Edwin Pozharski

Crystal structure of human thymine DNA glycosylase bound to DNA elucidates sequence-specific mismatch recognition

Cytosine methylation at CpG dinucleotides produces m5CpG, an epigenetic modification that is important for transcriptional regulation and genomic stability in vertebrate cells. However, m5C deamination yields mutagenic G·T mispairs, which are implicated in genetic disease, cancer, and aging. Human thymine DNA glycosylase (hTDG) removes T from G·T mispairs, producing an abasic (or AP) site, and follow-on base excision repair proteins restore the G·C pair. hTDG is inactive against normal A·T pairs, and is most effective for G·T mispairs and other damage located in a CpG context. The molecular basis of these important catalytic properties has remained unknown. Here, we report a crystal structure of hTDG (catalytic domain, hTDGcat) in complex with abasic DNA, at 2.8 Å resolution. Surprisingly, the enzyme crystallized in a 2:1 complex with DNA, one subunit bound at the abasic site, as anticipated, and the other at an undamaged (nonspecific) site. Isothermal titration calorimetry and electrophoretic mobility-shift experiments indicate that hTDG and hTDGcat can bind abasic DNA with 1:1 or 2:1 stoichiometry. Kinetics experiments show that the 1:1 complex is sufficient for full catalytic (base excision) activity, suggesting that the 2:1 complex, if adopted in vivo, might be important for some other activity of hTDG, perhaps binding interactions with other proteins. Our structure reveals interactions that promote the stringent specificity for guanine versus adenine as the pairing partner of the target base and interactions that likely confer CpG sequence specificity. We find striking differences between hTDG and its prokaryotic ortholog (MUG), despite the relatively high (32%) sequence identity.

Cysteine pKa depression by a protonated glutamic acid in human DJ-1.

Human DJ-1, a disease-associated protein that protects cells from oxidative stress, contains an oxidation-sensitive cysteine (C106) that is essential for its cytoprotective activity. The origin of C106 reactivity is obscure, due in part to the absence of an experimentally determined p K a value for this residue. We have used atomic-resolution X-ray crystallography and UV spectroscopy to show that C106 has a depressed p K a of 5.4 +/- 0.1 and that the C106 thiolate accepts a hydrogen bond from a protonated glutamic acid side chain (E18). X-ray crystal structures and cysteine p K a analysis of several site-directed substitutions at residue 18 demonstrate that the protonated carboxylic acid side chain of E18 is required for the maximal stabilization of the C106 thiolate. A nearby arginine residue (R48) participates in a guanidinium stacking interaction with R28 from the other monomer in the DJ-1 dimer and elevates the p K a of C106 by binding an anion that electrostatically suppresses thiol ionization. Our results show that the ionizable residues (E18, R48, and R28) surrounding C106 affect its p K a in a way that is contrary to expectations based on the typical ionization behavior of glutamic acid and arginine. Lastly, a search of the Protein Data Bank (PDB) produces several candidate hydrogen-bonded aspartic/glutamic acid-cysteine interactions, which we propose are particularly common in the DJ-1 superfamily.

Techniques, tools and best practices for ligand electron-density analysis and results from their application to deposited crystal structures.

As a result of substantial instrumental automation and the continuing improvement of software, crystallographic studies of biomolecules are conducted by non-experts in increasing numbers. While improved validation almost ensures that major mistakes in the protein part of structure models are exceedingly rare, in ligand-protein complex structures, which in general are most interesting to the scientist, ambiguous ligand electron density is often difficult to interpret and the modelled ligands are generally more difficult to properly validate. Here, (i) the primary technical reasons and potential human factors leading to problems in ligand structure models are presented; (ii) the most common categories of building errors or overinterpretation are classified; (iii) a few instructive and specific examples are discussed in detail, including an electron-density-based analysis of ligand structures that do not contain any ligands; (iv) means of avoiding such mistakes are suggested and the implications for database validity are discussed and (v) a user-friendly software tool that allows non-expert users to conveniently inspect ligand density is provided.

Consolidation of glycosyl hydrolase family 30: a dual domain 4/7 hydrolase family consisting of two structurally distinct groups.

In this work glycosyl hydrolase (GH) family 30 (GH30) is analyzed and shown to consist of its currently classified member sequences as well as several homologous sequence groups currently assigned within family GH5. A large scale amino acid sequence alignment and a phylogenetic tree were generated and GH30 groups and subgroups were designated. A partial rearrangement in the GH30 defining side‐associated β‐domain contributes to the differentiation of two major groups that contain up to eight subgroups. For this CAZy family of Clan A enzymes the dual domain fold is conserved, suggesting that it may be a requirement for evolved function. This work redefines GH family 30 and serves as a guide for future efforts regarding enzymes classified within this family.

Divalent metal ion complexes of S100B in the absence and presence of pentamidine.

As part of an effort to inhibit S100B, structures of pentamidine (Pnt) bound to Ca(2+)-loaded and Zn(2+),Ca(2+)-loaded S100B were determined by X-ray crystallography at 2.15 A (R(free)=0.266) and 1.85 A (R(free)=0.243) resolution, respectively. These data were compared to X-ray structures solved in the absence of Pnt, including Ca(2+)-loaded S100B and Zn(2+),Ca(2+)-loaded S100B determined here (1.88 A; R(free)=0.267). In the presence and absence of Zn(2+), electron density corresponding to two Pnt molecules per S100B subunit was mapped for both drug-bound structures. One Pnt binding site (site 1) was adjacent to a p53 peptide binding site on S100B (+/-Zn(2+)), and the second Pnt molecule was mapped to the dimer interface (site 2; +/-Zn(2+)) and in a pocket near residues that define the Zn(2+) binding site on S100B. In addition, a conformational change in S100B was observed upon the addition of Zn(2+) to Ca(2+)-S100B, which changed the conformation and orientation of Pnt bound to sites 1 and 2 of Pnt-Zn(2+),Ca(2+)-S100B when compared to Pnt-Ca(2+)-S100B. That Pnt can adapt to this Zn(2+)-dependent conformational change was unexpected and provides a new mode for S100B inhibition by this drug. These data will be useful for developing novel inhibitors of both Ca(2+)- and Ca(2+),Zn(2+)-bound S100B.

Ligand bound structures of a glycosyl hydrolase family 30 glucuronoxylan xylanohydrolase.

Xylanases of glycosyl hydrolase family 30 (GH30) have been shown to cleave β-1,4 linkages of 4-O-methylglucuronoxylan (MeGX(n)) as directed by the position along the xylan chain of an α-1,2-linked 4-O-methylglucuronate (MeGA) moiety. Complete hydrolysis of MeGX(n) by these enzymes results in singly substituted aldouronates having a 4-O-methylglucuronate moiety linked to a xylose penultimate from the reducing terminal xylose and some number of xylose residues toward the nonreducing terminus. This novel mode of action distinguishes GH30 xylanases from the more common xylanase families that cleave MeGX(n) in accessible regions. To help understand this unique biochemical function, we have determined the structure of XynC in its native and ligand-bound forms. XynC structure models derived from diffraction data of XynC crystal soaks with the simple sugar glucuronate (GA) and the tetrameric sugar 4-O-methyl-aldotetrauronate resulted in models containing GA and 4-O-methyl-aldotriuronate, respectively. Each is observed in two locations within XynC surface openings. Ligand coordination occurs within the XynC catalytic substrate binding cleft and on the structurally fused side β-domain, demonstrating a substrate targeting role for this putative carbohydrate binding module. Structural data reveal that GA acts as a primary functional appendage for recognition and hydrolysis of the MeGX(n) polymer by the protein. This work compares the structure of XynC with a previously reported homologous enzyme, XynA, from Erwinia chrysanthemi and analyzes the ligand binding sites. Our results identify the molecular interactions that define the unique function of XynC and homologous GH30 enzymes.

Visualizing ligand molecules in Twilight electron density.

Three-dimensional models of protein structures determined by X-ray crystallography are based on the interpretation of experimentally derived electron-density maps. The real-space correlation coefficient (RSCC) provides an easily comprehensible, objective measure of the residue-based fit of atom coordinates to electron density. Among protein structure models, protein-ligand complexes are of special interest, given their contribution to understanding the molecular underpinnings of biological activity and to drug design. For consumers of such models, it is not trivial to determine the degree to which ligand-structure modelling is biased by subjective electron-density interpretation. A standalone script, Twilight, is presented for the analysis, visualization and annotation of a pre-filtered set of 2815 protein-ligand complexes deposited with the PDB as of 15 January 2012 with ligand RSCC values that are below a threshold of 0.6. It also provides simplified access to the visualization of any protein-ligand complex available from the PDB and annotated by the Uppsala Electron Density Server. The script runs on various platforms and is available for download at http://www.ruppweb.org/twilight/.

The effects of CapZ peptide (TRTK-12) binding to S100B-Ca2+ as examined by NMR and X-ray crystallography.

Structure-based drug design is underway to inhibit the S100B-p53 interaction as a strategy for treating malignant melanoma. X-ray crystallography was used here to characterize an interaction between Ca(2)(+)-S100B and TRTK-12, a target that binds to the p53-binding site on S100B. The structures of Ca(2+)-S100B (1.5-A resolution) and S100B-Ca(2)(+)-TRTK-12 (2.0-A resolution) determined here indicate that the S100B-Ca(2+)-TRTK-12 complex is dominated by an interaction between Trp7 of TRTK-12 and a hydrophobic binding pocket exposed on Ca(2+)-S100B involving residues in helices 2 and 3 and loop 2. As with an S100B-Ca(2)(+)-p53 peptide complex, TRTK-12 binding to Ca(2+)-S100B was found to increase the proteins Ca(2)(+)-binding affinity. One explanation for this effect was that peptide binding introduced a structural change that increased the number of Ca(2+) ligands and/or improved the Ca(2+) coordination geometry of S100B. This possibility was ruled out when the structures of S100B-Ca(2+)-TRTK-12 and S100B-Ca(2+) were compared and calcium ion coordination by the protein was found to be nearly identical in both EF-hand calcium-binding domains (RMSD=0.19). On the other hand, B-factors for residues in EF2 of Ca(2+)-S100B were found to be significantly lowered with TRTK-12 bound. This result is consistent with NMR (15)N relaxation studies that showed that TRTK-12 binding eliminated dynamic properties observed in Ca(2+)-S100B. Such a loss of protein motion may also provide an explanation for how calcium-ion-binding affinity is increased upon binding a target. Lastly, it follows that any small-molecule inhibitor bound to Ca(2+)-S100B would also have to cause an increase in calcium-ion-binding affinity to be effective therapeutically inside a cell, so these data need to be considered in future drug design studies involving S100B.

Lesion processing by a repair enzyme is severely curtailed by residues needed to prevent aberrant activity on undamaged DNA

DNA base excision repair is essential for maintaining genomic integrity and for active DNA demethylation, a central element of epigenetic regulation. A key player is thymine DNA glycosylase (TDG), which excises thymine from mutagenic G·T mispairs that arise by deamination of 5-methylcytosine (mC). TDG also removes 5-formylcytosine and 5-carboxylcytosine, oxidized forms of mC produced by Tet enzymes. Recent studies show that the glycosylase activity of TDG is essential for active DNA demethylation and for embryonic development. Our understanding of how repair enzymes excise modified bases without acting on undamaged DNA remains incomplete, particularly for mismatch glycosylases such as TDG. We solved a crystal structure of TDG (catalytic domain) bound to a substrate analog and characterized active-site residues by mutagenesis, kinetics, and molecular dynamics simulations. The studies reveal how TDG binds and positions the nucleophile (water) and uncover a previously unrecognized catalytic residue (Thr197). Remarkably, mutation of two active-site residues (Ala145 and His151) causes a dramatic enhancement in G·T glycosylase activity but confers even greater increases in the aberrant removal of thymine from normal A·T base pairs. The strict conservation of these residues may reflect a mechanism used to strike a tolerable balance between the requirement for efficient repair of G·T lesions and the need to minimize aberrant action on undamaged DNA, which can be mutagenic and cytotoxic. Such a compromise in G·T activity can account in part for the relatively weak G·T activity of TDG, a trait that could potentially contribute to the hypermutability of CpG sites in cancer and genetic disease.

Ni(II) coordination to mixed sites modulates DNA binding of HpNikR via a long-range effect

Helicobacter pylori NikR (HpNikR) is a nickel-dependent transcription factor that regulates multiple genes in the H. pylori pathogen. There are conflicting data regarding the locations of the Ni(II) sites and the role of Ni(II) coordination in DNA recognition. Herein, we report crystal structures of (i) the metal-binding domain (MBD) of HpNikR (3.08 Å) and (ii) a mutant, H74A (2.04 Å), designed to disrupt native Ni(II) coordination. In the MBD structure, four nickel ions are coordinated to two different types of nickel sites (4-coordinate, square planar, and 5/6-coordinate, square pyramidal/octahedral). In the H74A structure, all four nickel ions are coordinated to 4-coordinate square-planar sites. DNA-binding studies reveal tighter binding for target DNA sequences for holo-HpNikR compared with the affinities of Ni(II) reconstituted apo-HpNikR and H74A for these same DNA targets, supporting a role for Ni(II) coordination to 5/6 sites in DNA recognition. Small-angle X-ray scattering studies of holo-HpNikR and H74A reveal a high degree of conformational flexibility centered at the DNA-binding domains of H74A, which is consistent with disorder observed in the crystal structure of the protein. A model of DNA recognition by HpNikR is proposed in which Ni(II) coordination to specific sites in the MBD have a long-range effect on the flexibility of the DNA-binding domains and, consequently, the DNA recognition properties.