Sung Chul Ha

Crystal structure of a junction between B-DNA and Z-DNA reveals two extruded bases

Left-handed Z-DNA is a higher-energy form of the double helix, stabilized by negative supercoiling generated by transcription or unwrapping nucleosomes. Regions near the transcription start site frequently contain sequence motifs favourable for forming Z-DNA, and formation of Z-DNA near the promoter region stimulates transcription. Z-DNA is also stabilized by specific protein binding; several proteins have been identified with low nanomolar binding constants. Z-DNA occurs in a dynamic state, forming as a result of physiological processes then relaxing to the right-handed B-DNA. Each time a DNA segment turns into Z-DNA, two B–Z junctions form. These have been examined extensively, but their structure was unknown. Here we describe the structure of a B–Z junction as revealed by X-ray crystallography at 2.6 Å resolution. A 15-base-pair segment of DNA is stabilized at one end in the Z conformation by Z-DNA binding proteins, while the other end remains B-DNA. Continuous stacking of bases between B-DNA and Z-DNA segments is found, with the breaking of one base pair at the junction and extrusion of the bases on each side (Fig. 1). These extruded bases may be sites for DNA modification.

Crystal structure of osmotin, a plant antifungal protein

However,the precise mechanism by which osmotin interacts withspecific fungal pathogens and mediates plasma membranepermeability has not been clearly elucidated. Recent ge-netic and biochemical data indicate that osmotin utilizes asignal transduction pathway in yeast to increase thesusceptibility of a target fungus to its cytotoxic effects.

The crystal structure of the second Z-DNA binding domain of human DAI (ZBP1) in complex with Z-DNA reveals an unusual binding mode to Z-DNA.

Mammalian DAI (DNA-dependent activator of IFN-regulatory factors), an activator of the innate immune response, senses cytosolic DNA by using 2 N-terminal Z-DNA binding domains (ZBDs) and a third putative DNA binding domain located next to the second ZBD. Compared with other previously known ZBDs, the second ZBD of human DAI (hZβDAI) shows significant variation in the sequence of the residues that are essential for DNA binding. In this article, the crystal structure of the hZβDAI/Z-DNA complex reveals that hZβDAI has a similar fold to that of other ZBDs, but adopts an unusual binding mode for recognition of Z-DNA. A residue in the first β-strand rather than residues in the β-loop contributes to DNA binding, and part of the (α3) recognition helix adopts a 310 helix conformation. The role of each residue that makes contact with DNA was confirmed by mutational analysis. The 2 ZBDs of DAI can together bind to DNA and both are necessary for full B-to-Z conversion. It is possible that binding 2 DAIs to 1 dsDNA brings about dimerization of DAI that might facilitate DNA-mediated innate immune activation.

Activation mechanism of HSP16.5 from Methanococcus jannaschii.

The small heat shock proteins are the ubiquitous proteins found in a wide range of organisms and function as molecular chaperones by binding to the folding intermediates of their substrates. Although the crystal structure of HSP16.5, a small heat shock protein from Methanococcus jannaschii, revealed that it is a hollow sphere composed of 24 identical subunits, its activation mechanism remains unclear. We found out that HSP16.5 is active only at high temperatures and forms a stable complex with substrate in a stoichiometric manner. We also observed that the conformational change of HSP16.5 is correlated with the increasing hydrophobic site and its activation as a molecular chaperone. However, it is revealed that the conformational change is not accompanied with the change of the secondary structure of a subunit, but correlated with the increasing diameter of HSP16.5. Therefore, it is proposed that the activation mechanism of HSP16.5 involves temperature induced conformational change with size increment of the complex resulting in the exposure of hydrophobic substrate-binding site.

Structural basis for the reaction mechanism of UDP-glucose pyrophosphorylase

UDP-glucose pyrophosphorylases (UGPase; EC 2.7.7.9) catalyze the conversion of UTP and glucose-1-phosphate to UDP-glucose and pyrophosphate and vice versa. Prokaryotic UGPases are distinct from their eukaryotic counterparts and are considered appropriate targets for the development of novel antibacterial agents since their product, UDP-glucose, is indispensable for the biosynthesis of virulence factors such as lipopolysaccharides and capsular polysaccharides. In this study, the crystal structures of UGPase from Helicobacter pylori (HpUGPase) were determined in apo- and UDP-glucose/Mg2+-bound forms at 2.9 Å and 2.3 Å resolutions, respectively. HpUGPase is a homotetramer and its active site is located in a deep pocket of each subunit. Magnesium ion is coordinated by Asp130, two oxygen atoms of phosphoryl groups, and three water molecules with octahedral geometry. Isothermal titration calorimetry analyses demonstrated that Mg2+ ion plays a key role in the enzymatic activity of UGPase by enhancing the binding of UGPase to UTP or UDP-glucose, suggesting that this reaction is catalyzed by an ordered sequential Bi Bi mechanism. Furthermore, the crystal structure explains the specificity for uracil bases. The current structural study combined with functional analyses provides essential information for understanding the reaction mechanism of bacterial UGPases, as well as a platform for the development of novel antibacterial agents.

Distinct Z-DNA binding mode of a PKR-like protein kinase containing a Z-DNA binding domain (PKZ)

Double-stranded ribonucleic acid-activated protein kinase (PKR) downregulates translation as a defense mechanism against viral infection. In fish species, PKZ, a PKR-like protein kinase containing left-handed deoxyribonucleic acid (Z-DNA) binding domains, performs a similar role in the antiviral response. To understand the role of PKZ in Z-DNA recognition and innate immune response, we performed structural and functional studies of the Z-DNA binding domain (Zα) of PKZ from Carassius auratus (caZαPKZ). The 1.7-Å resolution crystal structure of caZαPKZ:Z-DNA revealed that caZαPKZ shares the overall fold with other Zα, but has discrete structural features that differentiate its DNA binding mode from others. Functional analyses of caZαPKZ and its mutants revealed that caZαPKZ mediates the fastest B-to-Z transition of DNA among Zα, and the minimal interaction for Z-DNA recognition is mediated by three backbone phosphates and six residues of caZαPKZ. Structure-based mutagenesis and B-to-Z transition assays confirmed that Lys56 located in the β-wing contributes to its fast B-to-Z transition kinetics. Investigation of the DNA binding kinetics of caZαPKZ further revealed that the B-to-Z transition rate is positively correlated with the association rate constant. Taking these results together, we conclude that the positive charge in the β-wing largely affects fast B-to-Z transition activity by enhancing the DNA binding rate.

Crystallization and preliminary X-ray crystallographic study of UDP-glucose pyrophosphorylase (UGPase) from Helicobacter pylori

UDP-glucose pyrophosphorylase (UGPase) catalyzes the synthesis of UDP-glucose, an essential metabolite in all living organisms. An X-ray crystallographic study of UGPase from Helicobacter pylori has been performed in order to elucidate its role in the regulation of this important metabolic pathway. UGPase was crystallized from 0.1 M sodium acetate trihydrate pH 4.6, 2.0 M ammonium sulfate and 0.1 M guanidine-HCl. According to diffraction data collected at a resolution of 2.9 A using a synchrotron-radiation source, the crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 91.47, b = 98.61, c = 245.70 A, alpha = beta = gamma = 90.0 degrees.

Structural and kinetic bases for the metal preference of the M18 aminopeptidase from Pseudomonas aeruginosa

The peptidases in clan MH are known as cocatalytic zinc peptidases that have two zinc ions in the active site, but their metal preference has not been rigorously investigated. In this study, the molecular basis for metal preference is provided from the structural and biochemical analyses. Kinetic studies of Pseudomonas aeruginosa aspartyl aminopeptidase (PaAP) which belongs to peptidase family M18 in clan MH revealed that its peptidase activity is dependent on Co(2+) rather than Zn(2+): the kcat (s(-1)) values of PaAP were 0.006, 5.10 and 0.43 in no-metal, Co(2+), and Zn(2+)conditions, respectively. Consistently, addition of low concentrations of Co(2+) to PaAP previously saturated with Zn(2+) greatly enhanced the enzymatic activity, suggesting that Co(2+)may be the physiologically relevant cocatalytic metal ion of PaAP. The crystal structures of PaAP complexes with Co(2+) or Zn(2+) commonly showed two metal ions in the active site coordinated with three conserved residues and a bicarbonate ion in a tetragonal geometry. However, Co(2+)- and Zn(2+)-bound structures showed no noticeable alterations relevant to differential effects of metal species, except the relative orientation of Glu-265, a general base in the active site. The characterization of mutant PaAP revealed that the first metal binding site is primarily responsible for metal preference. Similar to PaAP, Streptococcus pneumonia glutamyl aminopeptidase (SpGP), belonging to aminopeptidase family M42 in clan MH, also showed requirement for Co(2+) for maximum activity. These results proposed that clan MH peptidases might be a cocatalytic cobalt peptidase rather than a zinc-dependent peptidase.

Cloning, purification, crystallization and preliminary X-ray studies of RFC boxes II-VIII of replication factor C from Methanococcus jannaschii.

Replication factor C (RFC) is the accessory protein required to load the proliferating cell nuclear antigen (PCNA) onto DNA in replication process. RFC is composed of several subunits and each subunit contains the highly conserved sequences RFC boxes II-VIII. RFC boxes II-VIII of the large subunit of replication factor C from Methanococcus jannaschii has been overexpressed in Escherichia coli, purified and crystallized at 295 K using ammonium sulfate as precipitant. Crystals belong to the space group R32, with unit-cell parameters a = b = 238.23 (5), c = 73.17 (12) A. Native data were collected at 100 K to a resolution of 3.2 A using a synchrotron-radiation source.

Crystallization and Preliminary X-Ray Studies of TON_0559, a Putative Member of the Haloacid Dehalogenase (HAD) Superfamily from Thermococcus onnurineus NA1

To elucidate the molecular basis underlying their broad substrate specificity and reaction mechanism of the enzymes belonging to the haloacid dehalogenase (HAD) superfamily, TON_0559, a putative HAD subfamily protein from a hyperthermophilic archaeon Thermococcus onnurineus NA1, was expressed, purified and crystallized. X-ray diffraction data were collected to 2.0 A resolution. The space group is C2, with unit cell parameters a = 121.2 A, b = 62.9 A, c = 37.5 A and beta= 106.5 degrees .