Browsing by Author "Lee, Seok-Yong"

Transient receptor potential channel subfamily A member 1 (TRPA1) is a Ca2+-permeable cation channel that serves as the primary sensor of environmental irritants, noxious substances, and temperature. Many TRPA1 agonists are electrophiles that are recognized by TRPA1 via covalent bond modifications of specific cysteine residues located in the cytoplasmic domains. TRPA1 is also a temperature activated channel displaying unique species-specific thermo sensitivity. Preceding this work, however, a mechanistic understanding of electrophile sensing by TRPA1 has been limited by a lack of structural information. Moreover, the mechanism by which TRPA1 sense temperature has been elusive. Using cryo-electron microscopy, we determined the structures of nanodisc-reconstituted human TRPA1 in ligand free state and in complex with the covalent agonists JT010 or BITC at 2.8, 2.9, and 3.1 Å, respectively. Our structural and functional studies provide the molecular basis for electrophile recognition by the extraordinarily reactive Cys621 in TRPA1 and grant mechanistic insights into electrophile-dependent conformational changes in TRPA1. This work illustrates the fundamental principles of irritant sensing in humans at the molecular level and provides a platform for future drug development targeting TRPA1. Moreover, we determined the cryo-EM structure of rattlesnake TRPA1 in nanodisc-reconstituted condition at 3.3 Å. This structural revealed a novel N-terminal ankyrin repeat domain that was not resolved in previous structures. Our structural and functional studies on rattlesnake TRPA1 provides a framework in understanding the principles of thermo sensitivity in TRPA1.

Bacterial peptidoglycan (PG) cell wall is an essential structural element, providing structural integrity and protection. The essentiality and uniqueness of PG make it an ideal target for antibiotics as over 50% of all antibiotics in clinical use target the PG biosynthesis pathway. PG synthesis involves precursor synthesis in the cytoplasm, transport across the membrane, and final assembly in the periplasmic space to form functional peptidoglycan. PG is also a dynamic structure, as it requires synthesis and degradation in a highly coordinated manner to prevent cell lysis. Despite decades of research, the cellular mechanisms regulating peptidoglycan synthesis and remodeling machineries have remained largely mysterious, particularly at the membrane-bound steps.To understand this essential process, we propose to investigate the structure, mechanism, and inhibition of the proteins and protein complexes involved in the PG pathway during cell division. My dissertation study focuses on key enzymes involved in PG precursor translocation (MraY), transport (MurJ), assembly (FtsBLQWI) and remodeling (FtsEX-EnvC). We employed single-particle cryo-electron microscopy (cryo- EM) to study the inhibition of MraY and MurJ, and to elucidate the regulatory mechanisms of protein complexes FtsBLQWI and FtsEX-EnvC from structural biology standpoint. We utilized in vitro biochemical assays to characterize the biochemical function of these protein complexes. The first part of the work studies PG precursor translocase and transport proteins MraY and MurJ, respectively. MraY catalyzes the reaction that anchors precursor on the membrane, while MurJ then flips the precursor across membrane to the periplasm. MraY has been a target for many naturally occurring nucleoside inhibitors. Rational design and synthesis of natural inhibitor analogs have been a strategy for discovering new and better antibiotics. In collaboration with Dr. Satoshi Ichikawa, we designed and constructed libraries of natural MraY inhibitor analogs. We identified promising MraY inhibitors from these libraries and several showed potent inhibitory and antibacterial activity. We reported two high-resolution cryo-EM structures of MraY bound to the new inhibitor analogs. The structures reveal a novel mode of inhibition, which provides a promising direction in antibiotics design. MurJ has been reported to be inhibited by chemically diverse compounds, but the mechanisms of MraY inhibition by these compounds have been elusive. Understanding MurJ inhibition is important because MurJ is considered a promising new drug target for antibiotic development. We attempted to elucidate the inhibition mechanism of MurJ by capturing MurJ with inhibitor(s) bound using X-ray crystallography. However, despite numerous attempts, the structural characterization of MurJ with inhibitors remained challenging. To overcome the challenges, we generated initial cryo-EM data and proposed strategies for future structural studies of MurJ using cryo-EM. The second part explores the core PG synthesis enzyme complex FtsWI and the accessory regulatory complex FtsBLQ, aiming to understand the interaction and regulation of these essential five proteins during cell division. The molecular regulation within the complex is unknown due to lack of structural information. We attempted to obtain the structure of FtsBLQWI using cryo-EM to elucidate the regulatory mechanism. I co-expressed and obtained homogeneous protein complex from an overexpression system. I developed a functional assay that circumvented the problem of substrate inaccessibility by in situ synthesis of the substrate and demonstrated that the purified complex had robust activity. While still in progress, we identified biochemical condition that yielded promising results for 3D reconstruction, laying the groundwork for future structural investigation on FtsBLQWI. The final part delves into the mechanism of FtsEX-EnvC complex function, which regulates the degradation of PG during cell division. FtsEX-EnvC complex controls PG remodeling by regulating the enzymes that hydrolyze PG. However, the molecular details of how this complex work is unknown. We obtained the cryo-EM structure of the full-length FtsEX-EnvC complex in different nucleotide states, revealing an unexpected conformation in the presence of ADP and providing a novel model on the activation mechanism through spatial regulation. Overall, the study provides structural, inhibitory, and mechanistic insights into several components of the PG pathway. Peptidoglycan synthesis and remodeling are not only fundamental processes in bacterial biology, but also a rich source of antibiotic drug targets. In light of rapidly emerging antimicrobial resistance, understanding the pathways governing PG synthesis and remodeling is crucial for discovering novel targets to develop new inhibitors.

The ability to sense and avoid detrimental harm from the environment is key to survival of organisms. In mammals, nociceptive neurons detect potentially damaging stimuli from the external environment, convert them into electrical signals, and elicit pain sensations in higher nerve center inside bodies. The detection of noxious stimuli at the beginning of this sensory circuit is mediated by receptors or ion channels expressed at peripheral nerve endings, which respond to specific chemical, thermal, or mechanical stimuli and trigger ion fluxes that lead to firing of action potentials for signal transduction. Understanding the mechanisms by which these receptor and ion channels transduce noxious signal for nociception would advance the development of analgesics for pain treatment.

A large group of such nociceptive transducers belongs to the transient receptor potential (TRP) channels superfamily. Several members of TRP channels have been found expressed in subsets of nociceptors and specialized in sensing diverse noxious chemical and thermal stimuli. The TRP melastatin subfamily member 8 (TRPM8) has been characterized as the principal molecular sensor in humans for detecting innocuous to noxious cold temperatures. It is also activated by the naturally occurring cooling compound menthol which induces cooling sensation. TRPM8 function requires the important signaling phospholipid phosphatidylinositol 4,5- bisphosphate (PIP2) as a cofactor, which allosterically couples with cooling agonists for channel activation. Since the cloning of trpm8 gene in 2002, substantial amount of functional studies has dedicated to understanding the molecular basis of ligand recognitions in TRPM8 and the underlying mechanisms of ligand- and temperature-dependent channel gating. However, a major barrier to achieve these goals has been the dearth of structural information of TRPM8, which would allow for direct visualization of ligand-channel interactions and for dissection of conformational pathways in channel gating.

We have attempted to address these gaps in knowledge from a structural biology standpoint. We employed single-particle cryo-electron microscopy (cryo-EM) and reported the first structure for a full-length TRPM8 channel form the collared flycatcher (Ficedula albicollis), which was resolved to an overall resolution of ~4.1 ångström. Our structure revealed a three-layered architecture for the homotetrameric TRPM8 channel and identified distinct features in channel assembly and interfacial contacts between subdomains. Combined with previous mutagenesis studies, we proposed the putative binding sites for menthol and PIP2. Our initial study provided a structural glimpse for the design principle of TRPM8 channel as the cold- and menthol-sensor. Next we examined the molecular basis of ligand recognition in the channel by determining additional cryo-EM structures of TRPM8 in complex with cooling compounds and PIP2. On the basis of our structural data and functional characterizations, we revealed the binding sites for cooling agonists and PIP2, and identified essential ligand-channel interactions, which corroborated with predictions from previous mutagenesis studies. More importantly, in comparison with other TRP channel structures, our TRPM8 complexes provided the structural basis of synergistic binding between cooling agonists and PIP2, which enables allosteric coupling between these two modulators for TRPM8 activation. Taken together, our study provided a platform for understanding the molecular mechanism of TRPM8 activation by cooling agents. It offered structural insights for development of novel analgesics targeting TRPM8 for pain treatment. Our study may also facilitate the sensory biology field to progress one step forward towards dissecting polymodal gating mechanisms of TRPM8 and other nociceptive TRP channels as well.

The biosynthesis of many important polysaccharides (including peptidoglycan, lipopolysaccharide, and N-linked glycans) necessitates membrane transport of oligosaccharide precursors from their cytoplasmic site of synthesis to their site of assembly outside the cytoplasm. To address this problem, cells utilize transporters such as those of the multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) superfamily to flip lipid-linked oligosaccharides across the cytoplasmic membrane. The MOP superfamily member MurJ has been shown to be the flippase that transports the lipid-linked peptidoglycan precursor lipid II, but the lack of structural information has limited our mechanistic understanding of the MurJ transport cycle. We determined the first crystal structure of MurJ (MurJTA from Thermosipho africanus) to 2.0-Å resolution, which assumed an inward-facing conformation unlike all other outward-facing structures of MOP transporters. Our structural and mutagenesis studies provide insight into a putative model of lipid II binding and an alternating-access mechanism of transport.

Nucleosides are small polar biomolecules that play important roles in every aspect of cellular life. Due to their impermeability to lipid bilayers, dedicated integral membrane proteins are tasked with selective transport of nucleosides across biological membranes. In humans, two genetically distinct protein families mediate nucleoside membrane transport: the concentrative and equilibrative nucleoside transporter families. Owing to the roles played by nucleoside transporters in physiology, pathophysiology, and pharmacology of nucleoside-analog therapeutics, a mechanistic understanding of human nucleoside transporters would not only advance our understanding of transporter biology in general but would also uncover exploitable pharmacological features for future drug development efforts. In this work, we first determined X-ray crystal structures of the human equilibrative nucleoside transporter in complex with two different adenosine reuptake inhibitors. Using our structural data as a starting point, we then designed and synthesized a series of novel transporter inhibitors with improved pharmacological properties. We also interrogated the role of both concentrative and equilibrative nucleoside transporters in nucleoside-analog antiviral drug pharmacological properties using biochemical experiments, viral assays, and cryo-electron microscopy.