Redefinition of the cleavage sites of DNase I on the nucleosome core particle

DNase I has been widely used for the footprinting of DNA-protein interactions including analyses of nucleosome core particle (NCP) structure. Our understanding of the relationship between the footprint and the structure of the nucleosome complex comes mainly from digestion studies of NCPs, since they have a well-defined quasi-symmetrical structure and have been widely investigated. However, several recent results suggest that the established consensus of opinion regarding the mode of digestion of NCPs by DNase I may be based on erroneous interpretation of results concerning the relationship between the NCP ends and the dyad axis. Here, we have used reconstituted NCPs with defined ends, bulk NCPs prepared with micrococcal nuclease and molecular modelling to reassess the mode of DNase I digestion. Our results indicate that DNase I cuts the two strands of the nucleosomal DNA independently with an average stagger of 4 nt with the 3'-ends protruding. The previously accepted value of 2 nt stagger is explained by the finding that micrococcal nuclease produces NCPs not with flush ends, but with approximately 1 nt 5'-recessed ends. Furthermore we explain why the DNA stagger is an even and not an odd number of nucleotides. These results are important for studies using DNase I to probe nucleosome structure in complex with other proteins or any DNA-protein complex containing B-form DNA. We also determine the origin of the 10n +/- 5 nt periodicity found in the internucleosomal ladder of DNase I digests of chromatin from various species. The explanation of the 10n +/- 5 nt ladder may have implications for the structure of the 30 nm fibre.     

Structure of the Drosophila nucleosome core particle highlights evolutionary constraints on the H2A-H2B histone dimer

We determined the 2.45 A crystal structure of the nucleosome core particle from Drosophila melanogaster and compared it to that of Xenopus laevis bound to the identical 147 base-pair DNA fragment derived from human alpha-satellite DNA. Differences between the two structures primarily reflect 16 amino acid substitutions between species, 15 of which are in histones H2A and H2B. Four of these involve histone tail residues, resulting in subtly altered protein-DNA interactions that exemplify the structural plasticity of these tails. Of the 12 substitutions occurring within the histone core regions, five involve small, solvent-exposed residues not involved in intraparticle interactions. The remaining seven involve buried hydrophobic residues, and appear to have coevolved so as to preserve the volume of side chains within the H2A hydrophobic core and H2A-H2B dimer interface. Thus, apart from variations in the histone tails, amino acid substitutions that differentiate Drosophila from Xenopus histones occur in mutually compensatory combinations. This highlights the tight evolutionary constraints exerted on histones since the vertebrate and invertebrate lineages diverged.     

Small-angle X-ray scattering studies of the structure of nucleosome core histone complexes in solution

The nucleosome core histone complex in solution at 2 M NaCl and pH 7 has a radius of gyration R_s, of 3.48 nm and a maximum dimension, L, of 12 nm. Its shape is disc-like with a mean thickness of 3 nm. The radius of gyration determined by us is of the same value as the radius of gyration of the complex in intact core particles (Braddock) et al., Biopolymers 1981, 20, 327). Thus, we conclude that the basic histone tails of the protein complex project about 2 nm from its central part.     

Structure of the nucleosome core particle at 8 A resolution

The x-ray crystallographic structure of the nucleosome core particle has been determined using 8 A resolution diffraction data. The particle has a mean diameter of 106 A and a maximum thickness of 65 A in the superhelical axis direction. The longest chord through the histone core measures 85 A and is in a non-axial direction. The 1.87 turn superhelix consists of B-DNA with about 78 base pairs or 7.6 helical repeats per superhelical turn. The mean DNA helical repeat contains 10.2 +/- 0.05 base pairs and spans 35 A, slightly more than standard B-DNA. The superhelix varies several Angstroms in radius and pitch, and has three distinct domains of curvature (with radii of curvature of 60, 45 and 51 A). These regions are separated by localized sharper bends +/- 10 and +/- 40 base pairs from the center of the particle, resulting in an overall radius of curvature about 43 A. Compression of superhelical DNA grooves on the inner surface and expansion on the outer surface can be seen throughout the DNA electron density. This density has been fit with a double helical ribbon model providing groove width estimates of 12 +/- 1 A inside vs. 19 +/- 1 A outside for the major groove, and 8 +/- 1 A inside vs. 13 +/- 1 A outside for the minor groove. The histone core is primarily contained within the bounds defined by the superhelical DNA, contacting the DNA where the phosphate backbone faces in toward the core. Possible extensions of density between the gyres have been located, but these are below the significance level of the electron density map. In cross-section, a tripartite organization of the histone octamer is apparent, with the tetramer occupying the central region and the dimers at the extremes. Several extensions of histone density are present which form contacts between nucleosomes in the crystal, perhaps representing flexible or "tail" histone regions. The radius of gyration of the histone portion of the electron density is calculated to be 30.4 A (in reasonable agreement with solution scattering values), and the histone core volume in the map is 93% of its theoretical volume.     

Probing nucleosome core secondary structure before and after α-chymotrypsin treatment by raman spectroscopy and thermal denaturation

Nucleosome cores were digested with α-chymotrypsin until histone H3 was degraded to a partial histone, CP1. As we reported previously, cleavage occurred at leucine 20 to H3 and resulted in an increase in circular dichroism between 265 to 285 nm. Some modest core unfolding was also observed as determined by a small decrease in the sedimentation coefficient. Studies reported here deal with the analysis of core secondary structure and subsequent perturbation caused by treatment with α-chymotrypsin. Raman spectroscopy indicated that chymotryptic treatment promoted a change in the conformational environment of a population of core histone tyrosines. In addition, a shift from B-form to an intermediate B- or A-form was observed for core DNA. High-resolution thermal denaturation was used to determine alterations in the stabilization of core DNA related to perturbation of the core histones. Brief chymotryptic treatment indicated changes in both pre-melt and irreversible transitions.     

Nonsequence-specific arginine interactions in the nucleosome core particle

We have analyzed the relative orientation of basic amino acid side chains towards DNA in the nucleosome core particle. The electric field created by DNA phosphates has no apparent preferential orientation: no favored orientation of the arginine guanidinium group is found. Arginine may be either directly hydrogen bonded to a phosphate oxygen or stabilized in the minor groove by van der Waals contacts and the local negative electric field. On the other hand, the phosphate oxygen atoms hydrogen bonded to arginines are always found close to the plane defined by the guanidinium group. Thus it can be concluded that the interactions of arginine are strongly directional, those of phosphate are not. We also find that a highly charged fragment of histone H2B, which is placed between two DNA turns, has a very variable conformation. An increase in protein positive charge density apparently allows multiple nonspecific protein conformations when interacting with DNA.     

Crystal structure of human coactosin-like protein at 1.9 A resolution

Human coactosin-like protein (CLP) shares high homology with coactosin, a filamentous (F)-actin binding protein, and interacts with 5LO and F-actin. As a tumor antigen, CLP is overexpressed in tumor tissue cells or cell lines, and the encoded epitopes can be recognized by cellular and humoral immune systems. To gain a better understanding of its various functions and interactions with related proteins, the crystal structure of CLP expressed in Escherichia coli has been determined to 1.9 A resolution. The structure features a central beta-sheet surrounded by helices, with two very tight hydrophobic cores on each side of the sheet. CLP belongs to the actin depolymerizing protein superfamily, and is similar to yeast cofilin and actophilin. Based on our structural analysis, we observed that CLP forms a polymer along the crystallographic b axis with the exact same repeat distance as F-actin. A model for the CLP polymer and F-actin binding has therefore been proposed.     

X-ray structure of the nucleosome core particle

Two monoclinic crystal forms (P2(1),C2) of chicken erythrocyte nucleosomes have been under study in this laboratory. The x-ray structure of the P2(1) crystal form has been solved to 15 A resolution. The B-DNA superhelix has a relatively uniform curvature, with only several local distortions observed in the superhelix. The individual histone domains have been localized and specific contacts between each histone and the DNA can be observed. Histone contacts to the inner surface of the DNA superhelix occur predominantly at the minor groove sites. Most of the histone core is contained within the inner surface of the superhelical DNA, except for part of H2A which extends between the DNA gyres near the terminus of the DNA. No part of H2A blocks the DNA terminus or would prevent a smooth exit of the DNA into the linker region. A similar extension of a portion of histone H4 between the DNA gyres occurs close to the dyad axis. Both unique nucleosomes in the P2(1) asymmetric unit demonstrate good dyad symmetry and are similar to each other throughout the histone core and DNA regions.     

Crystal structure of a proteolytically generated functional monoferric C-lobe of bovine lactoferrin at 1.9A resolution

This is the first crystal structure of a proteolytically generated functional C-lobe of lactoferrin. The purified samples of iron-saturated C-lobe were crystallized in 0.1 M Mes buffer (pH 6.5) containing 25% (v/v) polyethyleneglycol monomethyl ether 550 M and 0.1 M zinc sulphate heptahydrate. The X-ray intensity data were collected with 300 mm imaging plate scanner mounted on a rotating anode generator. The structure was determined by the molecular replacement method using the coordinates of the C-terminal half of bovine lactoferrin as a search model and refined to an R-factor of 0.193 for all data to 1.9A resolution. The final model comprises 2593 protein atoms (residues 342-676 and 681-685), 124 carbohydrate atoms (from ten monosaccharide units, in three glycan chains), one Fe(3+), one CO(3)(2-), two Zn(2+) and 230 water molecules. The overall folding of the C-lobe is essentially the same as that of C-terminal half of bovine lactoferrin but differs slightly in conformations of some of the loops and reveals a number of new interactions. There are 20 Cys residues in the C-lobe forming ten disulphide links. Out of these, one involving Cys481-Cys675 provides an inter-domain link at 2.01A while another Cys405-Cys684 is formed between the main C-lobe 342-676 and the hydrolyzed pentapeptide 681-685 fragment. Six inter-domain hydrogen bonds have been observed in the structure whereas only four were reported in the structure of intact lactoferrin, although domain orientations have been found similar in the two structures. The good quality of electron density has also revealed all the ten oligosaccharide units in the structure. The observation of two metal ions at sites other than the iron-binding cleft is another novel feature of the present structure. These zinc ions stabilize the crystal packing. This structure is also notable for extensive inter-molecular hydrogen bonding in the crystals. Therefore, the present structure appears to be one of the best packed crystal structures among the proteins of the transferrin superfamily.     

The structure of duplex DNA in the nucleosome core particle: An alternative model

Many studies indirectly indicate that the conformation ofin vivo duplex DNA is the double helix. The most direct view, from the X-ray analysis of the nucleosome core particle, has also been interpreted in terms of the double helix structure. However, an alternative possibility exists; that the duplex adopts a metastable side-by-side conformation which readily converts to the double helix on removal of protein. Evidence for the existence of this conformation has been obtained from a reanalysis of the electron density map for the nucleosome particle.     

Nucleosomal locations of dominant DNA sequence motifs for histone-DNA interactions and nucleosome positioning

DNA sequence is an important determinant of the positioning, stability, and activity of nucleosomes, yet the molecular basis of these effects remains elusive. A "consensus DNA sequence" for nucleosome positioning has not been reported and, while certain DNA sequence preferences or motifs for nucleosome positioning have been discovered, how they function is not known. Here, we report that an unexpected observation concerning the reassembly of nucleosomes during salt gradient dialysis has allowed a breakthrough in our efforts to identify the nucleosomal locations of the DNA sequence motifs that dominate histone-DNA interactions and nucleosome positioning. We conclude that a previous selection experiment for high-affinity, nucleosome-forming DNA sequences exerted selective pressure chiefly on the central stretch of the nucleosomal DNA. This observation implies that algorithms for aligning the selected DNA sequences should seek to optimize the alignment over much less than the full 147 bp of nucleosomal DNA. A new alignment calculation implemented these ideas and successfully aligned 19 of the 41 sequences in a non-redundant database of selected high-affinity, nucleosome-positioning sequences. The resulting alignment reveals strong conservation of several stretches within a central 71 bp of the nucleosomal DNA. The alignment further reveals an inherent palindromic symmetry in the selected DNAs; it makes testable predictions of nucleosome positioning on the aligned sequences and for the creation of new positioning sequences, both of which are upheld experimentally; and it suggests new signals that may be important in translational nucleosome positioning.     

Crystal structures of nucleosome core particles in complex with minor groove DNA-binding ligands

We determined the crystal structures of three nucleosome core particles in complex with site-specific DNA-binding ligands, the pyrrole-imidazole polyamides. While the structure of the histone octamer and its interaction with the DNA remain unaffected by ligand binding, nucleosomal DNA undergoes significant structural changes at the ligand-binding sites and in adjacent regions to accommodate the ligands. Our findings suggest that twist diffusion occurs over long distances through tightly bound nucleosomal DNA. This may be relevant to the mechanism of ATP-dependent and spontaneous nucleosome translocation, and to the effect of bound factors on nucleosome dynamics.     

Elucidation of binding preferences of YEATS domains to site-specific acetylated nucleosome core particles

Acetylated lysine residues (Kac) in histones are recognized by epigenetic reader proteins, such as Yaf9, ENL, AF9, Taf14, and Sas5 (YEATS) domain-containing proteins. Human YEATS domains bind to the acetylated N-terminal tail of histone H3; however, their Kac-binding preferences at the level of the nucleosome are unknown. Through genetic code reprogramming, here, we established a nucleosome core particle (NCP) array containing histones that were acetylated at specific residues and used it to compare the Kac-binding preferences of human YEATS domains. We found that AF9-YEATS showed basal binding to the unmodified NCP and that it bound stronger to the NCP containing a single acetylation at one of K4, K9, K14, or K27 of H3, or to histone H4 multi-acetylated between K5 and K16. Crystal structures of AF9-YEATS in complex with an H4 peptide diacetylated either at K5/K8 or K8/K12 revealed that the aromatic cage of the YEATS domain recognized the acetylated K8 residue. Interestingly, E57 and D103 of AF9, both located outside of the aromatic cage, were shown to interact with acetylated K5 and K12 of H4, respectively, consistent with the increase in AF9-YEATS binding to the H4K8-acetylated NCP upon additional acetylation at K5 or K12. Finally, we show that a mutation of E57 to alanine in AF9-YEATS reduced the binding affinity for H4 multiacetylated NCPs containing H4K5ac. Our data suggest that the Kac-binding affinity of AF9-YEATS increases additively with the number of Kac in the histone tail.     

Structural flexibility of the nucleosome core particle at atomic resolution studied by molecular dynamics simulation

Comparative explicit solvent molecular dynamics (MD) simulations have been performed on a complete nucleosome core particle with and without N-terminal histone tails for more than 20 ns. Main purpose of the simulations was to study the dynamics of mobile elements such as histone N-terminal tails and how packing and DNA-bending influences the fine structure and dynamics of DNA. Except for the tails, histone and DNA molecules stayed on average close to the crystallographic start structure supporting the quality of the current force field approach. Despite the packing strain, no increase of transitions to noncanonical nucleic acid backbone conformations compared to regular B-DNA was observed. The pattern of kinks and bends along the DNA remained close to the experiment overall. In addition to the local dynamics, the simulations allowed the analysis of the superhelical mobility indicating a limited relative mobility of DNA segments separated by one superhelical turn (mean relative displacement of approximately +/-0.2 nm, mainly along the superhelical axis). An even higher rigidity was found for relative motions (distance fluctuations) of segments separated by half a superhelical turn (approximately +/-0.1 nm). The N-terminal tails underwent dramatic conformational rearrangements on the nanosecond time scale toward partially and transiently wrapped states around the DNA. Many of the histone tail changes corresponded to coupled association and folding events from fully solvent-exposed states toward complexes with the major and minor grooves of DNA. The simulations indicate that the rapid conformational changes of the tails can modulate the DNA accessibility within a few nanoseconds.     

Possible nucleosome arrangements in the higher-order structure of chromatin

Consideration has been given to possible sequences of nucleosomes which can produce a ‘thick fibre’-like structure. Only a few basic requirements were imposed: (i) the thick fibre is a regular single helix with about 7 nucleosomes per turn; (ii) the nucleosomes are equidistant along the polynuclesome chain; (iii) the helix is flexible having variable pitch. It was found that in addition to the straightforward sequential arrangement there is only one other nonsequential arrangement which satisfies these requirements. This is a helix with around 8 nucleosomes per turn in which all nucleosomes are identically placed. It is possible in the region of 200 to 218 ± 10 base pairs (b.p.) DNA repeats lengths. The linker DNA is straight or almost straight and crosses the internal ‘hollow’ cylinder which is not occupied by nucleosomes. This structure satisfies the experimental data for the distance distribution function, and the observed mass per unit length and changes noted in the mass per unit length. Further, if it is assumed that the core particle axis of symmetry is in the plane of the two linkers and bisects them then this makes the core particles oblique to the thick fibre radii with alternate angles of ± 20 to 30°. This orientation of the nucleosomes can explain the DNA digestion patterns obtained with DNase II and with DNase I.     

The right place at the right time: chaperoning core histone variants

Histone proteins dynamically regulate chromatin structure and epigenetic signaling to maintain cell homeostasis. These processes require controlled spatial and temporal deposition and eviction of histones by their dedicated chaperones. With the evolution of histone variants, a network of functionally specific histone chaperones has emerged. Molecular details of the determinants of chaperone specificity for different histone variants are only slowly being resolved. A complete understanding of these processes is essential to shed light on the genuine biological roles of histone variants, their chaperones, and their impact on chromatin dynamics.     

The X-ray structure of a snake venom Gln48 phospholipase A2 at 1.9A resolution reveals anion-binding sites

Phospholipase A2 is an "interfacial" enzyme and its binding to negatively charged surfaces is an important step during catalysis. The Gln48 phospholipase A2 from the venom of Vipera ammodytes meridionalis plays the role of chaperone and directs a toxic His48 PLA2 onto its acceptor. In the venom the two phospholipases A2 exist as a postsynaptic neurotoxic complex, Vipoxin. The X-ray structure of Gln48 PLA2, complexed to sulphate ions, which mimic the negatively charged groups of anionic membranes, has been determined by the molecular replacement method and refined to 1.9A resolution. The protein forms a homodimer stabilized by ionic, hydrophobic, and hydrogen-bond interactions. The structure reveals two anion-binding sites per subunit. These sites are probably involved in interactions with the negatively charged membrane surface and, in this way, in the "targeting" of the toxic component to the receptors of the postsynaptic membranes. In the absence of the chaperone subunit the toxin changes the target of the physiological attack. A comparison of the homodimeric Gln48 PLA2 structure with that of the heterodimeric Vipoxin reveals differences in regions involved in the pharmacological activity of the toxin. This fact, except the active site histidine substitution, can explain the absence of toxicity in the Gln48 protein in comparison to the His48 phospholipase A2.     

Multifunctional xylooligosaccharide/cephalosporin C deacetylase revealed by the hexameric structure of the Bacillus subtilis enzyme at 1.9A resolution

Esterases and deacetylases active on carbohydrate ligands have been classified into 14 families based upon amino acid sequence similarities. Enzymes from carbohydrate esterase family seven (CE-7) are unusual in that they display activity towards both acetylated xylooligosaccharides and the antibiotic, cephalosporin C. The 1.9A structure of the multifunctional CE-7 esterase (hereinafter CAH) from Bacillus subtilis 168 reveals a classical alpha/beta hydrolase fold encased within a 32 hexamer. This is the first example of a hexameric alpha/beta hydrolase and is further evidence of the versatility of this particular fold, which is used in a wide variety of biological contexts. A narrow entrance tunnel leads to the centre of the molecule, where the six active-centre catalytic triads point towards the tunnel interior and thus are sequestered away from cytoplasmic contents. By analogy to self-compartmentalising proteases, the tunnel entrance may function to hinder access of large substrates to the poly-specific active centre. This would explain the observation that the enzyme is active on a variety of small, acetylated molecules. The structure of an active site mutant in complex with the reaction product, acetate, reveals details of the putative oxyanion binding site, and suggests that substrates bind predominantly through non-specific contacts with protein hydrophobic residues. Protein residues involved in catalysis are tethered by interactions with protein excursions from the canonical alpha/beta hydrolase fold. These excursions also mediate quaternary structure maintenance, so it would appear that catalytic competence is only achieved on protein multimerisation. We suggest that the acetyl xylan esterase (EC 3.1.1.72) and cephalosporin C deacetylase (EC 3.1.1.41) enzymes of the CE-7 family represent a single class of proteins with a multifunctional deacetylase activity against a range of small substrates.