Biology subjects

Lee, B.-Y.

Publications and source records attributed to Lee, B.-Y..

5 recordsLinked to original sources

bioRxiv2025

Discovery and CryoEM Structure of FPM13, a Periplasmic Metalloprotein Unique to Francisella

We report the cryoEM structure of the Francisella protein FTN_1118, identifying it as a novel 13 kDa periplasmic protein unique to the Francisella genus, which we now designate FPM13 (Francisella Periplasmic Metalloprotein, 13 kDa) based on its structural and biochemical properties. FPM13 was serendipitously identified during purification of Francisella type VI secretion system (T6SS) effector proteins, co-purifying with them. Its identity, initially unknown, was established using the novel cryoID method. The structure reveals a symmetrical, donut-shaped 18-mer with 9-fold dihedral symmetry, formed by two stacked nonamers head-to-head. It measures [~]8 nm both in height and in outer diameter, and has a 3.5 nm central channel. The complex features a double-layered wall with an inner {beta}-sheet core and an outer -helical shell. Each monomer adopts a compact fold comprising an N-terminus {beta}-strand, an -helix and two additional {beta} strands at the C-terminus. The assembly is stabilized by inter-ring loop interactions and hydrophobic and electrostatic contacts between neighboring subunits. Biochemical analyses, as shown by APEX-biotinylation and Triton X-114 phase partitioning, confirmed that FPM13 is a soluble periplasmic protein. ICP-MS demonstrated that FPM13 binds iron and copper. Deletion of FPM13 in Francisella novicida strains caused no growth defects in macrophages or mice but show increased copper sensitivity under iron-depleted conditions, suggesting a role for FPM13 in metal transport or detoxification.

microbiology↗

bioRxiv2025

Atomic Structure and Phospholipid Binding Properties of the Francisella Type VI Secretion System Effector Protein PdpC

Francisella tularensis, a gram-negative facultative intracellular pathogen, causes tularemia, a potentially fatal infection. The Francisella pathogenicity island (FPI) encodes a type VI secretion system (T6SS), critical for the bacteriums ability to replicate within host cells and cause disease. PdpC, an FPI-encoded T6SS effector protein, is essential for phagosomal escape, intracellular replication, and virulence in animals, yet its structure and function remain poorly understood. Here, we expressed PdpC recombinantly and determined the cryoEM structure of PdpC at 3.4 [A] resolution, revealing a monomeric 156 kDa protein with a seahorse-shaped architecture (80 [A] x 75 [A] x 120 [A]). PdpC comprises five domains: an N-terminal domain (NTD) forming a prominent lateral head lobe, a central body domain, a C-terminal tail domain, a small wedge domain at the groove between the body and tail, and an unmodeled "mouth" domain that completes the seahorse-like shape. Functional studies demonstrate that PdpC binds specific host cell phospholipids, including phosphatidylinositol-3-phosphate. These findings provide a foundation for elucidating PdpCs mechanism of action and developing strategies to prevent or treat tularemia.

microbiology↗

bioRxiv2025

Structure, identification and characterization of the RibD-enolase complex in Francisella

Francisella tularensis is a highly infectious bacterium, a Tier 1-select agent, and the causative agent of tularemia, a potentially fatal zoonotic disease. In this study originally aiming to identify anti-tularemia drug targets, we serendipitously determined the atomic structures and identified their components of the native RibD-enolase protein complex in Francisella novicida; and subsequently systematically characterized the catalytic functions of the RibD-enolase complex. Originally discovered as individually protein in Escherichia coli and yeast, respectively, RibD and enolase are two essential enzymes involved in distinct metabolic pathways, both of which could serve as potential therapeutic targets for tularemia treatment and prevention. Our biochemical validation using pull-down assays confirmed the formation of this complex in vivo, revealing that all eluted RibD is bound to enolase, while the majority of enolase remained uncomplexed. Structural analysis reveals unique features of the Francisella complex, including key RibD-enolase interactions that mediate complex assembly and {beta}-strand swapping between RibD subunits. Furthermore, molecular dynamics simulations of the ligand-bound RibD-enolase complex highlight localized conformational changes within the substrate-binding sites and suggest a gating mechanism between RibDs substrate and cofactor-binding sites to ensure efficient uptake and turnover. Despite the physical association between RibD and enolase, enzymatic assays indicated their catalytic activities are independent of each other, thus the complex may have alternative functional roles that warrant further exploration. Our study provides the first structural and biochemical characterization of the RibD-enolase complex, establishing a foundation for further investigations into its functional significance in Francisella and potential antibacterial development. SignificanceFrancisella tularensis, a dangerous bacterium classified as a high-priority bioterrorism threat, causes tularemia, a severe and sometimes deadly disease spread from animals to humans. In a study originally aimed at finding new treatments, we accidentally uncovered a unique partnership between two enzymatic proteins--RibD and enolase--in Francisella. These proteins, previously known to work alone in other non-pathologic organisms, were found to form a heterodimer of RibD and enolase dimers in Francisella. Biochemical experiments confirmed that enolase works both alone and together with RibD in living cells. In addition to the novel heterodimer-of-homodimer configuration, the RibD subunits feature swapped {beta} strands for dimerization as compared to E. coli RibD homodimer. The results offer clues for designing future antibiotics against tularemia and beyond.

microbiology↗

bioRxiv2019

Two-way migration of Lychnis wilfordii caused by the circular landform of Japan-Korea-northeast China-Russian Far East region and its suggestion for conservation in northeast Asia

In northeast Asia, substantial portion of the floras, including endangered species, are shared among its component countries in the continental, peninsula, and island parts largely through Quaternary migration. To effectively conserve nationally endangered plants in Northeast Asia, transnational conservation studies are vitally needed. Lychnis wilfordii (Caryophyllaceae) has disjunct distribution in Russian Far East (Primorsky Krai), northeast China (Jilin), Korea (Gangwon-do) and Japan (Hokkaido, Aomori, Nagano), surrounding the sea, and this is designated as an endangered species in Japan and Korea. Population genetic and molecular dating analyses were conducted 1) to elucidate geographic genetic structure covering the species range, 2) to test possible scenarios of migration, and 3) to develop logical plans for effective conservation. Population genetic analyses indicated the continent and peninsula parts (north and south Primorsky Krai, Jilin, and Gangwon-do) had higher genetic diversity compared to those in the Japanese Archipelago (Hokkaido and Nagano). Five genetically distinct groups were recognized, namely, Nagano, Gangwon-do, Jilin, north and south Primorsky Krai plus Aomori, and Hokkaido. Genetic distance between Hokkaido and Nagano was larger than between Hokkaido and north Primorsky Krai, and between Nagano and Gangwon-do, crossing national borders and the natural barrier of the sea. Considering these results, L. wilfordii likely migrated from the Asian continent to the Japanese Archipelago using two routes: north route from Russian Far East to Hokkaido and Aomori, and south route from the Korean Peninsula to Nagano. Based on molecular dating, migration from the continent to the islands likely occurred from the middle Pleistocene to the Holocene. For effective conservation of L. wilfordii, Hokkaido and Nagano populations should be distinguished as different evolutionary significant units, although these two regions belong to the same country, because Hokkaido and Nagano populations are at the different ends of the two migratory routes based on the migration scenario.

evolutionary biology↗

bioRxiv2019

Construction of High-Resolution RAD-Seq Based Linkage Map, Anchoring Reference Genome, and QTL Mapping of the Sex Chromosome in the Marine Medaka Oryzias melastigma

Medaka (Oryzias spp.) is an important fish species in ecotoxicology and considered as a model species due to its biological features including small body size and short generation time. Since Japanese medaka Oryzias latipes is a freshwater species with access to an excellent genome resources, the marine medaka Oryzias melastigma is also applicable for marine ecotoxicology. In genome era, a high-density genetic linkage map is a very useful resource in genomic research, providing a means for comparative genomic analysis and verification of de novo genome assembly. In this study, we developed a high-density genetic linkage map for O. melastigma using restriction-site associated DNA sequencing (RAD-seq). The genetic map consisted of 24 linkage groups with 2,481 RAD-tag markers. The total map length was 1,784 cM with an average marker space of 0.72 cM. The genetic map was integrated with the reference-assisted chromosome assembly (RACA) of O. melastigma, which anchored 90.7% of the assembled sequence onto the linkage map. The values of complete Benchmarking Universal Single-Copy Orthologs (BUSCO) were similar to RACA assembly but N50 (23.74 Mb; total genome length 779.4 Mb; gap 5.29%) increased to 29.99 Mb (total genome length 778.7 Mb; gap 5.2%). Using MapQTL analysis with a single nucleotide polymorphism markers, we identified a major quantitative trait locus for sex traits on the Om10. The integration of the genetic map with the reference genome of marine medaka will serve as a good resource for studies in molecular toxicology, genomics, CRISPR/Cas9, and epigenetics.

bioinformatics↗