Dietrich Suck

Sequence-dependent bending propensity of DNA as revealed by DNase I: parameters for trinucleotides.

Structural parameters characterizing the bending propensity of trinucleotides were deduced from DNase I digestion data using simple probabilistic models. In contrast to dinucleotide‐based models of DNA bending and/or bendability, the trinucleotide parameters are in good agreement with X‐ray crystallographic data on bent DNA. This improvement may be due to the fact that the trinucleotide model incorporates more sequence context information than do dinucleotide‐based descriptions.

Crystal structure of Penicillium citrinum P1 nuclease at 2.8 A resolution.

P1 nuclease from Penicillium citrinum is a zinc dependent glyco‐enzyme consisting of 270 amino acid residues which cleaves single‐stranded RNA and DNA into 5′‐mononucleotides. The X‐ray structure of a tetragonal crystal form of the enzyme with two molecules per asymmetric unit has been solved at 3.3 and refined at 2.8 A resolution to a crystallographic R‐factor of 21.6%. The current model consists of 269 amino acid residues, three Zn ions and two N‐acetyl glucosamines per subunit. The enzyme is folded very similarly to phospholipase C from Bacillus cereus, with 56% of the structure displaying an alpha‐helical conformation. The three Zn ions are located at the bottom of a cleft and appear to be rather inaccessible for any phosphate group in double‐stranded RNA or DNA substrates. A crystal soaking experiment with a dinucleotide gives clear evidence for two mononucleotide binding sites separated by approximately 20 A. One site shows binding of the phosphate group to one of the zinc ions. At both sites there is a hydrophobic binding pocket for the base, but no direct interaction between the protein and the deoxyribose. A cleavage mechanism is proposed involving nucleophilic attack by a Zn activated water molecule.

RNA binding in an Sm core domain: X-ray structure and functional analysis of an archaeal Sm protein complex.

Eukaryotic Sm and Sm‐like proteins associate with RNA to form the core domain of ribonucleoprotein particles involved in pre‐mRNA splicing and other processes. Recently, putative Sm proteins of unknown function have been identified in Archaea. We show by immunoprecipitation experiments that the two Sm proteins present in Archaeoglobus fulgidus (AF‐Sm1 and AF‐Sm2) associate with RNase P RNA in vivo, suggesting a role in tRNA processing. The AF‐Sm1 protein also interacts specifically with oligouridylate in vitro. We have solved the crystal structures of this protein and a complex with RNA. AF‐Sm1 forms a seven‐membered ring, with the RNA interacting inside the central cavity on one face of the doughnut‐shaped complex. The bases are bound via stacking and specific hydrogen bonding contacts in pockets lined by residues highly conserved in archaeal and eukaryotic Sm proteins, while the phosphates remain solvent accessible. A comparison with the structures of human Sm protein dimers reveals closely related monomer folds and intersubunit contacts, indicating that the architecture of the Sm core domain and RNA binding have been conserved during evolution.

X‐ray structure of T4 endonuclease VII: a DNA junction resolvase with a novel fold and unusual domain‐swapped dimer architecture

Phage T4 endonuclease VII (Endo VII), the first enzyme shown to resolve Holliday junctions, recognizes a broad spectrum of DNA substrates ranging from branched DNAs to single base mismatches. We have determined the crystal structures of the Ca2+‐bound wild‐type and the inactive N62D mutant enzymes at 2.4 and 2.1 Å, respectively. The Endo VII monomers form an elongated, highly intertwined molecular dimer exhibiting extreme domain swapping. The major dimerization elements are two pairs of antiparallel helices forming a novel ‘four‐helix cross’ motif. The unique monomer fold, almost completely lacking β‐sheet structure and containing a zinc ion tetrahedrally coordinated to four cysteines, does not resemble any of the known junction‐resolving enzymes, including the Escherichia coli RuvC and λ integrase‐type recombinases. The S‐shaped dimer has two ‘binding bays’ separated by ∼25 Å which are lined by positively charged residues and contain near their base residues known to be essential for activity. These include Asp40 and Asn62, which function as ligands for the bound calcium ions. A pronounced bipolar charge distribution suggests that branched DNA substrates bind to the positively charged face with the scissile phosphates located near the divalent cations. A model for the complex with a four‐way DNA junction is presented.

The crystal structure of an intact human Max–DNA complex: new insights into mechanisms of transcriptional control

BACKGROUND Max belongs to the basic helix-loop-helix leucine zipper (bHLHZ) family of transcription factors. Max is able to form homodimers and heterodimers with other members of this family, which include Mad, Mxi1 and Myc; Myc is an oncoprotein implicated in cell proliferation, differentiation and apoptosis. The homodimers and heterodimers compete for a common DNA target site (the E box) and rearrangement amongst these dimer forms provides a complex system of transcriptional regulation. Max is also regulated by phosphorylation at a site preceding the basic region. We report here the first crystal structure of an intact bHLHZ protein bound to its target site. RESULTS The X-ray crystal structure of the intact human Max protein homodimer in complex with a 13-mer DNA duplex was determined to 2.8 A resolution and refined to an R factor of 0.213. The C-terminal domains in both chains of the Max dimer are disordered. In contrast to the DNA observed in complex with other bHLH and bHLHZ proteins, the DNA in the Max complex is bent by about 25 degrees, directed towards the protein. Intimate contacts with interdigitating sidechains give rise to the formation of tetramers in the crystal. CONCLUSIONS The structure confirms the importance of the HLH and leucine zipper motifs in dimerization as well as the mode of E box recognition which was previously analyzed by X-ray crystallography of shortened constructs. The disorder observed in the C-terminal domain suggests that contacts with additional protein components of the transcription machinery are necessary for ordering the secondary structure. The tetramers seen in the crystal are consistent with the tendency of Max and other bHLHZ and HLH proteins to form higher order oligomers in solution and may play a role in DNA looping. The location of the two phosphorylation sites at Ser1 and Ser10 (the latter is the N-cap of the basic helix) suggests how phosphorylation could disrupt DNA binding.

X-ray structure and activity of the yeast Pop2 protein: a nuclease subunit of the mRNA deadenylase complex.

In Saccharomyces cerevisiae, a large complex, known as the Ccr4–Not complex, containing two nucleases, is responsible for mRNA deadenylation. One of these nucleases is called Pop2 and has been identified by similarity with PARN, a human poly(A) nuclease. Here, we present the crystal structure of the nuclease domain of Pop2 at 2.3 Å resolution. The domain has the fold of the DnaQ family and represents the first structure of an RNase from the DEDD superfamily. Despite the presence of two non‐canonical residues in the active site, the domain displays RNase activity on a broad range of RNA substrates. Site‐directed mutagenesis of active‐site residues demonstrates the intrinsic ability of the Pop2 RNase D domain to digest RNA. This first structure of a nuclease involved in the 3′–5′ deadenylation of mRNA in yeast provides information for the understanding of the mechanism by which the Ccr4–Not complex achieves its functions.

Crystal structure of tRNA-guanine transglycosylase: RNA modification by base exchange.

tRNA‐guanine transglycosylases (TGT) are enzymes involved in the modification of the anticodon of tRNAs specific for Asn, Asp, His and Tyr, leading to the replacement of guanine‐34 at the wobble position by the hypermodified base queuine. In prokaryotes TGT catalyzes the exchange of guanine‐34 with the queuine (.)precursor 7‐aminomethyl‐7‐deazaguanine (preQ1). The crystal structure of TGT from Zymomonas mobilis was solved by multiple isomorphous replacement and refined to a crystallographic R‐factor of 19% at 1.85 angstrom resolution. The structure consists of an irregular (beta/alpha)8‐barrel with a tightly attached C‐terminal zinc‐containing subdomain. The packing of the subdomain against the barrel is mediated by an alpha‐helix, located close to the C‐terminus, which displaces the eighth helix of the barrel. The structure of TGT in complex with preQ1 suggests a binding mode for tRNA where the phosphate backbone interacts with the zinc subdomain and the U33G34U35 sequence is recognized by the barrel. This model for tRNA binding is consistent with a base exchange mechanism involving a covalent tRNA‐enzyme intermediate. This structure is the first example of a (beta/alpha)‐barrel protein interacting specifically with a nucleic acid.

Crystal structure of T4 endonuclease VII resolving a Holliday junction.

Holliday proposed a four-way DNA junction as an intermediate in homologous recombination, and such Holliday junctions have since been identified as a central component in DNA recombination and repair. Phage T4 endonuclease VII (endo VII) was the first enzyme shown to resolve Holliday junctions into duplex DNAs by introducing symmetrical nicks in equivalent strands. Several Holliday junction resolvases have since been characterized, but an atomic structure of a resolvase complex with a Holliday junction remained elusive. Here we report the crystal structure of an inactive T4 endo VII(N62D) complexed with an immobile four-way junction with alternating arm lengths of 10 and 14 base pairs. The junction is a hybrid of the conventional square-planar and stacked-X conformation. Endo VII protrudes into the junction point from the minor groove side, opening it to a 14 Å × 32 Å parallelogram. This interaction interrupts the coaxial stacking, yet every base pair surrounding the junction remains intact. Additional interactions involve the positively charged protein and DNA phosphate backbones. Each scissile phosphate that is two base pairs from the crossover interacts with a Mg2+ ion in the active site. The similar overall shape and surface charge potential of the Holliday junction resolvases endo VII, RuvC, Ydc2, Hjc and RecU, despite having different folds, active site composition and DNA sequence preference, suggest a conserved binding mode for Holliday junctions.

Trinucleotide Models for DNA Bending Propensity: Comparison of Models Based on DNaseI Digestion and Nucleosome Packaging Data

DNaseI digestion studies (Brukner et al, EMBO J 14, 1812-1818 1995) and nucleosomebinding data (Satchwell et al, J. Mol. Biol. 191, 639-659 1986, Goodsell and Dickerson, Nucleic trinucleotides. A detailed comparison of the two models suggests that while both of them represent improvements with respect to dinucleotide based descriptions, the individual trinucleotide parameters are not highly correlated (linear correlation coefficient is 0.53), and a number of motifs such as TA-elements and CCA/TGG motifs are more realistically described in the DNaseI-based model. This may be due to the fact that the DNaseI-based model does not rely on a static geometry but rather captures a dynamic ability of ds DNA to bend towards the major grove. Future refinement of both models of both models on larger experimental data sets is expected to further improve the prediction of macroscopic DNA-curvature.

Three-dimensional structure of bovine pancreatic DNase I at 2.5 A resolution.

The three‐dimensional structure of bovine pancreatic deoxyribonuclease I (DNase I) has been determined at 2.5 A resolution by X‐ray diffraction from single crystals. An atomic model was fitted into the electron density using a graphics display system. DNase I is an alpha, beta‐protein with two 6‐stranded beta‐pleated sheets packed against each other forming the core of a ‘sandwich’‐type structure. The two predominantly anti‐parallel beta‐sheets are flanked by three longer alpha‐helices and extensive loop regions. The carbohydrate side chain attached to Asn 18 is protruding by approximately 15 A from the otherwise compact molecule of approximate dimensions 45 A X 40 A. The binding site of CA2+‐deoxythymidine‐3′,5′‐biphosphate (Ca‐pdTp) has been determined by difference Fourier techniques confirming biochemical results that the active centre is close to His 131. Ca‐pdTp binds at the surface of the enzyme between the two beta‐pleated sheets and seems to interact with several charged amino acid side chains. Active site geometry and folding pattern of DNase I are quite different from staphylococcal nuclease, the only other Ca2+‐dependent deoxyribonuclease whose structure is known at high resolution. The electron density map indicates that two Ca2+ ions are bound to the enzyme under crystallization conditions.