Thymine DNA Glycosylase (TDG) performs essential functions in maintaining genetic integrity and epigenetic regulation. Initiating base excision repair, TDG removes thymine from mutagenic G ·: T ...mispairs caused by 5-methylcytosine (mC) deamination and other lesions including uracil (U) and 5-hydroxymethyluracil (hmU). In DNA demethylation, TDG excises 5-formylcytosine (fC) and 5-carboxylcytosine (caC), which are generated from mC by Tet (ten-eleven translocation) enzymes. Using improved crystallization conditions, we solved high-resolution (up to 1.45 Å) structures of TDG enzyme-product complexes generated from substrates including G·U, G·T, G·hmU, G·fC and G·caC. The structures reveal many new features, including key water-mediated enzyme-substrate interactions. Together with nuclear magnetic resonance experiments, the structures demonstrate that TDG releases the excised base from its tight product complex with abasic DNA, contrary to previous reports. Moreover, DNA-free TDG exhibits no significant binding to free nucleobases (U, T, hmU), indicating a Kd >> 10 mM. The structures reveal a solvent-filled channel to the active site, which might facilitate dissociation of the excised base and enable caC excision, which involves solvent-mediated acid catalysis. Dissociation of the excised base allows TDG to bind the beta rather than the alpha anomer of the abasic sugar, which might stabilize the enzyme-product complex.
Apurinic/apyrimidinic endonuclease 1 (APE1) mediates the repair of abasic sites and other DNA lesions and is essential for base‐excision repair and strand‐break repair pathways. APE1 hydrolyzes the ...phosphodiester bond at abasic sites, producing 5′‐deoxyribose phosphate and the 3′‐OH primer needed for repair synthesis. It also has additional repair activities, including the removal of 3′‐blocking groups. APE1 is a powerful enzyme that absolutely requires Mg2+, but the stoichiometry and catalytic function of the divalent cation remain unresolved for APE1 and for other enzymes in the DNase I superfamily. Previously reported structures of DNA‐free APE1 contained either Sm3+ or Pb2+ in the active site. However, these are poor surrogates for Mg2+ because Sm3+ is not a cofactor and Pb2+ inhibits APE1, and their coordination geometry is expected to differ from that of Mg2+. A crystal structure of human APE1 was solved at 1.92 Å resolution with a single Mg2+ ion in the active site. The structure reveals ideal octahedral coordination of Mg2+via two carboxylate groups and four water molecules. One residue that coordinates Mg2+ directly and two that bind inner‐sphere water molecules are strictly conserved in the DNase I superfamily. This structure, together with a recent structure of the enzyme–product complex, inform on the stoichiometry and the role of Mg2+ in APE1‐catalyzed reactions.
Thymine DNA glycosylase (TDG) is a base excision repair enzyme with key functions in epigenetic regulation. Performing a critical step in a pathway for active DNA demethylation, TDG removes ...5-formylcytosine and 5-carboxylcytosine, oxidized derivatives of 5-methylcytosine that are generated by TET (ten–eleven translocation) enzymes. We determined a crystal structure of TDG bound to DNA with a noncleavable (2′-fluoroarabino) analogue of 5-formyldeoxycytidine flipped into its active site, revealing how it recognizes and hydrolytically excises fC. Together with previous structural and biochemical findings, the results illustrate how TDG employs an adaptable active site to excise a broad variety of nucleobases from DNA.
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•Ca2+-bound Calmodulin (CaM) interacts with the BP2 helix of STRA6.•CaM exhibits distinct Ca2+-dependent and independent interactions with BP2 peptide.•The CaM C-lobe interacts with ...STRA6 BP2 at Ca2+ concentrations ∼100 nM.•As free Ca2+ increases to 1000 nM, BP2 interacts with the N- and C-lobes of CaM.•Ca2+ signaling regulates vitamin A transport via structural changes in CaM-STRA6.
The interaction of calmodulin (CaM) with the receptor for retinol uptake, STRA6, involves an α-helix termed BP2 that is located on the intracellular side of this homodimeric transporter (Chen et al., 2016 1). In the absence of Ca2+, NMR data showed that a peptide derived from BP2 bound to the C-terminal lobe (C-lobe) of Mg2+-bound CaM (MgCaM). Upon titration of Ca2+ into MgCaM-BP2, NMR chemical shift perturbations (CSPs) were observed for residues in the C-lobe, including those in the EF-hand Ca2+-binding domains, EF3 and EF4 (CaKD = 60 ± 7 nM). As higher concentrations of free Ca2+ were achieved, CSPs occurred for residues in the N-terminal lobe (N-lobe) including those in EF1 and EF2 (CaKD = 1000 ± 160 nM). Thermodynamic and kinetic Ca2+ binding studies showed that BP2 addition increased the Ca2+-binding affinity of CaM and slowed its Ca2+ dissociation rates (koff) in both the C- and N-lobe EF-hand domains, respectively. These data are consistent with BP2 binding to the C-lobe of CaM at low free Ca2+ concentrations (<100 nM) like those found at resting intracellular levels. As free Ca2+ levels approach 1000 nM, which is typical inside a cell upon an intracellular Ca2+-signaling event, BP2 is shown here to interact with both the N- and C-lobes of Ca2+-loaded CaM (CaCaM-BP2). Because this structural rearrangement observed for the CaCaM-BP2 complex occurs as intracellular free Ca2+ concentrations approach those typical of a Ca2+-signaling event (CaKD = 1000 ± 160 nM), this conformational change could be relevant to vitamin A transport by full-length CaCaM-STRA6.
Xylanases of glycosyl hydrolase family 30 (GH30) have been shown to cleave β-1,4 linkages of 4-O-methylglucuronoxylan (MeGXn) as directed by the position along the xylan chain of an α-1,2-linked ...4-O-methylglucuronate (MeGA) moiety. Complete hydrolysis of MeGXn 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 MeGXn 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 MeGXn 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.
Targeting Clostridium difficile infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent C. difficile strains ...often have a binary toxin termed the C. difficile toxin, in addition to the enterotoxins TsdA and TsdB. The C. difficile toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single-particle cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (1.0 MDa) were solved at atomic resolution, including a symmetric (SymCDTb; 3.14 Å) and an asymmetric form (AsymCDTb; 2.84 Å). Roles played by 2 receptor-binding domains of activated CDTb were of particular interest since the receptor-binding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). For AsymCDTb, a Ca2+ binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the di-heptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to target the most severe strains of C. difficile.
This study demonstrates the application of metal-assisted and microwave-accelerated evaporative crystallization (MA-MAEC) technique to rapid crystallization of l-alanine on surface-engineered silver ...nanostructures. In this regard, silver island films (SIFs) were modified with hexamethylenediamine (HMA), 1-undecanethiol (UDET), and 11-mercaptoundecanoic acid (MUDA), which introduced -NH2, -CH3, and -COOH functional groups to SIFs, respectively. l-Alanine was crystallized on these engineered surfaces and blank SIFs at room temperature by the MA-MAEC technique. Significant improvements in crystal size, shape, and quality were observed on HMA-, MUDA- and UDET-modified SIFs at room temperature (crystallization time = 144, 40, and 147 min, respectively) as compared to those crystals grown on blank SIFs. By use of the MA-MAEC technique, the crystallization time of l-alanine on engineered surfaces was reduced to 17 s for microwave power level 10 (i.e., duty cycle 100%) and 7 min for microwave power level 1 (duty cycle 10%). Raman spectroscopy and powder X-ray diffraction (XRD) measurements showed that l-alanine crystals grown on engineered surfaces by the MA-MAEC technique had identical characteristic peaks to l-alanine crystals grown by traditional evaporative crystallization.
Crystal Structure of a Bivalent Antibody Fab Fragment Shahid, Salman; Gao, Mingming; Travis Gallagher, D. ...
Journal of Molecular Biology/Journal of molecular biology,
01/2021, Letnik:
433, Številka:
2
Journal Article
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•The Fab of human anti-HCV antibody HC84.26.5D forms bivalent dimers in solution.•Crystal structure revealed that Fab HC84.26.5D is a domain-swapped dimer.•Dimerization is mediated by ...deletion of one residue in the H chain elbow region.•Domain-swapped Fab dimers are useful as fiducial markers for cryoEM.
We determined the crystal structure to 1.8 Å resolution of the Fab fragment of an affinity-matured human monoclonal antibody (HC84.26.5D) that recognizes the E2 envelope glycoprotein of hepatitis C virus (HCV). Unlike conventional Fabs, which are monovalent monomers, Fab HC84.26.5D assembles into a bivalent domain-swapped dimer in which the two VL/VH modules are separated by ~25 Å. In solution, Fab HC84.26.5D exists predominantly as a dimer (~80%) in equilibrium with the monomeric form of the Fab (~20%). Dimerization is mediated entirely by deletion of a single residue, VHSer113 (Kabat numbering), in the elbow region linking the VH and CH1 domains. In agreement with the crystal structure, dimeric Fab HC84.26.5D is able to bind two HCV E2 molecules in solution. This is only the second example of a domain-swapped Fab dimer from among >3000 Fab crystal structures determined to date. Moreover, the architecture of the doughnut-shaped Fab HC84.26.5D dimer is completely different from that of the previously reported Fab 2G12 dimer. We demonstrate that the highly identifiable shape of dimeric Fab HC84.26.5D makes it useful as a fiducial marker for single-particle cryoEM analysis of HCV E2. Bivalent domain-swapped Fab dimers engineered on the basis of HC84.26.5D may also serve as a means of doubling the effective size of conventional Fab–protein complexes for cryoEM.
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 pK a value for this residue. We have used atomic-resolution X-ray crystallography and UV spectroscopy to show that C106 has a depressed pK 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 pK 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 pK 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 pK 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.
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The DNA base excision repair (BER) pathway is essential for maintaining genomic integrity and is implicated in active DNA demethylation, a key element of epigenetic transcriptional ...regulation. Thymine DNA glycosylase (TDG) excises thymine from mutagenic G·T mispairs, initiating repair of deaminated 5‐methylcytosine (mC). TDG also excises 5‐formylcytosine (fC) and 5‐carboxylcytosine (caC), oxidation products of mC produced by Tet enzymes. These seemingly disparate activities are consistent with TDG specificity for acting at CpG sites and its essential role in active DNA demethylation and embryonic development. Understanding how glycosylases excise lesions and avoid acting on undamaged DNA is an important problem in DNA repair. Structural and biochemical results here reveal how TDG attains broad specificity for G·T and G·fC lesions while avoiding A·T pairs. A crystal structure of TDG (catalytic domain) bound to substrate analogue suggests G·T glycosylase activity is suboptimal owing to unfavorable interactions between flipped dT substrate and two active‐site residues. Remarkably, mutating these residues greatly increases G·T activity and confers substantial activity for normal A·T base pairs. The results suggest TDG evolved with suboptimal G·T repair capability in order to minimize aberrant activity on undamaged DNA, an unprecedented finding for a repair enzyme. Supported by NIH (R01‐GM072711).
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