Structural insights into the repair mechanism of AGT for methyl-induced DNA damage

Methylation induced DNA base-pairing damage is one of the major causes of cancer. O6-alkylguanine-DNA alkyltransferase (AGT) is considered a demethylation agent of the methylated DNA. Structural investigations with thermodynamic properties of the AGT-DNA complex are still lacking. In this report, we modeled two catalytic states of AGT-DNA interactions and an AGT-DNA covalent complex and explored structural features using molecular dynamics (MD) simulations. We utilized the umbrella sampling method to investigate the changes in the free energy of the interactions in two different AGT-DNA catalytic states, one with methylated GUA in DNA and the other with methylated CYS145 in AGT. These non-covalent complexes represent the pre- A nd post-repair complexes. Therefore, our study encompasses the process of recognition, complex formation, and separation of the AGT and the damaged (methylated) DNA base. We believe that the use of parameters for the amino acid and nucleotide modifications and for the protein-DNA covalent bond will allow investigations of the DNA repair mechanism as well as the exploration of cancer therapeutics targeting the AGT-DNA complexes at various functional states as well as explorations via stabilization of the complex

Covalent Complex of DNA and Bacterial Topoisomerase: Implications in Antibacterial Drug Development

A topoisomerase-DNA transient covalent complex can be a druggable target for novel topoisomerase poison inhibitors that represent a new class of antibacterial or anticancer drugs. Herein, we have investigated molecular features of the functionally important Escherichia coli topoisomerase I (EctopoI)-DNA covalent complex (EctopoIcc) for molecular simulations, which is very useful in the development of new antibacterial drugs. To demonstrate the usefulness of our approach, we used a model small molecule (SM), NSC76027, obtained from virtual screening. We examined the direct binding of NSC76027 to EctopoI as well as inhibition of EctopoI relaxation activity of this SM via experimental techniques. We then performed molecular dynamics (MD) simulations to investigate the dynamics and stability of EctopoIcc and EctopoI-NSC76027-DNA ternary complex. Our simulation results show that NSC76027 forms a stable ternary complex with EctopoIcc. EctopoI investigated here also serves as a model system for investigating a complex of topoisomerase and DNA in which DNA is covalently attached to the protein

SPRD: a surface plasmon resonance database of common factors for better experimental planning

Background: Surface plasmon resonance is a label-free biophysical technique that is widely used in investigating biomolecular interactions, including protein-protein, protein-DNA, and protein-small molecule binding. Surface plasmon resonance is a very powerful tool in different stages of small molecule drug development and antibody characterization. Both academic institutions and pharmaceutical industry extensively utilize this method for screening and validation studies involving direct molecular interactions. In most applications of the surface plasmon resonance technology, one of the studied molecules is immobilized on a microchip, while the second molecule is delivered through a microfluidic system over the immobilized molecules. Changes in total mass on the chip surface is recorded in real time as an indicator of the molecular interactions. Main body: Quality and accuracy of the surface plasmon resonance data depend on experimental variables, including buffer composition, type of sensor chip, coupling chemistry of molecules on the sensor surface, and surface regeneration conditions. These technical details are generally included in materials and methods sections of published manuscripts and are not easily accessible using the common internet browser search engines or PubMed. Herein, we introduce a surface plasmon resonance database, www.sprdatabase.info that contains technical details extracted from 5140 publications with surface plasmon resonance data. We also provide an analysis of experimental conditions preferred by different laboratories. These experimental variables can be searched within the database and help future users of this technology to design better experiments. Conclusion: Amine coupling and CM5 chips were the most common methods used for immobilizing proteins in surface plasmon resonance experiments. However, number of different chips, capture methods and buffer conditions were used by multiple investigators. We predict that the database will significantly help the scientific community using this technology and hope that users will provide feedback to improve and expand the database indefinitely. Publicly available information in the database can save a great amount of time and resources by assisting initial optimization and troubleshooting of surface plasmon resonance experiments

Localization of Mycobacterium tuberculosis topoisomerase I C-terminal sequence motif required for inhibition by endogenous toxin MazF4

Only about half the multi-drug resistant tuberculosis (MDR-TB) cases are successfully cured. Thus, there is an urgent need of new TB treatment against a novel target. Mycobacterium tuberculosis (Mtb) topoisomerase I (TopA) is the only type IA topoisomerase in this organism and has been validated as an essential target for TB drug discovery. Toxin-antitoxin (TA) systems participate as gene regulators within bacteria. The TA systems contribute to the long-term dormancy of Mtb within the host-cell environment. Mtb’s toxin MazF4 (Rv1495) that is part of the MazEF4 TA system has been shown to have dual activities as endoribonuclease and topoisomerase I inhibitor. We have developed a complementary assay using an Escherichia coli strain with temperature-sensitive topA mutation to provide new insights into the MazF4 action. The assay showed that E. coli is not sensitive to the endoribonuclease activity of Mtb MazF4 but became vulnerable to MazF4 growth inhibition when recombinant Mtb TopA relaxation activity is required for growth. Results from the complementation by Mtb TopA mutants with C-terminal deletions showed that the lysine-rich C-terminal tail is required for interaction with MazF4. Site-directed mutagenesis is utilized to identify two lysine residues within a conserved motif in this C-terminal tail that are critical for MazF4 inhibition. We performed molecular dynamics simulations to predict the Mtb TopA-MazF4 complex. Our simulation results show that the complex is stabilized by hydrogen bonds and electrostatic interactions established by residues in the TopA C-terminal tail including the two conserved lysines. The mechanism of Mtb TopA inhibition by MazF4 could be useful for the discovery of novel inhibitors against a new antibacterial target in pathogenic mycobacteria for treatment of both TB and diseases caused by the non-tuberculosis mycobacteria (NTM)

Thermodynamic Study of Mo(II)-Penicillins

200-20

Investigating cyclic nucleotide and cyclic dinucleotide binding to HCN channels by surface plasmon resonance

<div><p>Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels control cardiac and neuronal rhythmicity. HCN channels contain cyclic nucleotide-binding domain (CNBD) in their C-terminal region linked to the pore-forming transmembrane segment with a C-linker. The C-linker couples the conformational changes caused by the direct binding of cyclic nucleotides to the HCN pore opening. Recently, cyclic dinucleotides were shown to antagonize the effect of cyclic nucleotides in HCN4 but not in HCN2 channels. Based on the structural analysis and mutational studies it has been proposed that cyclic dinucleotides affect HCN4 channels by binding to the C-linker pocket (CLP). Here, we first show that surface plasmon resonance (SPR) can be used to accurately measure cyclic nucleotide binding affinity to the C-linker/CNBD of HCN2 and HCN4 channels. We then used SPR to investigate cyclic dinucleotide binding in HCN channels. To our surprise, we detected no binding of cyclic dinucleotides to the isolated monomeric C-linker/CNBDs of HCN4 channels with SPR. The binding of cyclic dinucleotides was further examined with isothermal calorimetry (ITC), which indicated no binding of cyclic dinucleotides to both monomeric and tetrameric C-linker/CNBDs of HCN4 channels. Taken together, our results suggest that interaction of the C-linker/CNBD with other parts of the channel is necessary for cyclic-dinucleotide binding in HCN4 channels.</p></div

Cyclic nucleotide and cyclic dinucleotide binding to the monomeric and tetrameric HCN4 C-linker/CNBD tested with ITC.

<p>Thermograms of successive injections of 1.25 μl of cAMP (A and D), c-di-GMP (B and E) and c-di-GMP in the presence of cAMP (C and F) (top panels) and the corresponding binding isotherms (bottom panels). For experiments in (C) 100 μM cAMP and in (F) 1 mM cAMP was present in both the protein and ligand solutions. Monomeric 6-His tagged hHCN4 C-linker/CNBDs at 6 μM concentration, purified in the same manner as for the SPR-based experiments, were used for experiments in (A-C). hHCN4 C-linker/CNBDs after the MBP tag cleavage at 80 μM concentration were used for experiments in (D-F). The binding isotherms were obtained by integrating the peaks in the top panels, normalizing the obtained values by the cAMP concentration and plotting them against the molar ratio of cAMP to the protein. The lines represent a nonlinear least-square fit to a single–site binding model for (A-C) and a two independent binding site model for (D-F). The binding affinities for cAMP were 1.1 ± 0.5 μM in (A), and 1.7 ± 0.3 μM and 0.03 ± 0.02 μM in (D).</p

Cyclic nucleotide and cyclic dinucleotide binding to the monomeric and tetrameric HCN4 C-linker/CNBD tested with ITC.

<p>Thermograms of successive injections of 1.25 μl of cAMP (A and D), c-di-GMP (B and E) and c-di-GMP in the presence of cAMP (C and F) (top panels) and the corresponding binding isotherms (bottom panels). For experiments in (C) 100 μM cAMP and in (F) 1 mM cAMP was present in both the protein and ligand solutions. Monomeric 6-His tagged hHCN4 C-linker/CNBDs at 6 μM concentration, purified in the same manner as for the SPR-based experiments, were used for experiments in (A-C). hHCN4 C-linker/CNBDs after the MBP tag cleavage at 80 μM concentration were used for experiments in (D-F). The binding isotherms were obtained by integrating the peaks in the top panels, normalizing the obtained values by the cAMP concentration and plotting them against the molar ratio of cAMP to the protein. The lines represent a nonlinear least-square fit to a single–site binding model for (A-C) and a two independent binding site model for (D-F). The binding affinities for cAMP were 1.1 ± 0.5 μM in (A), and 1.7 ± 0.3 μM and 0.03 ± 0.02 μM in (D).</p

Structural model of the c-di-GMP regulation.

<p>(A) Ribbon representation of transmembrane segments of two opposing subunits and the C-linker/CNBDs of the two subunits adjacent to them from the full-length structure of HCN1 channels [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185359#pone.0185359.ref045" target="_blank">45</a>]. The transmembrane segments of the subunits containing the C-linker/CNBDs shown in the figure and the C-linker/CNBDs of the subunits containing transmembrane segments shown in the figure are omitted for clarity. In the crystal structure C-linkers are making direct contacts with S4-S5 linkers and HCN domains from only the adjacent subunits. Transmembrane segments (TM) are shown in grey, S4-S5 linkers and HCN domains are orange, C-linkers are red, β-roll and helices A, P and B are blue, and the C-helix and the distal C-terminus are green. cAMP bound inside the β-roll cavity is yellow. c-di-GMP placed in the proposed CLP site is cyan. (B) Structural alignment of the C-linker/CNBD from the full-length HCN1 structure [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185359#pone.0185359.ref045" target="_blank">45</a>] shown in gray and isolated C-linker/CNBD of HCN2 channels [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185359#pone.0185359.ref015" target="_blank">15</a>] shown in magenta.</p