Methylation of histone H3 by coactivator-associated arginine methyltransferase 1.

The preferential in vitro methylation of histone H3 by coactivator-associated arginine methyltransferase 1 (CARM1) has been proposed as a basis for its ability to enhance gene transcription [Chen, D., et al. (1999) Science 284, 2174-2177]. To further evaluate the significance of H3 methylation, we studied the kinetics and site specificity of its modification by CARM1. Affinity-purified CARM1 methylated recombinant chick H3, which is free of posttranslational modifications, and calf thymus H3, which is heterogeneous with regard to preexisting modifications, equally well, exhibiting a V(max) of 4500 pmol min(-1) (mg of enzyme)(-1) and an apparent K(m) for H3 of < or = 0.2 microM. The catalytic efficiency (k(cat)/K(m)) of CARM1 toward H3 was at least 1000 times that toward R1 (GGFGGRGGFGG-amide), a highly effective substrate for protein arginine methyltransferase 1. Peptide mapping of 3H-methyl-labeled H3 indicated methylation at Arg-2, Arg-17, and Arg-26 in the N-terminal region and at one or more of four arginines (128/129/131/134) at the C-terminus. Two of the N-terminal sites, Arg-17 and Arg-26, occur in the sequence KAXRK and appear to be more efficiently methylated than Arg-2. CARM1 catalyzed formation of N(G),N(G)-dimethylarginine (asymmetric) but little or no N(G),N'(G)-dimethylarginine (symmetric) and no form of methyllysine. Amino acid analysis of untreated calf thymus H3 revealed that 3.7% of the molecules naturally contain asymmetric dimethylarginine and/or monomethylarginine. Our findings support the hypothesis that methylation of H3 may be involved in the mechanism of transcriptional coactivation by CARM1 of genes whose expression is under the control of nuclear receptors.

Asymmetries in the nucleosome core particle at 2.5 A resolution.

The 2.5 A X-ray crystal structure of the nucleosome core particle presented here provides significant additions to the understanding of the nucleosome, the fundamental unit of chromatin structure. Extensions are made to the structure of the N-terminal histone tails and details are provided on hydration and ion binding. The structure is composed of twofold symmetric molecules, native chicken histone octamer cores and the DNA palindrome, which were expected to form a perfectly twofold symmetric nucleosome core particle. In fact, the result is asymmetric owing to the binding of the DNA to the protein surface and to the packing of the particles in the crystal lattice. An analysis is made of the asymmetries by comparisons both within the nucleosome core particle and to the structure of the histone octamer core of the nucleosome.

Crystal structure and mechanism of a carbon-carbon bond hydrolase.

BACKGROUND: Fumarylacetoacetate hydrolase (FAH) catalyzes the final step of tyrosine and phenylalanine catabolism, the hydrolytic cleavage of a carbon-carbon bond in fumarylacetoacetate, to yield fumarate and acetoacetate. FAH has no known sequence homologs and functions by an unknown mechanism. Carbon-carbon hydrolysis reactions are essential for the human metabolism of aromatic amino acids. FAH deficiency causes the fatal metabolic disease hereditary tyrosinemia type I. Carbon-carbon bond hydrolysis is also important in the microbial metabolism of aromatic compounds as part of the global carbon cycle. RESULTS: The FAH crystal structure has been determined by rapid, automated analysis of multiwavelength anomalous diffraction data. The FAH polypeptide folds into a 120-residue N-terminal domain and a 300-residue C-terminal domain. The C-terminal domain defines an unusual beta-strand topology and a novel 'mixed beta-sandwich roll' structure. The structure of FAH complexed with its physiological products was also determined. This structure reveals fumarate binding near the entrance to the active site and acetoacetate binding to an octahedrally coordinated calcium ion located in close proximity to a Glu-His dyad. CONCLUSIONS: FAH represents the first structure of a hydrolase that acts specifically on carbon-carbon bonds. FAH also defines a new class of metalloenzymes characterized by a unique alpha/beta fold. A mechanism involving a Glu-His-water catalytic triad is suggested based on structural observations, sequence conservation and mutational analysis. The histidine imidazole group is proposed to function as a general base. The Ca(2+) is proposed to function in binding substrate, activating the nucleophile and stabilizing a carbanion leaving group. An oxyanion hole formed from sidechains is proposed to stabilize a tetrahedral alkoxide transition state. The proton transferred to the carbanion leaving group is proposed to originate from a lysine sidechain. The results also reveal the molecular basis for mutations causing the hereditary tyrosinemia type 1.

Macromolecular crystal annealing: evaluation of techniques and variables.

Additional examples of successful application of macromolecular crystal annealing are presented. A qualitative evaluation of variables related to the annealing process was conducted using a variety of macromolecular crystals to determine in which cases parameters may be varied and in which cases the original macromolecular crystal annealing protocol is preferred. A hypothesis is presented relating the solvent content of the crystal to the specific protocol necessary for the successful application of annealing.

Macromolecular crystal annealing: overcoming increased mosaicity associated with cryocrystallography.

Although cryogenic data collection has become the method of choice for macromolecular crystallography, the flash-cooling step can dramatically increase the mosaicity of some crystals. Macromolecular crystal annealing significantly reduces the mosaicity of flash-cooled crystals without affecting molecular structure. The process, which cycles a flash-cooled crystal to ambient temperature and back to cryogenic temperature, is simple, quick and requires no special equipment. The annealing process has been applied to crystals of several different macromolecules grown from different precipitants and using a variety of cryoprotectants. The protocol for macromolecular crystal annealing also has been applied to restore diffraction from flash-cooled crystals that were mishandled during transfer to or from cryogenic storage. These results will be discussed in relation to crystal mosaicity and effects of radiation damage in flash-cooled crystals.

X-ray diffraction analysis of crystals containing twofold symmetric nucleosome core particles.

Nucleosome core particles containing a DNA palindrome and purified chicken erythrocyte histone octamer have been reconstituted and crystallized. The dyad symmetry of the palindrome extends the dyad symmetry of the histone octamer to result in a twofold symmetric particle. This ensures that the structure determined by X-ray diffraction will yield a true representation of the DNA strand rather than the twofold averaged structure which would result from using a non-palindromic DNA sequence. The crystals provide isotropic diffraction to 3.2 A with observed reflections extending to d spacings of about 2.8 A using a rotating-anode Cu Kalpha X-ray source. Although the DNA palindrome is a factor contributing to the quality of the diffraction data, another significant factor is an improved preparative technique which enriches for correctly phased nucleosome core particles.

Large-scale production of palindrome DNA fragments.

Our structural studies of nucleosomes necessitated the production of over 100 mg of a 146-bp perfect palindrome DNA for use in the reconstitution of perfectly symmetrical nucleosome core particles for detailed X-ray crystallographic analysis. The propagation of palindromic DNA DNA sequences by bacterial culture is hindered by the instability of these sequences during bacterial replication and recombination. While the loss of some palindrome sequences can be eliminated by the use of sbcB or sbcC mutants of Escherichia coli, not all palindrome-containing plasmids are faithfully maintained by these strains. The production of large quantities of palindrome DNA that involves production of plasmid containing multiple copies of the repeating unit of the palindrome which are isolated by restriction digestion and ligated in vitro to form the palindrome DNA. The procedure has resulted in the production of over 20 mg of a 146-bp DNA fragment in 2 weeks.

Preparative separation of nucleosome core particles containing defined-sequence DNA in multiple translational phases.

The nucleosome core particle is composed of an octamer of core histone proteins and about 146 bp of DNA. When reconstituted from purified histone octamer and defined-sequence, nucleosome positioning DNA fragments, the DNA will bind to the histone core in a number of translational phases with respect to the dyad symmetry axis of the histone octamer. Only one of these phases contains symmetrically bound DNA, and it is this species which is required for crystallization and X-ray diffraction studies. We have developed a technique for separating nucleosome core particles, containing defined-sequence 146 bp DNA, which differ only in translational phasing of the DNA with respect to the histone octamer core.

Conformational changes in yeast phosphoglycerate kinase upon substrate binding.

Small-angle neutron scattering (SANS) was used to measure the radius of gyration (Rg) of solutions of phosphoglycerate kinase (PGK) in a variety of substrate environments in D2O. The Rg of 24.0 A was measured for native PGK. A decrease in Rg was observed for the following: 23.7 A for PGK+sulphate; 23.5 A for PGK+ beta, gamma-bidentate Cr(H2O)4ATP (CrATP); 23.3 A for PGK + 3-phospho-D-glycerate (PGA)+CrATP; 22.9 A for PGK+CrATP+sulphate; 22.6 A for PGK+PGA+CrATP+sulphate. The statistical error was about +/- 0.3 A, which is less than systematic effects in this system. These results are consistent with catalysis by a hinge-bending motion of the enzyme. Since CrATP is not hydrolyzed, these results represent the conformational states of the bound substrates in the catalytically relevant ternary complex in the absence of product formation. The second virial coefficient is also measured for this system and this is consistent with that calculated from the protein volume only.

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.

DNA sequence patterns in precisely positioned nucleosomes.

Several investigators have recognized the importance of non-periodic DNA sequence information in determining the translational position of precisely positioned nucleosomes. The purpose of this study is to determine the extent of such information, in addition to the character of periodic information present. This is accomplished by examining the half-nucleosome DNA sequences of a considerable number of precisely positioned nucleosomes, and determining the probability of occurrence of each dinucleotide type as a function of position from the nucleosome center to the terminus (positions 0 to 72). By the nature of this procedure, no assumptions of periodicity are made. The results show the importance of several DNA sequence periodicities including 6-7, 10, and 21 base pairs, in addition to significant nonperiodic information. The results demonstrate that each dinucleotide type is unique in terms of its positional preference in precisely positioned nucleosomes (for example AA not equal to TT). The probabilities of occurrence for the dinucleotide types can be used to predict the translational positions of a number of observed nucleosomes.

Rapid, large-scale purification and characterization of 'Ada protein' (O6 methylguanine-DNA methyltransferase) of E. coli.

The E. coli Ada protein (O6-methylguanine-DNA methyltransferase) has been purified using a high-level expression vector with a yield of about 3 mg per liter of E. coli culture. The 39-kDa protein has an extinction coefficient (E280 nm (1%)) of 5.3. Its isoelectric point of 7.1 is lower than that predicted from the amino acid content. The homogeneous Ada protein is fully active as a methyl acceptor from O6-methylguanine in DNA. Its reaction with O6-methylguanine in a synthetic DNA has a second-order rate constant of 1.1 x 10(9) M-1 min-1 at O degree C. Both the native form and the protein methylated at Cys-69 are monomeric. The CD spectrum suggests a low alpha-helical content and the radius of gyration of 23 A indicates a compact, globular shape. The middle region of the protein is sensitive to a variety of proteases, including an endogenous activity in E. coli, suggesting that the protein is composed of N-terminal and C-terminal domains connected by a hinge region. E. coli B has a higher level of this protease than does K12.

Domain interaction in rabbit muscle pyruvate kinase. II. Small angle neutron scattering and computer simulation.

The effects of ligands on the structure of rabbit muscle pyruvate kinase were studied by small angle neutron scattering. The radius of gyration, RG, decreases by about 1 A in the presence of the substrate phosphoenolpyruvate, but increases by about the same magnitude in the presence of the allosteric inhibitor phenylalanine. With increasing pH or in the absence of Mg2+ and K+, the RG of pyruvate kinase increases. Hence, there is a 2-A difference in RG between two alternative conformations. Length distribution analysis indicates that, under all experimental conditions which increase the radius of gyration, there is a pronounced increase observed in the probability for interatomic distance between 80 and 110 A. These small angle neutron scattering results indicate a "contraction" and "expansion" of the enzyme when it transforms between its active and inactive forms. Using the alpha-carbon coordinates of crystalline cat muscle pyruvate kinase, a length distribution profile was calculated, and it matches the scattering profile of the inactive form. These observations are expected since the crystals were grown in the absence of divalent cations (Stuart, D. I., Levine, M., Muirhead, H., and Stammers, D. K. (1979) J. Mol. Biol. 134, 109-142). Hence, results from neutron scattering, x-ray crystallographic, and sedimentation studies (Oberfelder, R. W., Lee, L. L.-Y., and Lee, J.C. (1984) Biochemistry 23, 3813-3821) are totally consistent with each other. With the aid of computer modeling, the crystal structure has been manipulated in order to effect changes that are consistent with the conformational change described by the solution scattering data. The structural manipulation involves the rotation of the B domain relative to the A domain, leading to the closure of the cleft between these domains. These manipulations resulted in the generation of new sets of atomic (C-alpha) coordinates, which were utilized in calculations, the result of which compared favorably with the solution data.

Small-angle neutron-scattering and electron microscope studies of the chicken liver fatty acid synthase.

A structural model for the chicken liver fatty acid synthase is proposed based on electron microscope and small-angle neutron-scattering studies of the enzyme. The model has the overall appearance of two side by side cylinders with dimensions of 160 X 146 X 73 A, with each subunit 160 A in length and 73 A in diameter. The model was constructed by dividing each cylinder into three domains having lengths of 32, 82, and 46 A, with the domain structures in the two subunits being related to each other by a dyad axis. The model is consistent with chemical cross-linking studies which indicated that the subunits are arranged in a head to tail fashion. The cross-linking studies further showed that the beta-ketoacyl synthase active site contains a cysteine and a pantetheine residue from adjacent subunits. It is proposed that the domains which catalyze the addition of C2 units from malonate to the growing fatty acid chain lie in the crevice between the two subunits and that the two independent sets of fatty acid-synthesizing centers lie on the major axis of the model on opposite ends of the molecular dyad.

Denaturation of a halophilic enzyme monitored by small-angle neutron scattering.

Malate dehydrogenase from Halobacterium maris mortui exists in 4 M-NaCl as a stable protein dimer, with which are associated unusually large amounts of salt and water. In 1 M-NaCl at 25 degrees C, it denatures with a time-constant of about 0.5 h-1. Small-angle neutron scattering data from the protein under these conditions were monitored regularly over more than 12 hours during denaturation. They are quantitatively consistent with a model in which the protein dimer loses its exceptional salt and water-binding properties, and dissociates into monomers that partially unfold and have the interactions with solvent expected from their relatively charged amino acid composition. The exceptional salt and water-binding by the native enzyme, therefore, is associated with the native structure of the dimer.

Shape analysis of the histone octamer in solution.

The conformation of the histone octamer is shown to depend upon the specific salt used to solubilize it. In 2M sodium chloride the octamer is similar in size and shape to the histone component of crystallized core nucleosomes. In contrast, in 3.5M ammonium sulfate the octamer is elongated, resembling an ellipsoid with approximate dimensions of 114 by 62 by 62 angstroms. These results indicate that the elongated conformation seen in the 3.3 angstroms electron density map of the histone octamer crystallized in ammonium sulfate is due to the particular salt conditions used.

Formation of actin dimers as studied by small angle neutron scattering.

Small angle neutron scattering has been used to study the dimensions of G-actin and the formation of low molecular weight actin oligomers under conditions where rapid polymerization does not take place. In the presence of 200 microM Ca2+, actin in solution consists of a single component with a radius of gyration (Rg) of 19.9 +/- 0.4 A, consistent with the known molecular dimensions of the G-actin molecule. In the presence of 50 microM Mg2+, however, formation of an actin species with a larger Rg occurs over a 4-h period. Multicomponent fits were tried and the data were best fit assuming two components, the monomer and a species with an Rg of 29 +/- 1 A. This latter value is consistent with the dimensions expected for certain actin dimers. The apparent dissociation constant for dimer formation is approximately 150 microM with forward and reverse rate constants of 6.0 X 10(-7) microM-1 s-1 and 8.8 X 10(-5) s-1, respectively. Kinetic fluorescence experiments show that the dimer formed in the presence of low levels of Mg2+ is a nonproductive complex which does not participate in the polymerization process. However, the addition of cytochalasin D to actin in the presence of 50 microM Mg2+ rapidly induces the formation of dimers, presumably related to cytochalasin's ability to nucleate actin polymerization.

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.

Neutron scattering studies of nucleosome structure at low ionic strength.

Ionic strength studies using homogeneous preparations of chicken erythrocyte nucleosomes containing either 146 or 175 base pairs of DNA show a single unfolding transition at about 1.5 mM ionic strength as determined by small-angle neutron scattering. The transition seen by some investigators at between 2.9 and 7.5 mM ionic strength is not observed by small-angle neutron scattering in either type of nucleosome particle. The two contrasts measured (H2O and D2O) indicate that only small conformational changes occur in the protein core, but the DNA is partially unfolded below the transition point. Patterson inversion of the data and analysis of models indicate that the DNA in both types of particle is unwinding from the ends, leaving about one turn of supercoiled DNA bound to the histone core in approximately its normal (compact) conformation. The mechanism of unfolding appears to be similar for both types of particles and in both cases occurs at the same ionic strength. The unfolding observed for nucleosomes in this study is in definite disagreement with extended superhelical models for the DNA and also disagrees with models incorporating an unfolded histone core.