Eunmi Hong

Chemical structure and biological activity of the Caenorhabditis elegans dauer-inducing pheromone

Pheromones are cell type-specific signals used for communication between individuals of the same species. When faced with overcrowding or starvation, Caenorhabditis elegans secrete the pheromone daumone, which facilitates communication between individuals for adaptation to adverse environmental stimuli. Daumone signals C. elegans to enter the dauer stage, an enduring and non-ageing stage of the nematode life cycle with distinctive adaptive features and extended life. Because daumone is a key regulator of chemosensory processes in development and ageing, the chemical identification of daumone is important for elucidating features of the daumone-mediated signalling pathway. Here we report the isolation of natural daumone from C. elegans by large-scale purification, as well as the total chemical synthesis of daumone. We present the stereospecific chemical structure of purified daumone, a fatty acid derivative. We demonstrate that both natural and chemically synthesized daumones equally induce dauer larva formation in C. elegans (N2 strain) and certain dauer mutants, and also result in competition between food and daumone. These results should help to elucidate the daumone-mediated signalling pathway, which might in turn influence ageing and obesity research and the development of antinematodal drugs.

Structure of an Atypical Orphan Response Regulator Protein Supports a New Phosphorylation-independent Regulatory Mechanism

Two-component signal transduction systems, commonly found in prokaryotes, typically regulate cellular functions in response to environmental conditions through a phosphorylation-dependent process. A new type of response regulator, hp1043 (HP-RR) from Helicobacter pylori, has been recently identified. HP-RR is essential for cell growth and does not require the well known phosphorelay scheme. Unphosphorylated HP-RR binds specifically to its own promoter (P1043) and autoregulates the promoter of the tlpB gene (PtlpB). We have determined the structure of HP-RR by NMR and x-ray crystallography, revealing a symmetrical dimer with two functional domains. The molecular topology resembles that of the OmpR/PhoB subfamily, however, the symmetrical dimer is stable even in the unphosphorylated state. The dimer interface, formed by three secondary structure elements (α4-β5-α5), resembles that of the active, phosphorylated forms of ArcA and PhoB. Several conserved residues of the HP-RR dimeric interface deviate from the OmpR/PhoB subfamily, although there are similar salt bridges and hydrophobic patches within the interface. Our findings reveal how a new type of response regulator protein could function as a cell growth-associated regulator in the absence of post-translational modification.

Backbone dynamics of an atypical orphan response regulator protein, Helicobacter pylori 1043

An atypical orphan response regulator protein, HP1043 (HP-RR) in Helicobacter pylori, is proven to be essential for cell growth and does not require the well known phosphorelay scheme. HP-RR was identified as a symmetric dimer with two functional domains, an N-terminal regulatory domain (HP-RRr) and a C-terminal effector domain (HP-RRe). HP-RR is a new class of response regulator, as a phosphorylation-independent regulator. Previously, we have presented a detailed three-dimensional structure of HP-RR using NMR spectroscopy and X-ray crystallography. In this study, in order to understand the functional importance of flexibilities in HP-RRr and HP-RRe, T1, T2, heteronuclear NOE experiments have been performed and backbone dynamics of HP-RRr and HP-RRe were investigated. HP-RRr is a symmetric dimer and the interface region, α4-β5-α5 of dimer, showed high rigidity (high S2 values). Site of rearrangements associated with phosphorylation of HP-RRr (Ser75: Rex = 3.382, Ile95: Rex = 5.228) showed slow chemical exchanges. HP-RRe is composed of three α-helices flanked on two sides by anti-parallel β-sheets. Low order parameters as well as conformational exchanges in the centers of loop regions known as the DNA binding site and transcription site of HP-RRe suggested that flexibility of HP-RRe is essential for interaction with DNA. In conclusion, backbone dynamics information for HP-RR implies that structural flexibilities in HP-RRr are necessary for the phosphorylation site and the dynamic nature of HP-RRe is essential for the regulation of interaction between protein and DNA.

NMR structure of the conserved novel-fold protein TA0743 from Thermoplasma acidophilum

Introduction. Thermoplasma acidophilum has one of the smallest known archaeal genomes. This organism is a thermo-acidophilic archaeon that is surrounded by only a plasma membrane. TA0743 is a 122-residue conserved hypothetical protein found in Thermoplasma acidophilum. Because neither functional nor structural information is available for this protein, the solution structure of TA0743 has been determined by NMR spectroscopy. Based on the solution structure and the amino acid sequence analysis, we propose that TA0743 is an uncharacterized protein with a novel structural fold. Therefore, our structure may serve as a starting point to study the structure– function of proteins with a similar fold in various species.

Solution structure of TA0895, a MoaD homologue from Thermoplasma acidophilum

Jinwon Jung, In Young Yeo, Eunmi Hong, Adelinda Yee, Cheryl H. Arrowsmith, and Weontae Lee* Department of Biochemistry, College of Science and National Research Laboratory, Yonsei University, Seoul 120-749, Korea Institute of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea Division of Molecular and Structural Biology, Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada M5G 2M9

Biophysical characterization of the basic cluster in the transcription repression domain of human MeCP2 with AT-rich DNA

MeCP2 is a chromatin associated protein which is highly expressed in brain and relevant with Rett syndrome (RTT). There are AT-hook motifs in MeCP2 which can bind with AT-rich DNA, suggesting a role in chromatin binding. Here, we report the identification and characterization of another AT-rich DNA binding motif (residues 295 to 313) from the C-terminal transcription repression domain of MeCP2 by nuclear magnetic resonance (NMR) and isothermal calorimetry (ITC). This motif shows a micromolar affinity to AT-rich DNA, and it binds to the minor groove of DNA like AT-hook motifs. Together with the previous studies, our results provide an insight into a critical role of this motif in chromatin structure and function.

Cost-effective isotope labeling technique developed for 15 N/ 13 C-labeled proteins

A newly developed cost-effective approach to prepare 15 N/ 13 C- labeled protein for NMR studies is presented. This method has been successfully applied to isotopically labeling of PTK6 SH2 domain and MTH 1880 protein. The production method generates cell density using a growing media containing 15 NH4Cl, 12 C6-D-glucose. Following a doubling time period for unlabeled metabolite exhaustion and then addition 13 C6-D- glucose into a M9 growing media, the cells are induced. Our results demonstrate that in order to get full incorporation of 13 C, the isotopes are not totally required during the initial growth phase before induction. The addition of small amounts of 13 C6-D-glucose to the induction phase is sufficient to obtain more than 95% incorporation of isotopes into the protein. Our optimized protocol is two-thirds less costly than the classical method using 13 C isotope during the entire growth phase.