Biology subjects

Lim, H.-H.

Publications and source records attributed to Lim, H.-H..

5 recordsLinked to original sources

bioRxiv2025

Cryo-EM structures of an anti-MLC1 Fab in apo and peptide-bound states reveal the structural basis of antigen recognition

Monoclonal antibodies are indispensable tools in structural biology and biomedical research, but defining the molecular basis of their specificity remains challenging. Here, we developed a novel monoclonal antibody (37E5) against the astrocytic membrane protein MLC1, a component of gliovascular signaling implicated in megalencephalic leukoencephalopathy with subcortical cysts. 37E5 demonstrated high specificity and versatility across biochemical, cellular, and histological assays, enabling reliable detection of MLC1 in both human and mouse tissue. Using single-particle cryo-EM, we determined [~]3 [A] resolution structures of the 37E5 Fab in apo and antigen-bound states, despite the small molecular mass ([~]50 kDa), close to the lower size limit of cryo-EM. The antigen-bound structure revealed continuous density for an MLC1-derived peptide and enabled atomic mapping of polar and non-polar interaction networks. Conformational changes in CDR-L1 and CDR-L2 indicated an induced-fit mechanism of recognition. Comparison with AlphaFold-predicted models underscored the accuracy of Fab backbone prediction but revealed major limitations in modeling epitope-paratope geometry. These findings establish 37E5 as a versatile antibody for mechanistic studies of gliovascular biology and MLC disease, while demonstrating that cryo-EM can achieve atomic-level characterization of small Fab-antigen complexes, thereby expanding the methodological frontier of antibody-antigen structural biology. SignificanceThis study defines the molecular basis of MLC1 recognition by a novel monoclonal antibody, establishes 37E5 as a versatile reagent for mechanistic and translational research, and demonstrates the feasibility of cryo-EM to resolve dynamic features of small Fab-antigen complexes.

biochemistry↗

bioRxiv2025

MEG3 Enhances Survival of Developing Human Neurons with CLCN4-Linked Autophagy Impairment

Genetic variations in CLCN4, encoding the H+/Cl- exchanger CLC-4, are associated with human neurodevelopmental disorders with highly variable phenotypes. A lack of physiologically relevant models has hampered molecular understanding of pathogenic mechanisms. We now establish engineered brain organoid and neuronal cell systems to examine impacts of patient-relevant CLCN4 genetic variations. We find that CLCN4 variants reduced excitatory neuron numbers due to early-stage cell death, accompanied by altered endo-lysosomal dynamics and disrupted autophagic flux. Transcriptomic profiling showed significant downregulation of long non-coding RNA MEG3 in CLCN4-variant neurons. Restoring MEG3 expression is sufficient to rescue cellular defects and improve survival of CLCN4-variant neurons. These findings link CLCN4 dysfunction with impaired autophagy and neuronal cell death, highlighting MEG3 as a potential therapeutic target for neurodevelopmental disorders involving autophagic dysfunction.

neuroscience↗

bioRxiv2025

High-resolution cryo-EM structures of small protein-ligand complexes near the theoretical size limit

Cryo-electron microscopy (cryo-EM) is widely used to determine macromolecular structures at atomic resolution. The theoretical size lower limit of particles for cryo-EM analysis is 38 kDa, limited by factors such as contrast and particle alignment accuracy. To date, no cryo-EM structures have been reported for proteins near this size limit. This study presents cryo-EM structures of two protein-ligand complexes around 40 kDa. The structure of maltose-binding protein (43 kDa) was determined at 2.32 [A] resolution, clearly revealing the bound maltose and water molecules. Additionally, the kinase domain of human PLK1 (37 kDa), slightly below the theoretical limit, was determined at 3.04 [A] resolution, allowing the identification of the bound ligand, onvansertib. These findings demonstrate that cryo-EM can be effectively employed for structure determination and structure-based drug screening of small proteins or domains.

biophysics↗

bioRxiv2020

Altering CLC stoichiometry by reducing non-polar side-chains at the dimerization interface

CLC-ec1 is a Cl-/H+ antiporter that forms stable homodimers in lipid bilayers, with a free energy of -10.9 kcal/mole relative to the 1 subunit/lipid standard state in 2:1 POPE/POPG lipid bilayers. The dimerization interface is formed by four transmembrane helices: H, I, P and Q, that are lined by non-polar side-chains that come in close contact, yet it is unclear as to whether their interactions drive dimerization. To investigate whether non-polar side-chains are required for dimer assembly, we designed a series of constructs where side-chain packing in the dimer state is significantly reduced by making 4-5 alanine substitutions along each helix (H-ala, I-ala, P-ala, Q-ala). All constructs are functional and three purify as stable dimers in detergent micelles despite the removal of significant side-chain interactions. On the other hand, H-ala shows the unique behavior of purifying as a mixture of monomers and dimers, followed by a rapid and complete conversion to monomers. In lipid bilayers, all four constructs are monomeric as examined by single-molecule photobleaching analysis. Further study of the H-helix shows that the single mutation L194A is sufficient to yield monomeric CLC-ec1 in detergent micelles and lipid bilayers. X-ray crystal structures of L194A reveal the protein re-assembles to form dimers, with a structure that is identical to wild-type. Altogether, these results demonstrate that non-polar membrane embedded side-chains play an important role in defining dimer stability, but the stoichiometry is highly contextual to the solvent environment. Furthermore, we discovered that L194 is a molecular hot-spot for defining dimerization of CLC-ec1.

biophysics↗

bioRxiv2018

Structural capture of an intermediate transport state of a CLC Cl-/H+ antiporter

The CLC family proteins are involved in a variety of cellular processes, where chloride homeostasis needs to be controlled. Two distinct classes of CLC proteins, Cl- channels and Cl-/H+ antiporters, have been functionally and structurally investigated over the last several decades. Recent studies have revealed that the conformational heterogeneity of the critical glutamate residue, Gluex could explain the transport cycle of CLC-type Cl-/H+ antiporters. However, the presence of multiple conformations of the Gluex has been suggested from combined structural snapshots of two different CLC antiporters. Thus, we aimed to investigate the presence of these three intermediate conformations in CLC-ec1, the most deeply studied CLC at both functional and structural levels. By comparing crystal structures of E148D, E148A mutant and wildtype CLC-ec1 with varying anion concentrations, we suggest that the Gluex indeed take at least three distinct conformational states in a single CLC antiporter, CLC-ec1.

biochemistry↗