Junsang Ko

Crystal structures of human DJ-1 and Escherichia coli Hsp31, which share an evolutionarily conserved domain.

Human DJ-1 and Escherichia coli Hsp31 belong to ThiJ/PfpI family, whose members contain a conserved domain. DJ-1 is associated with autosomal recessive early onset parkinsonism and Hsp31 is a molecular chaperone. Structural comparisons between DJ-1, Hsp31, and an Archaea protease, a member of ThiJ/PfpI family, lead to the identification of the chaperone activity of DJ-1 and the proteolytic activity of Hsp31. Moreover, the comparisons provide insights into how the functional diversity is realized in proteins that share an evolutionarily conserved domain. On the basis of the chaperone activity the possible role of DJ-1 in the pathogenesis of Parkinsons disease is discussed.

Conversion of Methylglyoxal to Acetol by Escherichia coli Aldo-Keto Reductases

Methylglyoxal (MG) is a toxic metabolite known to accumulate in various cell types. We detected in vivo conversion of MG to acetol in MG-accumulating Escherichia coli cells by use of (1)H nuclear magnetic resonance ((1)H-NMR) spectroscopy. A search for homologs of the mammalian aldo-keto reductases (AKRs), which are known to exhibit activity to MG, revealed nine open reading frames from the E. coli genome. Based on both sequence similarities and preliminary characterization with (1)H-NMR for crude extracts of the corresponding mutant strains, we chose five genes, yafB, yqhE, yeaE, yghZ, and yajO, for further study. Quantitative assessment of the metabolites produced in vitro from the crude extracts of these mutants and biochemical study with purified AKRs indicated that the yafB, yqhE, yeaE, and yghZ genes are involved in the conversion of MG to acetol in the presence of NADPH. When we assessed their in vivo catalytic activities by creating double mutants, all of these genes except for yqhE exhibited further sensitivities to MG in a glyoxalase-deficient strain. The results imply that the glutathione-independent detoxification of MG can occur through multiple pathways, consisting of yafB, yqhE, yeaE, and yghZ genes, leading to the generation of acetol.

Structure of PP4397 Reveals the Molecular Basis for Different c-di-GMP Binding Modes by Pilz Domain Proteins.

Cyclic diguanylate (c-di-GMP) is a global regulator that modulates pathogen virulence and biofilm formation in bacteria. Although a bioinformatic study revealed that PilZ domain proteins are the long-sought c-di-GMP binding proteins, the mechanism by which c-di-GMP regulates them is uncertain. Pseudomonas putida PP4397 is one such protein that contains YcgR-N and PilZ domains and the apo-PP4397 structure was solved earlier by the Joint Center for Structural Genomics. We determined the crystal structure of holo-PP4397 and found that two intercalated c-di-GMPs fit into the junction of its YcgR-N and PilZ domains. Moreover, c-di-GMP binding induces PP4397 to undergo a dimer-to-monomer transition. Interestingly, another PilZ domain protein, VCA0042, binds to a single molecule of c-di-GMP, and both its apo and holo forms are dimeric. Mutational studies and the additional crystal structure of holo-VCA0042 (L135R) showed that the Arg122 residue of PP4397 is crucial for the recognition of two molecules of c-di-GMP. Thus, PilZ domain proteins exhibit different c-di-GMP binding stoichiometry and quaternary structure, and these differences are expected to play a role in generating diverse forms of c-di-GMP-mediated regulation.

Structural Studies of Potassium Transport Protein KtrA Regulator of Conductance of K+ (RCK) C domain in Complex with Cyclic Diadenosine Monophosphate (c-di-AMP)

Background: Cyclic di-AMP inactivates the potassium transport activity of KtrA. Results: Cyclic di-AMP binding to KrtA induced conformational changes. Conclusion: Cyclic di-AMP selectively binds to the KtrA RCK_C domain and signals the inactivation of potassium transport. Significance: The molecular basis for the role of cyclic di-AMP in potassium channel activity was investigated. Although it was only recently identified as a second messenger, c-di-AMP was found to have fundamental importance in numerous bacterial functions such as ion transport. The potassium transporter protein, KtrA, was identified as a c-di-AMP receptor. However, the co-crystallization of c-di-AMP with the protein has not been studied. Here, we determined the crystal structure of the KtrA RCK_C domain in complex with c-di-AMP. The c-di-AMP nucleotide, which adopts a U-shaped conformation, is bound at the dimer interface of RCK_C close to helices α3 and α4. c-di-AMP interacts with KtrA RCK_C mainly by forming hydrogen bonds and hydrophobic interactions. c-di-AMP binding induces the contraction of the dimer, bringing the two monomers of KtrA RCK_C into close proximity. The KtrA RCK_C was able to interact with only c-di-AMP, but not with c-di-GMP, 3′,3-cGAMP, ATP, and ADP. The structure of the KtrA RCK_C domain and c-di-AMP complex would expand our understanding about the mechanism of inactivation in Ktr transporters governed by c-di-AMP.

Structural characterization reveals that a PilZ domain protein undergoes substantial conformational change upon binding to cyclic dimeric guanosine monophosphate

PA4608 is a single PilZ domain protein from Pseudomonas aeruginosa that binds to cyclic dimeric guanosine monophosphate (c‐di‐GMP). Although the monomeric structure of unbound PA4608 has been studied in detail, the molecular details of c‐di‐GMP binding to this protein are still uncharacterized. Hence, we determined the solution structure of c‐di‐GMP bound PA4608. We found that PA4608 undergoes conformational changes to expose the c‐di‐GMP binding site by ejection of the C‐terminal 310 helix. A dislocation of the C‐terminal tail in the presence of c‐di‐GMP implies that this region acts as a lid that alternately covers and exposes the hydrophobic surface of the binding site. In addition, mutagenesis and NOE data for PA4608 revealed that conserved residues are in contact with the c‐di‐GMP molecule. The unique structural characteristics of PA4608, including its monomeric state and its ligand binding characteristics, yield insight into its function as a c‐di‐GMP receptor.

Insights into the regulation of human Rev1 for translesion synthesis polymerases revealed by the structural studies on its polymerase-interacting domain

Dinan Liu, Kyoung-Seok Ryu, Junsang Ko, Dawei Sun, Kyungeun Lim, Jie-Oh Lee, Jung me Hwang, Zee-won Lee, and Byong-Seok Choi Department of Chemistry, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejon 305-701, Republic of Korea Division of Magnetic Resonance, Korea Basic Science Institute Ochang Campus, CheongwonGun, Ochang-Eup, Yangcheong-Ri 804-1, Chungcheongbuk-Do 363-883, Republic of Korea Department of Bio-Analytical Science, University of Science and Technology, Daejeon 305-333, Republic of Korea Division of Life Science, Korea Basic Science Institute, Daejeon 305-333, Republic of Korea

Aminoglycoside antibiotics bind to the influenza A virus RNA promoter

Aminoglycosides bind to the influenza A virus promoter (vRNA) at submicromolar concentration. The complex structure between the vRNA and neomycin illustrates that binding of neomycin causes a conformational change which would affect further transcription processes. Thus, aminoglycosides represent lead compounds for the discovery of antiviral therapeutics against influenza A virus.

Effect of divalent cations on the ATPase activity of Escherichia coli SecA

It was found that Ca2+ stimulates the intrinsic SecA ATPase activity in the absence as well as in the presence of liposome. On the other hand, Mg2+, the general cofactor for ATPase, did not affect the intrinsic SecA ATPase but reduced the portion of ATPase activity enhanced by Ca2+. The enhancement of SecA ATPase activity correlated well with the increase in 8‐anilino‐1‐naphthalene‐sulfonic acid binding of SecA, suggesting that increased exposure of hydrophobic residues stimulates the enzyme activity.

Solution structure of the RecQ C-terminal domain of human Bloom syndrome protein

RecQ C-terminal (RQC) domain is known as the main DNA binding module of RecQ helicases such as Bloom syndrome protein (BLM) and Werner syndrome protein (WRN) that recognizes various DNA structures. Even though BLM is able to resolve various DNA structures similarly to WRN, BLM has different binding preferences for DNA substrates from WRN. In this study, we determined the solution structure of the RQC domain of human BLM. The structure shares the common winged-helix motif with other RQC domains. However, half of the N-terminal has unstructured regions (α1–α2 loop and α3 region), and the aromatic side chain on the top of the β-hairpin, which is important for DNA duplex strand separation in other RQC domains, is substituted with a negatively charged residue (D1165) followed by the polar residue (Q1166). The structurally distinctive features of the RQC domain of human BLM suggest that the DNA binding modes of the BLM RQC domain may be different from those of other RQC domains.

Biophysical characterization of sites of host adaptive mutation in the influenza A virus RNA polymerase PB2 RNA-binding domain

Influenza RNA polymerase is composed of three subunits, PA, PB1, and PB2, which interact with each other for transcription and replication of the viral RNA genome in the nucleus of infected cells. PB2 RNA-binding 627-domain (residues 535-693), located in the C-terminus, presents a highly basic surface around residue lysine 627 and has been proposed to interact with viral or cellular factors, resulting in host adaptation. However, the function of this domain is not yet characterized in detail. In this study, we identified RNA-binding activity and RNA-binding surfaces in both the N-terminal and basic C-terminal regions of PB2 627-domain using NMR experiments. Through mutagenesis studies, we confirmed which residues directly interact with RNA and mapped their locations on the RNA-binding surface. In addition, by luciferase activity assays, we showed that influenza virus polymerase activity may correlate with the interaction between PB2 and RNA. Representative host adaptive mutations (residues 591 and 627) were found to be located on the RNA-binding surface and were confirmed to directly interact with RNA and to affect polymerase activity. From these results, we suggest that influenza virus polymerase activity may be regulated through the interaction between PB2 627-domain and RNA and that consequently host adaptation of the virus may be influenced.