Dynamic condensation of linker histone C-terminal domain regulates chromatin structure

The basic and intrinsically disordered C-terminal domain (CTD) of the linker histone (LH) is essential for chromatin compaction. However, its conformation upon nucleosome binding and its impact on chromatin organization remain unknown. Our mesoscale chromatin model with a flexible LH CTD captures a dynamic, salt-dependent condensation mechanism driven by charge neutralization between the LH and linker DNA. Namely, at low salt concentration, CTD condenses, but LH only interacts with the nucleosome and one linker DNA, resulting in a semi-open nucleosome configuration; at higher salt, LH interacts with the nucleosome and two linker DNAs, promoting stem formation and chromatin compaction. CTD charge reduction unfolds the domain and decondenses chromatin, a mechanism in consonance with reduced counterion screening in vitro and phosphorylated LH in vivo. Divalent ions counteract this decondensation effect by maintaining nucleosome stems and expelling the CTDs to the fiber exterior. Additionally, we explain that the CTD folding depends on the chromatin fiber size, and we show that the asymmetric structure of the LH globular head is responsible for the uneven interaction observed between the LH and the linker DNAs. All these mechanisms may impact epigenetic regulation and higher levels of chromatin folding.     

Effect of sequence and metal ions on UVB-induced anti cyclobutane pyrimidine dimer formation in human telomeric DNA sequences

Irradiation of G-quadruplex forming human telomeric DNA with ultraviolet B (UVB) light results in the formation of anti cyclobutane pyrimidine dimers (CPDs) between loop 1 and loop 3 in the presence of potassium ions but not sodium ions. This was unexpected because the sequences involved favor the nonphotoreactive hybrid conformations in K⁺ solution, whereas a potentially photoreactive basket conformation is favored in Na⁺ solution. To account for these contradictory results, it was proposed that the loops are too far apart in the basket conformation in Na⁺ solution but close enough in a two G-tetrad basket-like form 3 conformation that can form in K⁺ solution. In the current study, Na⁺ was still found to inhibit anti CPD formation in sequences designed to stabilize the form 3 conformation. Furthermore, anti CPD formation in K⁺ solution was slower for the sequence previously shown to exist primarily in the proposed photoreactive form 3 conformation than the sequence shown to exist primarily in a nonphotoreactive hybrid conformation. These results suggest that the form 3 conformation is not the principal photoreactive conformation, and that G-quadruplexes in K⁺ solution are dynamic and able to access photoreactive conformations more easily than in Na⁺ solution.     

Compaction of Single-Molecule Megabase-Long Chromatin under the Influence of Macromolecular Crowding

The megabase-sized length of chromatin is highly relevant to the state of chromatin in vivo, where it is subject to a highly crowded environment and is organized in topologically associating domains of similar dimension. We developed an in vitro experimental chromatin model system reconstituted from T4 DNA (approximately 166 kbp) and histone octamers and studied the monomolecular compaction of this megabase-sized chromatin fiber under the influence of macromolecular crowding. We used single-molecule fluorescence microscopy and observed compaction in aqueous solutions containing poly(ethylene glycol) in the presence of monovalent (Na⁺ and K⁺) and divalent (Mg²⁺) cations. Both DNA and chromatin demonstrated compaction under comparable conditions in the presence of poly(ethylene glycol) and Na⁺ or Mg²⁺ salt. However, the mechanism of the compaction changed from a first-order phase transition for DNA to a continuous folding for megabase-sized chromatin fibers. A more efficient and pronounced chromatin compaction was observed in the presence of Na⁺ compared to K⁺. A flow-stretching technique to unfold DNA and chromatin coils was used to gain further insight into the morphology of partially folded chromatin fibers. The results revealed a distribution of partially folded chromatin fibers. This variability is likely the result of the heterogeneous distribution of nucleosomes on the DNA chain. The packaging of DNA in the form of chromatin in the crowded nuclear environment appears essential to ensure gradual conformational changes of DNA.     

Protein–DNA and ion–DNA interactions revealed through contrast variation SAXS

Understanding how DNA carries out its biological roles requires knowledge of its interactions with biological partners. Since DNA is a polyanionic polymer, electrostatic interactions contribute significantly. These interactions are mediated by positively charged protein residues or charge compensating cations. Direct detection of these partners and/or their effect on DNA conformation poses challenges, especially for monitoring conformational dynamics in real time. Small-angle x-ray scattering (SAXS) is uniquely sensitive to both the conformation and local environment (i.e. protein partner and associated ions) of the DNA. The primary challenge of studying multi-component systems with SAXS lies in resolving how each component contributes to the measured scattering. Here, we review two contrast variation (CV) strategies that enable targeted studies of the structures of DNA or its associated partners. First, solution contrast variation enables measurement of DNA conformation within a protein–DNA complex by masking out the protein contribution to the scattering profile. We review a specific example, in which the real-time unwrapping of DNA from a nucleosome core particle is measured during salt-induced disassembly. The second method, heavy atom isomorphous replacement, reports the spatial distribution of the cation cloud around duplex DNA by exploiting changes in the scattering strength of cations with varying atomic numbers. We demonstrate the application of this approach to provide the spatial distribution of monovalent cations (Na⁺, K⁺, Rb⁺, Cs⁺) around a standard 25-base pair DNA. The CV strategies presented here are valuable tools for understanding DNA interactions with its biological partners.     

Single-Molecule TPM Studies on the Conversion of Human Telomeric DNA

Human telomere contains guanine-rich (G-rich) tandem repeats of single-stranded DNA sequences at its 3′ tail. The G-rich sequences can be folded into various secondary structures, termed G-quadruplexes (G4s), by Hoogsteen basepairing in the presence of monovalent cations (such as Na⁺ and K⁺). We developed a single-molecule tethered particle motion (TPM) method to investigate the unfolding process of G4s in the human telomeric sequence AGGG(TTAGGG)₃ in real time. The TPM method monitors the DNA tether length change caused by formation of the G4, thus allowing the unfolding process and structural conversion to be monitored at the single-molecule level. In the presence of its antisense sequence, the folded G4 structure can be disrupted and converted to the unfolded conformation, with apparent unfolding time constants of 82 s and 3152 s. We also observed that the stability of the G4 is greatly affected by different monovalent cations. The folding equilibrium constant of G4 is strongly dependent on the salt concentration, ranging from 1.75 at 5 mM Na⁺ to 3.40 at 15 mM Na⁺. Earlier spectral studies of Na⁺- and K⁺-folded states suggested that the spectral conversion between these two different folded structures may go through a structurally unfolded intermediate state. However, our single-molecule TPM experiments did not detect any totally unfolded intermediate within our experimental resolution when sodium-folded G4 DNA molecules were titrated with high-concentration, excess potassium ions. This observation suggests that a totally unfolding pathway is likely not the major pathway for spectral conversion on the timescale of minutes, and that interconversion among folded states can be achieved by the loop rearrangement. This study also demonstrates that TPM experiments can be used to study conformational changes in single-stranded DNA molecules.     

Changing Chromatin Fiber Conformation by Nucleosome Repositioning

Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ∼10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell.     

Optical Anisotropy of Chromatin. Flow Linear Dichroism and Electric Dichroism Studies

Abstract

The optical anisotropy of chromatin with different length of the linker DNA isolated from a variety of sources (Frend erythroleukemia cells, calf thymus, hen erythrocytes and sea urchin sperm) has been studied in a large range of mono- and bivalent cations concentraitons by the use of flow linear dichroism (LD) and electric dichroism.

We have found that all chromatins studied displayed negative LD values in the range of 0.25 mM EDTA—2 mM NaCl and close positive values in the range of 2–100 mM NaCl. Mg²⁺ cations, in contrast to Na⁺ cations, induce optically isotropic chromatin fibers. All chromatin samples exhibit positive form effect amounting to 5–10% of LD amplitude observed at 260 nm. This form effect is determined by the anisotropic scattering of polarized light by single chromatin fibers.

The conformational transition at 2 mM NaCl leads to the distortion of chromatin filament structure. The reversibility of this distortion depends on the length of the linker DNA—for chromatins with the linker DNA of 10–30 b.p. it is parially reversible, while for preparations with longer linker DNA it is irreversible.

Relatively low electric field does not affect chromatin structure, while higher electric field (more than 7 kV/cm) distorts the structure of chromatin.

Presented resutls explain the contradictory data obtained by electrooptical and hydrooptical methods.     

Confining the sodium pump in a phosphoenzyme form: the effect of lead(II) ions

The effect of Pb²⁺ ions on the Na⁺,K⁺-ATPase was investigated in detail by means of steady-state fluorescence spectroscopy. Experiments were performed by using the electrochromic styryl dye RH421. It is shown that Pb²⁺ ions can bind reversibly to the protein and do not affect the Na⁺ and K⁺ binding affinities in the E₁ and P-E₂ conformations of the enzyme. The pH titrations indicate that lead(II) favors binding of one H⁺ to the P-E₂ conformation in the absence of K⁺. A model scheme is proposed that accounts for the experimental results obtained for backdoor phosphorylation of the enzyme in the presence of Pb²⁺ ions. Taken together, our results clearly indicate that Pb²⁺ bound to the enzyme stabilizes an E₂-type conformation. In particular, under conditions that promote enzyme phosphorylation, Pb²⁺ ions are able to confine the Na⁺,K⁺-ATPase into a phosphorylated E₂ state.     

Nucleosome geometry and internucleosomal interactions control the chromatin fiber conformation

Based on model structures with atomic resolution, a coarse-grained model for the nucleosome geometry was implemented. The dependence of the chromatin fiber conformation on the spatial orientation of nucleosomes and the path and length of the linker DNA was systematically explored by Monte Carlo simulations. Two fiber types were analyzed in detail that represent nucleosome chains without and with linker histones, respectively: two-start helices with crossed-linker DNA (CL conformation) and interdigitated one-start helices (ID conformation) with different nucleosome tilt angles. The CL conformation was derived from a tetranucleosome crystal structure that was extended into a fiber. At thermal equilibrium, the fiber shape persisted but relaxed into a structure with a somewhat lower linear mass density of 3.1 ± 0.1 nucleosomes/11 nm fiber. Stable ID fibers required local nucleosome tilt angles between 40° and 60°. For these configurations, much higher mass densities of up to 7.9 ± 0.2 nucleosomes/11 nm fiber were obtained. A model is proposed, in which the transition between a CL and ID fiber is mediated by relatively small changes of the local nucleosome geometry. These were found to be in very good agreement with changes induced by linker histone H1 binding as predicted from the high resolution model structures.     

Structure of the human telomere in Na+ solution: an antiparallel (2+2) G-quadruplex scaffold reveals additional diversity

Single-stranded DNA overhangs at the ends of human telomeric repeats are capable of adopting four-stranded G-quadruplex structures, which could serve as potential anticancer targets. Out of the five reported intramolecular human telomeric G-quadruplex structures, four were formed in the presence of K⁺ ions and only one in the presence of Na⁺ ions, leading often to a perception that this structural polymorphism occurs exclusively in the presence of K⁺ but not Na⁺. Here we present the structure of a new antiparallel (2+2) G-quadruplex formed by a derivative of a 27-nt human telomeric sequence in Na⁺ solution, which comprises a novel core arrangement distinct from the known topologies. This structure complements the previously elucidated basket-type human telomeric G-quadruplex to serve as reference structures in Na⁺-containing environment. These structures, together with the coexistence of other conformations in Na⁺ solution as observed by nuclear magnetic resonance spectroscopy, establish the polymorphic nature of human telomeric repeats beyond the influence of K⁺ ions.     

Conformational change of a G-quadruplex under molecular crowding conditions

Abstract

This study examined the influence of the molecular crowding condition induced by polyethylene glycol (PEG) on the G-quadruplex structure of the thrombin-binding aptamer sequence, 5′-GGGTTGGGTGTGGGTTGGG (G3), in a solution containing a sufficient concentration of mono cations (K⁺ and Na⁺). Although the G3 sequence preferably formed the antiparallel type G-quadruplex structure in a Na⁺ solution, conversion to the parallel type occurred when PEG was added. The antiparallel type was maintained at low PEG concentrations. When the PEG concentration reached 30%, the antiparallel type and parallel type coexist. At PEG concentrations above 40%, the G-quadruplex structure adopted the parallel type completely. In the presence of K⁺ ions, G3 showed a parallel conformation and remained as a parallel conformation with increasing PEG concentration. The dissociation temperature increased with increasing PEG concentration in all cases, suggesting that the G-quadruplex conformation is more stable under molecular crowding conditions.

Communicated by Ramaswamy H. Sarma     

K+-Dependent Selectivity and External Ca2+ Block of Shab K+ Channels

Potassium channels allow the selective flux of K⁺ excluding the smaller, and more abundant in the extracellular solution, Na⁺ ions. Here we show that Shab is a typical K⁺ channel that excludes Na⁺ under bi-ionic, Na_o/K_i or Na_o/Rb_i, conditions. However, when internal K⁺ is replaced by Cs⁺ (Na_o/Cs_i), stable inward Na⁺ and outward Cs⁺ currents are observed. These currents show that Shab selectivity is not accounted for by protein structural elements alone, as implicit in the snug-fit model of selectivity. Additionally, here we report the block of Shab channels by external Ca²⁺ ions, and compare the effect that internal K⁺ replacement exerts on both Ca²⁺ and TEA block. Our observations indicate that Ca²⁺ blocks the channels at a site located near the external TEA binding site, and that this pore region changes conformation under conditions that allow Na⁺ permeation. In contrast, the latter ion conditions do not significantly affect the binding of quinidine to the pore central cavity. Based on our observations and the structural information derived from the NaK bacterial channel, we hypothesize that Ca²⁺ is probably coordinated by main chain carbonyls of the pore´s first K⁺-binding site.     

NaK-dependent conformation change of proteins of excitable membranes

The K⁺, Na⁺ and Ca²⁺ form of excitable membranes of rat brain were investigated by infrared, ORD and CD spectroscopy. It is shown that with the K⁺ form the conformatio of relatively large parts of the membrane proteins occurs as an antiparallel β structure. No β structure is found with the Na⁺ and Ca²⁺ form. In the presence of these ions the proteins are largely helical. This suggests that during the action potential, membrane proteins change their conformation depending on the cations shifted.     

Changing Chromatin Fiber Conformation by Nucleosome Repositioning

Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ∼10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell.     

Human telomeric sequence forms a hybrid-type intramolecular G-quadruplex structure with mixed parallel/antiparallel strands in potassium solution

Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG). The formation and stabilization of DNA G-quadruplexes in the human telomeric sequence have been shown to inhibit the activity of telomerase, thus the telomeric DNA G-quadruplex has been considered as an attractive target for cancer therapeutic intervention. However, knowledge of the intact human telomeric G-quadruplex structure(s) formed under physiological conditions is a prerequisite for structure-based rational drug design. Here we report the folding structure of the human telomeric sequence in K⁺ solution determined by NMR. Our results demonstrate a novel, unprecedented intramolecular G-quadruplex folding topology with hybrid-type mixed parallel/antiparallel G-strands. This telomeric G-quadruplex structure contains three G-tetrads with mixed G-arrangements, which are connected consecutively with a double-chain-reversal side loop and two lateral loops, each consisting of three nucleotides TTA. This intramolecular hybrid-type telomeric G-quadruplex structure formed in K⁺ solution is distinct from those reported on the 22 nt Tel22 in Na⁺ solution and in crystalline state in the presence of K⁺, and appears to be the predominant conformation for the extended 26 nt telomeric sequence Tel26 in the presence of K⁺, regardless of the presence or absence of Na⁺. Furthermore, the addition of K⁺ readily converts the Na⁺-form conformation to the K⁺-form hybrid-type G-quadruplex. Our results explain all the reported experimental data on the human telomeric G-quadruplexes formed in the presence of K⁺, and provide important insights for understanding the polymorphism and interconversion of various G-quadruplex structures formed within the human telomeric sequence, as well as the effects of sequence and cations. This hybrid-type G-quadruplex topology suggests a straightforward pathway for the secondary structure formation with effective packing within the extended human telomeric DNA. The hybrid-type telomeric G-quadruplex is most likely to be of pharmacological relevance, and the distinct folding topology of this G-quadruplex suggests that it can be specifically targeted by G-quadruplex interactive small molecule drugs.     

Different domain organization of two main conformational states of Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase

The Na⁺/K⁺-ATPase generates an electrochemical gradient of Na⁺ and K⁺, which is necessary for the functioning of animal cells. During the catalytic act, the enzyme passes through two principal conformational states, E₁ and E₂. To assess the domain organization of the protein in these conformations, thermal denaturation of Na⁺/K⁺-ATPases from duck salt gland and from rabbit kidney has been studied in the absence and in the presence of Na⁺ or K⁺, which induce the transition to E₁ or E₂. The melting curves for the ion-free forms of the two ATPases have different shapes: the rabbit protein shows one transition at 56.1°C, whereas the duck protein shows two transitions, at 49.8 and 56.9°C. Addition of Na⁺ or K⁺ ions abolishes the difference in thermal behavior between these enzymes, but through opposite effects. The melting curves for the E₂ conformation (K⁺ bound) in both cases exhibit a single peak of heat absorption at ∼63°C. For the E₁ conformation (Na⁺ bound), each melting curve has three peaks, indicating denaturation of three domains. The difference in the domain organization of Na⁺/K⁺-ATPase in the E1 and E2 states may account for the different sensitivity to temperature, proteolysis, and oxidative stress observed for the two enzyme conformations.     

Selectivity and permeation of alkali metal ions in K+-channels

Ion conduction in K⁺-channels is usually described in terms of concerted movements of K⁺ progressing in a single file through a narrow pore. Permeation is driven by an incoming ion knocking on those ions already inside the protein. A fine-tuned balance between high-affinity binding and electrostatic repulsive forces between permeant ions is needed to achieve efficient conduction. While K⁺-channels are known to be highly selective for K⁺ over Na⁺, some K⁺ channels conduct Na⁺ in the absence of K⁺. Other ions are known to permeate K⁺-channels with a more moderate preference and unusual conduction features. We describe an extensive computational study on ion conduction in K⁺-channels rendering free energy profiles for the translocation of three different alkali ions and some of their mixtures. The free energy maps for Rb⁺ translocation show at atomic level why experimental Rb⁺ conductance is slightly lower than that of K⁺. In contrast to K⁺ or Rb⁺, external Na⁺ block K⁺ currents, and the sites where Na⁺ transport is hindered are characterized. Translocation of K⁺/Na⁺ mixtures is energetically unfavorable owing to the absence of equally spaced ion-binding sites for Na⁺, excluding Na⁺ from a channel already loaded with K⁺.     

Identification of Electric-Field-Dependent Steps in the Na+,K+-Pump Cycle

The charge-transporting activity of the Na⁺,K⁺-ATPase depends on its surrounding electric field. To isolate which steps of the enzyme’s reaction cycle involve charge movement, we have investigated the response of the voltage-sensitive fluorescent probe RH421 to interaction of the protein with BTEA (benzyltriethylammonium), which binds from the extracellular medium to the Na⁺,K⁺-ATPase’s transport sites in competition with Na⁺ and K⁺, but is not occluded within the protein. We find that only the occludable ions Na⁺, K⁺, Rb⁺, and Cs⁺ cause a drop in RH421 fluorescence. We conclude that RH421 detects intramembrane electric field strength changes arising from charge transport associated with conformational changes occluding the transported ions within the protein, not the electric fields of the bound ions themselves. This appears at first to conflict with electrophysiological studies suggesting extracellular Na⁺ or K⁺ binding in a high field access channel is a major electrogenic reaction of the Na⁺,K⁺-ATPase. All results can be explained consistently if ion occlusion involves local deformations in the lipid membrane surrounding the protein occurring simultaneously with conformational changes necessary for ion occlusion. The most likely origin of the RH421 fluorescence response is a change in membrane dipole potential caused by membrane deformation.