Hee-Chul Ahn

NMR Spectroscopic Elucidation of the B−Z Transition of a DNA Double Helix Induced by the Zα Domain of Human ADAR1

The human RNA editing enzyme ADAR1 (double-stranded RNA deaminase I) deaminates adenine in pre-mRNA to yield inosine, which codes as guanine. ADAR1 has two left-handed Z-DNA binding domains, Z alpha and Z beta, at its NH(2)-terminus and preferentially binds Z-DNA, rather than B-DNA, with high binding affinity. The cocrystal structure of Z alpha(ADAR1) complexed to Z-DNA showed that one monomeric Z alpha(ADAR1) domain binds to one strand of double-stranded DNA and a second Z alpha(ADAR1) monomer binds to the opposite strand with 2-fold symmetry with respect to DNA helical axis. It remains unclear how Z alpha(ADAR1) protein specifically recognizes Z-DNA sequence in a sea of B-DNA to produce the stable Z alpha(ADAR1)-Z-DNA complex during the B-Z transition induced by Z alpha(ADAR1). In order to characterize the molecular recognition of Z-DNA by Z alpha(ADAR1), we performed circular dichroism (CD) and NMR experiments with complexes of Zalpha(ADAR1) bound to d(CGCGCG)(2) (referred to as CG6) produced at a variety of protein-to-DNA molar ratios. From this study, we identified the intermediate states of the CG6-Z alpha(ADAR1) complex and calculated their relative populations as a function of the Z alpha(ADAR1) concentration. These findings support an active B-Z transition mechanism in which the Z alpha(ADAR1) protein first binds to B-DNA and then converts it to left-handed Z-DNA, a conformation that is then stabilized by the additional binding of a second Z alpha(ADAR1) molecule.

Structural Basis for Ufm1 Processing by UfSP1

Ubiquitin-fold modifier 1 (Ufm1) is a newly identified ubiquitin-like protein. Like ubiquitin and other ubiquitin-like proteins, Ufm1 is synthesized as a precursor that needs to be processed to expose the conserved C-terminal glycine prior to its conjugation to target proteins. Two novel proteases, named UfSP1 and UfSP2, have been shown to be responsible for the release of Ufm1 from Ufm1-conjugated cellular proteins as well as for the processing of its precursor. They show no sequence homology with known proteases. Here, we describe the 1.7Å resolution crystal structure of mouse UfSP1, consisting of 217 amino acids. The structure reveals that it is a novel cysteine protease having a papain-like fold, with Cys53, Asp175, and His177 that form a catalytic triad, and Tyr41 that participates in the formation of the oxyanion hole. This differs from the canonical catalytic triad of papain-like proteases in that the aspartate and the histidine residues are from the “Asp-Pro-His” box. The Asp-Pro-His configuration seen in UfSP1, together with Atg4B and M48USP, seem to form a new subfamily of the cysteine protease superfamily. The mutagenesis study of the active site residues confirms structural basis for catalysis. The interaction between UfSP1 and Ufm1 appears quite substantial, since the KD value was estimated to be 1.6 μm by the isothermal titration calorimetry analysis. Furthermore, the NMR data shows that the loop between β3 and α2 in addition to the C-terminal region of Ufm1 plays a role in binding to UfSP1.

Comparative study of the tribological behavior of thermal sprayed quasicrystalline coating layers

To investigate the role of tribological reactions on the friction and wear of quasicrystalline materials, coatings with two alloy compositions have been prepared by plasma and HVOF spraying techniques. The tribolayers were characterized by the formation of a transfer film on the counterface and densification of the coating subsurfaces. It was observed that the thickness of the transfer film and pore-free region were dependent on the composition and process used for the deposition of the coatings as well as the sliding velocity. As the sliding velocity increased, the growth rate of the transfer film decreased, resulting in a decrease of the coefficient of friction. On the other hand, the wear rate appeared to be controlled by the thickness of the pore-free region formed within the coating surface zone.

Functional identification of toxin-antitoxin molecules from Helicobacter pylori 26695 and structural elucidation of the molecular interactions.

Bacterial toxin-antitoxin (TA) systems are associated with many important cellular processes including antibiotic resistance and microorganism virulence. Here, we identify and structurally characterize TA molecules from the gastric pathogen, Helicobacter pylori. The HP0894 protein had been previously suggested, through our structural genomics approach, to be a putative toxin molecule. In this study, the intrinsic RNase activity and the bacterial cell growth-arresting activity of HP0894 were established. The RNA-binding surface was identified at three residue clusters: (Lys8 and Ser9), (Lys50–Lys54 and Glu58), and (Arg80 and His84–Phe88). In particular, the -UA- and -CA- sequences in RNA were preferentially cleaved by HP0894, and residues Lys52, Trp53, and Ser85–Lys87 were observed to be the main contributors to sequence recognition. The action of HP0894 could be inhibited by the HP0895 protein, and the HP0894-HP0895 complex formed an oligomer with a binding stoichiometry of 1:1. The N and C termini of HP0894 constituted the binding sites to HP0895. In contrast, the unstructured C-terminal region of HP0895 was responsible for binding to HP0894 and underwent a conformational change in the process. Finally, DNA binding activity was observed for both HP0895 and the HP0894-0895 complex but not for HP0894 alone. Taken together, it is concluded that the HP0894-HP0895 protein couple is a TA system in H. pylori, where HP0894 is a toxin with an RNase function, whereas HP0895 is an antitoxin functioning by binding to both the toxin and DNA.

NMR study on the B-Z junction formation of DNA duplexes induced by Z-DNA binding domain of human ADAR1.

Z-DNA is produced in a long genomic DNA by Z-DNA binding proteins, through formation of two B-Z junctions with the extrusion of one base pair from each junction. To answer the question of how Z-DNA binding proteins induce B-Z transitions in CG-rich segments while maintaining the B-conformation of surrounding segments, we investigated the kinetics and thermodynamics of base-pair openings of a 13-bp DNA in complex with the Z-DNA binding protein, Zα(ADAR1). We also studied perturbations in the backbone of Zα(ADAR1) upon binding to DNA. Our study demonstrates the initial contact conformation as an intermediate structure during B-Z junction formation induced by Zα(ADAR1), in which the Zα(ADAR1) protein displays unique backbone conformational changes, but the 13-bp DNA duplex maintains the B-form helix. We also found the unique structural features of the 13-bp DNA duplex in the initial contact conformation: (i) instability of the AT-rich region II and (ii) longer lifetime for the opening state of the CG-rich region I. Our findings suggest a three-step mechanism of B-Z junction formation: (i) Zα(ADAR1) specifically interacts with a CG-rich DNA segment maintaining B-form helix via a unique conformation; (ii) the neighboring AT-rich region becomes very unstable, and the CG-rich DNA segment is easily converted to Z-DNA; and (iii) the AT-rich regions are base-paired again, and the B-Z junction structure is formed.

Inhibition of VEGF transcription through blockade of the hypoxia inducible factor-1α–p300 interaction by a small molecule

Vascular endothelial growth factor (VEGF) plays a pro-angiogenic role in tumor progression. Stabilization of a key regulator termed the hypoxia inducible factor (HIF)-1α under oxygen deficient environment around tumor is known to elicit expression of VEGF through binding to p300. Thus, inhibition of the HIF-1α-p300 interaction would lead to down-regulation of VEGF expression, thereby providing potential cancer therapeutics. Here, we have screened a chemical library against the interaction of the HIF-1α-derived peptide with p300 employing a fluorescence polarization-based assay. We have identified a compound as the most prominent inhibitor against the protein-protein interaction. Further, we have observed suppression of the mRNA level of VEGF upon treatment of HeLa cells with the compound, demonstrating its inhibitory effect at the cellular level.

Solution and solid-state NMR structural studies of antimicrobial peptides LPcin-I and LPcin-II.

Lactophoricin (LPcin-I) is an antimicrobial, amphiphatic, cationic peptide with 23-amino acid residues isolated from bovine milk. Its analogous peptide, LPcin-II, lacks six N-terminal amino acids compared to LPcin-I. Interestingly, LPcin-II does not display any antimicrobial activity, whereas LPcin-I inhibits the growth of both Gram-negative and Gram-positive bacteria without exhibiting any hemolytic activity. Uniformly (15)N-labeled LPcin peptides were prepared by the recombinant expression of fusion proteins in Escherichia coli, and their properties were characterized by electrospray ionization mass spectrometry, circular dichroism spectroscopy, and antimicrobial activity tests. To understand the structure-activity relationship of these two peptides, they were studied in model membrane environments by a combination of solution and solid-state NMR spectroscopy. We determined the tertiary structure of LPcin-I and LPcin-II in the presence of dodecylphosphorylcholine micelles by solution NMR spectroscopy. Magnetically aligned unflipped bicelle samples were used to investigate the structure and topology of LPcin-I and LPcin-II by solid-state NMR spectroscopy.

Sequence discrimination of the Zα domain of human ADAR1 during B–Z transition of DNA duplexes

The Zα domain of human ADAR1 (ZαADAR1) preferentially binds Z‐DNA rather than B‐DNA with high binding affinity. ZαADAR1 binds to the Z‐conformation of both non‐CG‐repeat DNA duplexes and a d(CGCGCG)2 duplex similarly. We performed NMR experiments on complexes between the ZαADAR1 and non‐CG‐repeat DNA duplexes, d(CACGTG)2 or d(CGTACG)2, with a variety of protein‐DNA molar ratios. Comparison of these results with those from the analysis of d(CGCGCG)2 in the previous study suggests that ZαADAR1 exhibits the sequence preference of d(CGCGCG)2 ≫ d(CACGTG)2 > d(CGTACG)2 through multiple sequence discrimination steps during the B–Z transition.

Tribological properties of Al–Ni–Co–Si quasicrystalline coatings against Cr-coated cast iron disc

Abstract The tribological behavior of Al–Ni–Co–Si quasicrystalline (QC) coatings prepared by air plasma spray (APS) and high velocity oxy-fuel (HVOF) techniques was investigated in dry sliding condition against a Cr-coated cast iron disc. Tests were performed for different parameters of the load, sliding velocity, and temperature under a reciprocal motion. Our results demonstrated the influence of the coating microstructure on the frictional and wear behavior. Values of the coefficient of friction measured for HVOF coatings against Cr-coated cast iron disc were found to be about 14% lower than those of APS coatings. Under the standard condition, HVOF coatings exhibited a better wear resistance than APS coatings, however, this difference was reduced for tests performed under the highest values of the load and temperature investigated in this study. The sliding conditions imposed in this study led to significant modifications of the contacting surfaces characterized by the formation of a transfer film on the counterpart material, densification of the coating subsurface, and generation of wear particles. The variations of the coefficient of friction with the testing parameters were explained by the third body concept. It was also shown that the wear performance was dependent on the local surface properties of the QC coatings.

Syntheses and biological evaluation of 1-heteroaryl-2-aryl-1H-benzimidazole derivatives as c-Jun N-terminal kinase inhibitors with neuroprotective effects.

1-Heteroaryl-2-aryl-1H-benzimidazole derivatives were synthesized as inhibitors of c-Jun N-terminal kinases, JNK3. Their activities were evaluated through measurement of Kd using SPR, JNK3 kinase assay, and cell-viability of human neuroblastoma cells. Most tested compounds showed high affinity (10 μM-46 nM) to JNK3. Among them, compound 16f exhibited potent activities (Kd=46 nM). Especially, 16f was also found to present a potent cell protective effect (IC50=1.09 μM) against toxicity induced by anisomycin, showing a possibility as protective therapeutics in neuronal cell apoptosis.