Structure of the ArgRS–GlnRS–AIMP1 complex and its implications for mammalian translation

Fu, Yaoyao,Kim, Youngran,Jin, Kyeong Sik,Kim, Hyun Sook,Kim, Jong Hyun,Wang, DongMing,Park, Minyoung,Jo, Chang Hwa,Kwon, Nam Hoon,Kim, Doyeun,Kim, Myung Hee,Jeon, Young Ho,Hwang, Kwang Yeon,Kim, Sungh National Academy of Sciences 2014 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.111 No.42

<P><B>Significance</B></P><P>In higher eukaryotes, aminoacyl-tRNA synthetases (ARSs) are assembled to form a multisynthetase complex (MSC), which plays critical roles in translation and nontranslation functions essential for cell growth and survival of organisms. The MSC complex is comprised of nine different ARSs and three accessary proteins. The crystal structure of the arginyl-tRNA synthetase (ArgRS)–glutaminyl-tRNA synthase–aminoacyl tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1) subcomplex reveals that the N-terminal domains of ArgRS and AIMP1 form an extended coiled-coil structure, which provides a central depot for the assembly of a ternary complex. The stability of the N-terminal helix of ArgRS is critical for its ARS activity and noncanonical function of the subcomplex, explaining the significance of the MSC structure in translation and cellular functions.</P><P>In higher eukaryotes, one of the two arginyl-tRNA synthetases (ArgRSs) has evolved to have an extended N-terminal domain that plays a crucial role in protein synthesis and cell growth and in integration into the multisynthetase complex (MSC). Here, we report a crystal structure of the MSC subcomplex comprising ArgRS, glutaminyl-tRNA synthetase (GlnRS), and the auxiliary factor aminoacyl tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1)/p43. In this complex, the N-terminal domain of ArgRS forms a long coiled-coil structure with the N-terminal helix of AIMP1 and anchors the C-terminal core of GlnRS, thereby playing a central role in assembly of the three components. Mutation of AIMP1 destabilized the N-terminal helix of ArgRS and abrogated its catalytic activity. Mutation of the N-terminal helix of ArgRS liberated GlnRS, which is known to control cell death. This ternary complex was further anchored to AIMP2/p38 through interaction with AIMP1. These findings demonstrate the importance of interactions between the N-terminal domains of ArgRS and AIMP1 for the catalytic and noncatalytic activities of ArgRS and for the assembly of the higher-order MSC protein complex.</P>

Novel enzymatic elimination method for the chromatographic purification of ginsenoside Rb₃ in an isomeric mixture

Cui, Chang-Hao,Fu, Yaoyao,Jeon, Byeong-Min,Kim, Sun-Chang,Im, Wan-Taek The Korean Society of Ginseng 2020 Journal of Ginseng Research Vol.44 No.6

Background: The separation of isomeric compounds from a mixture is a recurring problem in chemistry and phytochemistry research. The purification of pharmacologically active ginsenoside Rb₃ from ginseng extracts is limited by the co-existence of its isomer Rb₂. The aim of the present study was to develop an enzymatic elimination-combined purification method to obtain pure Rb₃ from a mixture of isomers. Methods: To isolate Rb₃ from the isomeric mixture, a simple enzymatic selective elimination method was used. A ginsenoside-transforming glycoside hydrolase (Bgp2) was employed to selectively hydrolyze Rb₂ into ginsenoside Rd. Ginsenoside Rb₃ was then efficiently separated from the mixture using a traditional chromatographic method. Results: Chromatographic purification of Rb₃ was achieved using this novel enzymatic elimination-combined method, with 58.6-times higher yield and 13.1% less time than those of the traditional chromatographic method, with a lower minimum column length for purification. The novelty of this study was the use of a recombinant glycosidase for the selective elimination of the isomer. The isolated ginsenoside Rb₃ can be used in further pharmaceutical studies. Conclusions: Herein, we demonstrated a novel enzymatic elimination-combined purification method for the chromatographic purification of ginsenoside Rb₃. This method can also be applied to purify other isomeric glycoconjugates in mixtures.

Leucine zipper-mediated targeting of multi-enzyme cascade reactions to inclusion bodies in <i>Escherichia coli</i> for enhanced production of 1-butanol

Han, Gui Hwan,Seong, Wonjae,Fu, Yaoyao,Yoon, Paul K.,Kim, Seong Keun,Yeom, Soo-Jin,Lee, Dae-Hee,Lee, Seung-Goo Academic Press 2017 Metabolic engineering Vol.40 No.-

<P><B>Abstract</B></P> <P>Metabolons in nature have evolved to facilitate more efficient catalysis of multistep reactions through the co-localization of functionally related enzymes to cellular organelles or membrane structures. To mimic the natural metabolon architecture, we present a novel artificial metabolon that was created by targeting multi-enzyme cascade reactions onto inclusion body (IB) in <I>Escherichia coli</I>. The utility of this system was examined by co-localizing four heterologous enzymes of the 1-butanol pathway onto an IB that was formed in <I>E. coli</I> through overexpression of the cellulose binding domain (CBD) of <I>Cellulomonas fimi</I> exoglucanase. To target the 1-butanol pathway enzymes to the CBD IB, we utilized a peptide-peptide interaction between leucine zipper (LZ) peptides. We genetically fused the LZ peptide to the N-termini of four heterologous genes involved in the synthetic 1-butanol pathway, whereas an antiparallel LZ peptide was fused to the CBD gene. The <I>in vivo</I> activity of the CBD IB-based metabolon was examined through the determination of 1-butanol synthesis using <I>E. coli</I> transformed with two plasmids containing the LZ-fused CBD and LZ-fused 1-butanol pathway genes, respectively. <I>In vivo</I> synthesis of 1-butanol using the engineered <I>E. coli</I> yielded 1.98g/L of 1-butanol from glucose, representing a 1.5-fold increase over that obtained from <I>E. coli</I> expressing the LZ-fused 1-butanol pathway genes alone. In an attempt to examine the <I>in vitro</I> 1-butanol productivity, we reconstituted CBD IB-based metabolon using CBD IB and individual enzymes of 1-butanol pathway. The 1-butanol productivity of <I>in vitro</I> reconstituted CBD IB-based metabolon using acetoacetyl-CoA as the starting material was 2.29mg/L/h, 7.9-fold higher than that obtained from metabolon-free enzymes of 1-butanol pathway. Therefore, this novel CBD-based artificial metabolon may prove useful in metabolic engineering both <I>in vivo</I> and <I>in vitro</I> for the efficient production of desired products.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Enzyme cascades via heterologous metabolons facilitate host cell product production </LI> <LI> We used leucine zipper (LZ) tags to target metabolon reactions onto inclusion body </LI> <LI> Enhanced production of 1-butanol occurred both <I>in vitro</I> and <I>in vivo</I> </LI> <LI> Enzyme localization via LZ binding gave 7.9-fold increase in 1-butanol productivity </LI> <LI> This strategy may be useful in metabolic engineering for multiple target production </LI> </UL> </P>

Measurement-Based Stochastic Cross-Correlation Models of a Multilink Channel in Cooperative Communication Environments

박재준,김명돈,권헌국,정현규,Xuefeng Yin,Yaoyao Fu 한국전자통신연구원 2012 ETRI Journal Vol.34 No.6

In this paper, stochastic models for the cross-correlation of multiple channels are established based on measurement data collected using a wideband multiple-input multiple-output relay Band Exploration and Channel Sounder system at 3.7 GHz. We propose models for the cross-correlation characteristics of large-scale parameters (LSPs) between two links, that is, the base station and mobile station (MS) link and the relay station and MS link. The LSPs include shadow fading, Rician K-factor, delay spread, angle spread of arrival, and angle spread of departure. Furthermore, models are established for the cross-correlation of the small-scale fading in the impulse responses of two links. The statistics of these model parameters are investigated as functions of geometrical features of the multilink. They are extracted from a large amount of cross-correlation observations, which are obtained in three measurement sites along more than one hundred measurement routes. These models can be used together with the standard single-link channel models for the generation of correlated components, for example, path clusters, in two separate channels.

Crystal structures of the structure-selective nuclease Mus81-Eme1 bound to flap DNA substrates.

Gwon, Gwang Hyeon,Jo, Aera,Baek, Kyuwon,Jin, Kyeong Sik,Fu, Yaoyao,Lee, Jong-Bong,Kim, Youngchang,Cho, Yunje Published for the European Molecular Biology Organ 2014 The EMBO journal Vol.33 No.9

<P>The Mus81-Eme1 complex is a structure-selective endonuclease with a critical role in the resolution of recombination intermediates during DNA repair after interstrand cross-links, replication fork collapse, or double-strand breaks. To explain the molecular basis of 3' flap substrate recognition and cleavage mechanism by Mus81-Eme1, we determined crystal structures of human Mus81-Eme1 bound to various flap DNA substrates. Mus81-Eme1 undergoes gross substrate-induced conformational changes that reveal two key features: (i) a hydrophobic wedge of Mus81 that separates pre- and post-nick duplex DNA and (ii) a '5' end binding pocket' that hosts the 5' nicked end of post-nick DNA. These features are crucial for comprehensive protein-DNA interaction, sharp bending of the 3' flap DNA substrate, and incision strand placement at the active site. While Mus81-Eme1 unexpectedly shares several common features with members of the 5' flap nuclease family, the combined structural, biochemical, and biophysical analyses explain why Mus81-Eme1 preferentially cleaves 3' flap DNA substrates with 5' nicked ends.</P>

Crystal structure of LRG1 and the functional significance of LRG1 glycan for LPHN2 activation

Yang Jimin,Yin Guo Nan,Kim Do-Kyun,Han Ah-reum,Lee Dong Sun,Min Kwang Wook,Fu Yaoyao,Yun Jeongwon,Suh Jun-Kyu,Ryu Ji-Kan,Kim Ho Min 생화학분자생물학회 2023 Experimental and molecular medicine Vol.55 No.-

The serum glycoprotein leucine-rich ɑ-2-glycoprotein 1 (LRG1), primarily produced by hepatocytes and neutrophils, is a multifunctional protein that modulates various signaling cascades, mainly TGFβ signaling. Serum LRG1 and neutrophil-derived LRG1 have different molecular weights due to differences in glycosylation, but the impact of the differential glycan composition in LRG1 on its cellular function is largely unknown. We previously reported that LRG1 can promote both angiogenic and neurotrophic processes under hyperglycemic conditions by interacting with LPHN2. Here, we determined the crystal structure of LRG1, identifying the horseshoe-like solenoid structure of LRG1 and its four N-glycosylation sites. In addition, our biochemical and cell-biological analyses found that the deglycosylation of LRG1, particularly the removal of glycans on N325, is critical for the high-affinity binding of LRG1 to LPHN2 and thus promotes LRG1/LPHN2-mediated angiogenic and neurotrophic processes in mouse tissue explants, even under normal glucose conditions. Moreover, the intracavernous administration of deglycosylated LRG1 in a diabetic mouse model ameliorated vascular and neurological abnormalities and restored erectile function. Collectively, these data indicate a novel role of LRG1 glycans as molecular switches that can tune the range of LRG1’s cellular functions, particularly the LRG1/LPHN2 signaling axis.