Structural studies of the nucleosome core particle

The 2.5 Å X-ray crystal structure of the nucleosome core particle presented here pro- vides 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 provided on hydration and ion binding. The structure is com- posed of 2-fold symmetric molecules, native chicken histone octamer, and the DNA palindrome, which were expected to form a perfectly 2-fold symmetric nucleosome core particle. In fact, the result is asymmetric due 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. Details of the discovery and investigation of macromolecular crystal annealing, M-CA, are also presented. This discovery was a direct consequence of studies directed toward obtaining high-resolution diffraction from nucleosome core particle crystals. The existence of the phenomenon was completely unexpected and thus no documenta- tion of its mechanism or application to macromolecular crystals was available. MCA has already proven to be an important contribution to the field of macromolecular cryocrystallography. Research sponsored by grants from NIH (GM-29818), NASA (NAG8-1568), the Office of Biological and Environmental Research, U.S Department of Energy, and the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory, managed by UT-Battelle, LLC, for the U.S. Department of Energy under Contract No. DE-AC05-00OR22725

Phosphorus SAD Phasing for Nucleic Acid Structures: Limitations and Potential

Phasing of nucleic acid crystal diffraction data using the anomalous signal of phosphorus, P-SAD, at Cukα wavelength has been previously demonstrated using Z-DNA. Since the original work on P-SAD with Z-DNA there has been, with a notable exception, a conspicuous absence of applications of the technique to additional nucleic acid crystal structures. We have reproduced the P-SAD phasing of Z-DNA using a rotating-anode source and have attempted to phase a variety of nucleic acid crystals using P-SAD without success. A comparison of P-SAD using Z-DNA and a representative nucleic acid, the Dickerson-Drew dodecamer, is presented along with a S-SAD using only two sulfurs to phase a 2’-thio modified DNA decamer. A theoretical explanation for the limitation of P-SAD applied to nucleic acids is presented to show that the relatively high atomic displacement parameter of phosphorus in the nucleic acid backbone is responsible for the lack of success in applying P-SAD to nucleic acid diffraction data

Phosphorus SAD Phasing for Nucleic Acid Structures: Limitations and Potential

Phasing of nucleic acid crystal diffraction data using the anomalous signal of phosphorus, P-SAD, at Cukα wavelength has been previously demonstrated using Z-DNA. Since the original work on P-SAD with Z-DNA there has been, with a notable exception, a conspicuous absence of applications of the technique to additional nucleic acid crystal structures. We have reproduced the P-SAD phasing of Z-DNA using a rotating-anode source and have attempted to phase a variety of nucleic acid crystals using P-SAD without success. A comparison of P-SAD using Z-DNA and a representative nucleic acid, the Dickerson-Drew dodecamer, is presented along with a S-SAD using only two sulfurs to phase a 2’-thio modified DNA decamer. A theoretical explanation for the limitation of P-SAD applied to nucleic acids is presented to show that the relatively high atomic displacement parameter of phosphorus in the nucleic acid backbone is responsible for the lack of success in applying P-SAD to nucleic acid diffraction data

Structure of the heterodimeric ecdysone receptor DNA-binding complex

Ecdysteroids initiate molting and metamorphosis in insects via a heterodimeric receptor consisting of the ecdysone receptor (EcR) and ultraspiracle (USP). The EcR–USP heterodimer preferentially mediates transcription through highly degenerate pseudo-palindromic response elements, resembling inverted repeats of 5′-AGGTCA-3′ separated by 1 bp (IR-1). The requirement for a heterodimeric arrangement of EcR–USP subunits to bind to a symmetric DNA is unusual within the nuclear receptor superfamily. We describe the 2.24 Å structure of the EcR–USP DNA-binding domain (DBD) heterodimer bound to an idealized IR-1 element. EcR and USP use similar surfaces, and rely on the deformed minor groove of the DNA to establish protein–protein contacts. As retinoid X receptor (RXR) is the mammalian homolog of USP, we also solved the 2.60 Å crystal structure of the EcR–RXR DBD heterodimer on IR-1 and found the dimerization and DNA-binding interfaces to be the same as in the EcR–USP complex. Sequence alignments indicate that the EcR–RXR heterodimer is an important model for understanding how the FXR–RXR heterodimer binds to IR-1 sites

The active site of the SET domain is constructed on a knot

The SET domain contains the catalytic center of lysine methyltransferases that target the N-terminal tails of histones and regulate chromatin function. Here we report the structure of the SET7/9 protein in the absence and presence of its cofactor product, S-adenosyl-L-homocysteine (AdoHcy). A knot within the SET domain helps form the methyltransferase active site, where AdoHcy binds and lysine methylation is likely to occur. A structure-guided comparison of sequences within the SET protein family suggests that the knot substructure and active site environment are conserved features of the SET domain.status: publishe

Crystal structure and mechanism of a carbon–carbon bond hydrolase

AbstractBackground: 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 β-strand topology and a novel ‘mixed β-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 α/β 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 Ca2+ 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

Structure, stability and function of 5-chlorouracil modified A:U and G:U base pairs

The thymine analog 5-chlorouridine, first reported in the 1950s as anti-tumor agent, is known as an effective mutagen, clastogen and toxicant as well as an effective inducer of sister-chromatid exchange. Recently, the first microorganism with a chemically different genome was reported; the selected Escherichia coli strain relies on the four building blocks 5-chloro-2'-deoxyuridine (ClU), A, C and G instead of the standard T, A, C, G alphabet [Marlière,P., Patrouix,J., Döring,V., Herdewijn,P., Tricot,S., Cruveiller,S., Bouzon,M. and Mutzel,R. (2011) Chemical evolution of a bacterium's genome. Angew. Chem. Int. Ed., 50, 7109-7114]. The residual fraction of T in the DNA of adapted bacteria was 1500 A to G or G to A transitions in a culture. The former is most likely due to wobble base pairing between ClU and G, which may be more common for ClU than T. To identify potential changes in the geometries of base pairs and duplexes as a result of replacement of T by ClU, we determined four crystal structures of a B-form DNA dodecamer duplex containing ClU:A or ClU:G base pairs. The structures reveal nearly identical geometries of these pairs compared with T:A or T:G, respectively, and no consequences for stability and cleavage by an endonuclease (EcoRI). The lack of significant changes in the geometry of ClU:A and ClU:G base pairs relative to the corresponding native pairs is consistent with the sustained unlimited self-reproduction of E. coli strains with virtually complete T→ClU genome substitution.status: publishe