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  1. Free, publicly-accessible full text available November 14, 2024
  2. Homodimeric class 1 cytokine receptors include the erythropoietin (EPOR), thrombopoietin (TPOR), granulocyte colony-stimulating factor 3 (CSF3R), growth hormone (GHR), and prolactin receptors (PRLR). These cell-surface single-pass transmembrane (TM) glycoproteins regulate cell growth, proliferation, and differentiation and induce oncogenesis. An active TM signaling complex consists of a receptor homodimer, one or two ligands bound to the receptor extracellular domains and two molecules of Janus Kinase 2 (JAK2) constitutively associated with the receptor intracellular domains. Although crystal structures of soluble extracellular domains with ligands have been obtained for all the receptors except TPOR, little is known about the structure and dynamics of the complete TM complexes that activate the downstream JAK-STAT signaling pathway. Three-dimensional models of five human receptor complexes with cytokines and JAK2 were generated here using AlphaFold Multimer. Given the large size of the complexes (from 3220 to 4074 residues), the modeling required a stepwise assembly from smaller parts with selection and validation of the models through comparisons with published experimental data. The modeling of active and inactive complexes supports a general activation mechanism that involves ligand binding to a monomeric receptor followed by receptor dimerization and rotational movement of the receptor TM α-helices causing proximity, dimerization, and activation of associated JAK2 subunits. The binding mode of two eltrombopag molecules to TM α-helices of the active TPOR dimer was proposed. The models also help elucidating the molecular basis of oncogenic mutations that may involve a non-canonical activation route. Models equilibrated in explicit lipids of the plasma membrane are publicly available. 
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    Free, publicly-accessible full text available September 11, 2024
  3. The B cell and T cell antigen receptors (BCR and TCR) share a common architecture in which variable dimeric antigen-binding modules assemble with invariant dimeric signaling modules to form functional receptor complexes. In the TCR, a highly conserved T cell receptor αβ (TCRαβ) transmembrane (TM) interface forms a rigid structure around which its three dimeric signaling modules assemble through well-characterized polar interactions. Noting that the key features stabilizing this TCRαβ TM interface also appear with high evolutionary conservation in the TM sequences of the membrane immunoglobulin (mIg) heavy chains that form the BCR’s homodimeric antigen-binding module, we asked whether the BCR contained an analogous TM structure. Using an unbiased biochemical and computational modeling approach, we found that the mouse IgM BCR forms a core TM structure that is remarkably similar to that of the TCR. This structure is reinforced by a network of interhelical hydrogen bonds, and our model is nearly identical to the arrangement observed in the just-released cryo-electron microscopy (cryo-EM) structures of intact human BCRs. Our biochemical analysis shows that the integrity of this TM structure is vital for stable assembly with the BCR signaling module CD79AB in the B cell endoplasmic reticulum, and molecular dynamics simulations indicate that BCRs of all five isotypes can form comparable structures. These results demonstrate that, despite their many differences in composition, complexity, and ligand type, TCRs and BCRs rely on a common core TM structure that has been shaped by evolution for optimal receptor assembly and stability in the cell membrane. 
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  4. Abstract

    G-protein coupled receptors (GPCRs) and ion channels serve as key molecular switches through which extracellular stimuli are transformed into intracellular effects, and it has long been postulated that ion channels are direct effector molecules of the alpha subunit of G-proteins (Gα). However, no complete structural evidence supporting the direct interaction between Gα and ion channels is available. Here, we present the cryo-electron microscopy structures of the human transient receptor potential canonical 5 (TRPC5)-Gαi3complexes with a 4:4 stoichiometry in lipid nanodiscs. Remarkably, Gαi3binds to the ankyrin repeat edge of TRPC5 ~ 50 Å away from the cell membrane. Electrophysiological analysis shows that Gαi3increases the sensitivity of TRPC5 to phosphatidylinositol 4,5-bisphosphate (PIP2), thereby rendering TRPC5 more easily opened in the cell membrane, where the concentration of PIP2is physiologically regulated. Our results demonstrate that ion channels are one of the direct effector molecules of Gα proteins triggered by GPCR activation–providing a structural framework for unraveling the crosstalk between two major classes of transmembrane proteins: GPCRs and ion channels.

     
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  5. Mycobacterium tuberculosis ( Mtb ) is the causative agent of tuberculosis (TB), a disease that claims ~1.6 million lives annually. The current treatment regime is long and expensive, and missed doses contribute to drug resistance. Therefore, development of new anti-TB drugs remains one of the highest public health priorities. Mtb has evolved a complex cell envelope that represents a formidable barrier to antibiotics. The Mtb cell envelop consists of four distinct layers enriched for Mtb specific lipids and glycans. Although the outer membrane, comprised of mycolic acid esters, has been extensively studied, less is known about the plasma membrane, which also plays a critical role in impacting antibiotic efficacy. The Mtb plasma membrane has a unique lipid composition, with mannosylated phosphatidylinositol lipids (phosphatidyl-myoinositol mannosides, PIMs) comprising more than 50% of the lipids. However, the role of PIMs in the structure and function of the membrane remains elusive. Here, we used multiscale molecular dynamics (MD) simulations to understand the structure-function relationship of the PIM lipid family and decipher how they self-organize to shape the biophysical properties of mycobacterial plasma membranes. We assess both symmetric and asymmetric assemblies of the Mtb plasma membrane and compare this with residue distributions of Mtb integral membrane protein structures. To further validate the model, we tested known anti-TB drugs and demonstrated that our models agree with experimental results. Thus, our work sheds new light on the organization of the mycobacterial plasma membrane. This paves the way for future studies on antibiotic development and understanding Mtb membrane protein function. 
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