerase from Sulfolobus acidocaldarius

A topoisomerase able to introduce positive supercoils in a closed circular DNA, has been isolated from the archaebacterium Sulfolobus acidocaldarius. This enzyme, fully active at 75 degrees C, performed in vitro positive supercoiling either from negatively supercoiled, or from relaxed DNA in a catalytic reaction. In the presence of polyethylene glycol (PEG 6000), this reaction became very fast and highly processive, and the product was positively supercoiled DNA with a high superhelical density (form I+). Very low (5 - 10 micromoles) ATP concentrations were sufficient to support full supercoiling; the nonhydrolyzable analogue adenosine-5' -0-(3-thiotriphosphate) also sustained the production of positive supercoils, but to a lesser extent, suggesting that ATP hydrolysis was necessary for efficient activity. Nevertheless, low residual of positive supercoiling occurred, even in the absence of ATP, when the substrate was negatively supercoiled. Finally, the different ATP-driven topoisomerizations observed, i.e., relaxation of negative supercoils and positive supercoiling, in all cases increased the linking number of DNA in steps of 1, suggesting the action of a type I, rather than a type II topoisomerase.=