Yeo-Jin Seo

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.

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.

NMR study of hydrogen exchange during the B–Z transition of a DNA duplex induced by the Zα domains of yatapoxvirus E3L

The Yaba‐like disease viruses (YLDV) are members of the Yatapoxvirus family and have double‐stranded DNA genomes. The E3L protein, which is essential for pathogenesis in the vaccinia virus, consists of two domains: an N‐terminal Z‐DNA binding domain and a C‐terminal RNA binding domain. The crystal structure of the E3L orthologue of YLDV (yabZαE3L) bound to Z‐DNA revealed that the overall structure of yabZαE3L and its interaction with Z‐DNA are very similar to those of hZαADAR1. Here we have performed NMR hydrogen exchange experiments on the complexes between yabZαE3L and d(CGCGCG)2 with a variety of protein‐to‐DNA molar ratios. This study revealed that yabZαE3L could efficiently change the B‐form helix of the d(CGCGCG)2 to left‐handed Z‐DNA via the active‐mono B–Z transition pathway like hZαADAR1.

The Zβ domain of human DAI binds to Z-DNA via a novel B–Z transition pathway

The human DNA‐dependent activator of IFN‐regulatory factor (DAI) protein, which activates the innate immune response in response to DNA, contains two tandem Z‐DNA binding domains (Zα and Zβ) at the NH2 terminus. The hZβDAI structure is similar to other Z‐DNA binding proteins, although it demonstrates an unusual Z‐DNA recognition. We performed NMR experiments on complexes of hZβDAI with DNA duplex, d(CGCGCG)2, at a variety of protein‐to‐DNA molar ratios. The results suggest that hZβDAI binds to Z‐DNA via an active‐di B–Z transition mechanism, where two hZβDAI proteins bind to B‐DNA to form the hZβDAI–B‐DNA complex; the B‐DNA is subsequently converted to left‐handed Z‐DNA. This novel mechanism of DNA binding and B–Z conversion is distinct from Z‐DNA binding of the human ADAR1 protein.

Comparison of backbone dynamics of the type III antifreeze protein and antifreeze-like domain of human sialic acid synthase

Antifreeze proteins (AFPs) are found in a variety of cold-adapted (psychrophilic) organisms to promote survival at subzero temperatures by binding to ice crystals and decreasing the freezing temperature of body fluids. The type III AFPs are small globular proteins that consist of one α-helix, three 310-helices, and two β-strands. Sialic acids play important roles in a variety of biological functions, such as development, recognition, and cell adhesion and are synthesized by conserved enzymatic pathways that include sialic acid synthase (SAS). SAS consists of an N-terminal catalytic domain and a C-terminal antifreeze-like (AFL) domain, which is similar to the type III AFPs. Despite having very similar structures, AFL and the type III AFPs exhibit very different temperature-dependent stability and activity. In this study, we have performed backbone dynamics analyses of a type III AFP (HPLC12 isoform) and the AFL domain of human SAS (hAFL) at various temperatures. We also characterized the structural/dynamic properties of the ice-binding surfaces by analyzing the temperature gradient of the amide proton chemical shift and its correlation with chemical shift deviation from random coil. The dynamic properties of the two proteins were very different from each other. While HPLC12 was mostly rigid with a few residues exhibiting slow motions, hAFL showed fast internal motions at low temperature. Our results provide insight into the molecular basis of thermostability and structural flexibility in homologous psychrophilic HPLC12 and mesophilic hAFL proteins.

Base pair opening kinetics study of the aegPNA:DNA hydrid duplex containing a site-specific GNA-like chiral PNA monomer

Peptide nucleic acids (PNA) are one of the most widely used synthetic DNA mimics where the four bases are attached to a N-(2-aminoethyl)glycine (aeg) backbone instead of the negative-charged phosphate backbone in DNA. We have developed a chimeric PNA (chiPNA), in which a chiral GNA-like γ3T monomer is incorporated into aegPNA backbone. The base pair opening kinetics of the aegPNA:DNA and chiPNA:DNA hybrid duplexes were studied by NMR hydrogen exchange experiments. This study revealed that the aegPNA:DNA hybrid is much more stable duplex and is less dynamic compared to DNA duplex, meaning that base pairs are opened and reclosed much more slowly. The site-specific incorporation of γ3T monomer in the aegPNA:DNA hybrid can destabilize a specific base pair and its neighbors, maintaining the thermal stabilities and dynamic properties of other base pairs. Our hydrogen exchange study firstly revealed the unique kinetic features of base pairs in the aegPNA:DNA and chiPNA:DNA hybrids, which will provide an insight into the development of methodology for specific DNA recognition using PNA fragments.

NMR study of the antifreeze activities of active and inactive isoforms of a type III antifreeze protein

The quaternary‐amino‐ethyl 1 (QAE1) isoforms of type III antifreeze proteins (AFPs) prevent the growth of ice crystals within organisms living in polar regions. We determined the antifreeze activity of wild‐type and mutant constructs of the Japanese notched‐fin eelpout (Zoarces elongates Kner) AFP8 (nfeAFP8) and characterized the structural and dynamics properties of their ice‐binding surface using NMR. We found that the three constructs containing the V20G mutation were incapable of stopping the growth of ice crystals and exhibited structural changes, as well as increased conformational flexibility, in the first 310 helix (residues 18–22) of the sequence. Our results suggest that the inactive nfeAFP8s are incapable of anchoring water molecules due to the unusual and flexible backbone conformation of their primary prism plane‐binding surface.

NMR investigation on the DNA binding and B-Z transition pathway of the Zα domain of human ADAR1.

Human ADAR1, which has two left-handed Z-DNA binding domains, preferentially binds Z-DNA rather than B-DNA with a high binding affinity. Z-DNA can be induced in long genomic DNA by Z-DNA binding proteins through the formation of two B-Z junctions with the extrusion of one base pair from each junction. We performed NMR experiments on complexes of Zα(ADAR1) with three DNA duplexes at a variety of protein-to-DNA molar ratios. This study confirmed that the Zα(ADAR1) first binds to an 8-bp CG-rich DNA segment via a unique conformation during B-Z transition and the neighboring AT-rich region becomes destabilized. We also found that, when DNA duplexes have only 6-bp CG-rich segment, the interaction with Zα(ADAR1) did not affect the thermal stabilities of the 6-bp CG-rich segment as well as the neighboring two A·T base pairs. These results indicate that four Zα(ADAR1) proteins interact with the 8-bp DNA sequence containing a 6-bp CG-repeat segment as well as a dinucleotide step, even though the dinucleotid step contains A∙T base pairs. Thus this study suggests that the length of the CG-rich region is more important than the specific DNA sequence for determining which base-pair is extruded from the B-Z junction structure. This study also found that the Zα(ADAR1) in complex with a 11-bp DNA duplex exhibits a Z-DNA-bound conformation distinct from that of free Zα(ADAR1) and the initial contact conformations of Zα(ADAR1) complexed with 13-bp DNA duplexes.

Expression and Purification of the Helicase-like Subdomains, H1 and H23, of Reverse Gyrase from A. fulgidus for Heteronuclear NMR study

Reverse gyrase is a hyperthermophile specific protein which introduces positive supercoils into DNA molecules. Reverse gyrase consists of an N-terminal helicase-like domain and a C-terminal topoisomerase domain. The helicase-like domain shares the three-dimensional structure with two tandem RecA-folds (H1 and H2), in which the subdomain H2 is interrupted by the latch domain (H3). To understand the physical property of the hyperthermophile-specific protein, two subdomains af_H1 and af_H23 have been cloned into E. coli expression vector, pET28a. The N-labeled af_H1 and af_H23 proteins were expressed and purified for heteronuclear NMR study. The af_H1 protein exhibits the well-dispersion of amide signals in its H/N-HSQC spectra and thus further NMR study continues to be progressed.

Thermodynamic Model for B-Z Transition of DNA Induced by Z-DNA Binding Proteins

Z-DNA is stabilized by various Z-DNA binding proteins (ZBPs) that play important roles in RNA editing, innate immune response, and viral infection. In this review, the structural and dynamics of various ZBPs complexed with Z-DNA are summarized to better understand the mechanisms by which ZBPs selectively recognize d(CG)-repeat DNA sequences in genomic DNA and efficiently convert them to left-handed Z-DNA to achieve their biological function. The intermolecular interaction of ZBPs with Z-DNA strands is mediated through a single continuous recognition surface which consists of an α3 helix and a β-hairpin. In the ZBP-Z-DNA complexes, three identical, conserved residues (N173, Y177, and W195 in the Zα domain of human ADAR1) play central roles in the interaction with Z-DNA. ZBPs convert a 6-base DNA pair to a Z-form helix via the B-Z transition mechanism in which the ZBP first binds to B-DNA and then shifts the equilibrium from B-DNA to Z-DNA, a conformation that is then selectively stabilized by the additional binding of a second ZBP molecule. During B-Z transition, ZBPs selectively recognize the alternating d(CG)n sequence and convert it to a Z-form helix in long genomic DNA through multiple sequence discrimination steps. In addition, the intermediate complex formed by ZBPs and B-DNA, which is modulated by varying conditions, determines the degree of B-Z transition.