Biology subjects

Huang, Y.-m. M.

Publications and source records attributed to Huang, Y.-m. M..

4 recordsLinked to original sources

bioRxiv2026

Structural and Energetic Determinants of Monobody Recognition of Oncogenic KRAS Variants

Monobodies are engineered binding proteins that recognize extended protein surfaces and offer advantages over small-molecule inhibitors for targeting challenging KRAS oncoproteins. Monobody 12D4 exhibits high affinity and selectivity for the oncogenic KRAS(G12D) mutant, but the molecular determinants governing its recognition and the basis for its mutant selectivity remain poorly understood. Here, we combined molecular dynamics simulations and energy calculations to characterize the interactions between monobody 12D4 and WT KRAS as well as four clinically relevant oncogenic variants (G12C, G12D, G12V, and G12R) in both GTP- and GDP-bound states. Our simulations revealed that 12D4 recognition depends on a conserved hydrophobic interaction network centered on the monobody FG loop (residues L77, F78, and W79). This network forms stable contacts with KARS Switch II and 3-helix. The energy calculations also showed that residue K75 of 12D4 formed a mutation-specific electrostatic interaction with KRAS G12D. This interaction contributed significantly to the affinity of 12D4 toward this mutant, whereas this interaction was absent in other variants. No monobody currently exists for targeting KRAS G12R in either nucleotide state, and no monobody selectively targets KRAS G12C and G12V in the GDP-bound inactive state. To address these, we performed computational redesign at residues 75. We identified mutations (K75Q, K75Y, and K75M) that enhanced predicted binding to G12C, G12R, and G12V variants through reorganization of interfacial contacts. Our work establishes a structural framework for understanding KRAS-monobody recognition and provides a rational foundation for engineering variant-selective monobodies with improved affinity toward previously untargetable KRAS mutants. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/737552v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1e3cbf1org.highwire.dtl.DTLVardef@62684borg.highwire.dtl.DTLVardef@ad2ab0org.highwire.dtl.DTLVardef@cdc29a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Multiscale Analysis of PNPLA2 and PNPLA3 Membrane Targeting

Lipid droplets (LDs) are dynamic organelles that regulate cellular lipid storage and mobilization through the coordinated action of LD-associated proteins. Patatin-like phospholipase domain-containing proteins PNPLA2 (ATGL) and PNPLA3 are central regulators of lipid metabolism, yet the molecular mechanisms underlying their membrane targeting and distinct enzymatic activities remain poorly understood. Here, we combine coarse-grained and all-atom molecular dynamics simulations with enhanced sampling to investigate how PNPLA2 and PNPLA3 associate with endoplasmic reticulum (ER) and LD membranes. Despite sharing a conserved N-terminal patatin domain, the two proteins exhibit distinct membrane-binding modes driven by divergent C-terminal amphipathic helices. In both proteins, membrane association is mediated primarily by deep insertion of C-terminal helices, while the patatin domain provides surface contact. PNPLA2 forms a deeply embedded U-shaped helical bundle on LDs that induce pronounced membrane curvature and promote opening of the catalytic dyad, consistent with its high triglyceride lipase activity. In contrast, PNPLA3 engages membranes through a more flexible helical arrangement that maintains a compact catalytic geometry and limits substrate accessibility. Membrane composition further modulates these interactions and leads to protein-specific lipid redistribution and curvature remodeling. Fluorescence microscopy experiments validate the computational predictions and demonstrate that mutation of a single arginine residue within the C-terminal region is sufficient to reduce LD targeting of both proteins. These results establish a mechanistic connection between membrane binding, conformational plasticity, and catalytic regulation in PNPLA2 and PNPLA3. Our work provides molecular insights into how lipid environments tune the function of LD-associated enzymes. Author SummaryLDs are essential cellular organelles that control how fats are stored and released, a process that relies on the precise recruitment and regulation of lipid-metabolizing enzymes. Our work focuses on two closely related enzymes, PNPLA2 (ATGL) and PNPLA3, which play central but distinct roles in lipid metabolism and metabolic diseases. Using a combination of multiscale modeling simulations and fluorescence microscopy, we examine how these proteins recognize and bind to ER and LD membranes. Although PNPLA2 and PNPLA3 share a conserved catalytic core, we show that they interact with membranes in different ways due to differences in their C-terminal amphipathic helices. We find that PNPLA2 forms a deeply embedded helical arrangement that reshapes the membrane and promotes access to its catalytic site, which explains why it typically shows strong lipase activity. In contrast, PNPLA3 adopts a more compact membrane-bound catalytic geometry that limits substrate access and enzymatic activity. We further applied fluorescence microscopy to experimentally validate the computational predictions. The results show that mutation of a single arginine residue within the membrane-binding helix reduces LD targeting. These findings reveal how membrane association and protein conformational dynamics jointly regulate catalytic accessibility and activity.

biophysics↗

bioRxiv2024

Lipid Droplet Targeting of ABHD5 and PNPLA3 I148M is required to promote liver steatosis

The storage and release of triacylglycerol (TAG) in lipid droplets (LDs) is regulated by dynamic protein interactions. /{beta} hydrolase domain-containing protein 5 (ABHD5; also known as CGI-58) is a membrane/LD bound protein that functions as a co-activator of Patatin Like Phospholipase Domain Containing 2 (PNPLA2; also known as Adipose triglyceride lipase, ATGL) the rate-limiting enzyme for TAG hydrolysis. The dysregulation of TAG hydrolysis is involved in various metabolic diseases such as metabolic dysfunction-associated steatotic liver disease (MASLD). We previously demonstrated that ABHD5 interacted with PNPLA3, a closely related family member to PNPLA2. Importantly, a common missense variant in PNPLA3 (I148M) is the greatest genetic risk factor for MASLD. PNPLA3 148M functions to sequester ABHD5 and prevent co-activation of PNPLA2, which has implications for initiating MASLD; however, the exact mechanisms involved are not understood. Here we demonstrate that LD targeting of both ABHD5 and PNPLA3 I148M is required for the interaction. Molecular modeling demonstrates important resides in the C-terminus of PNPLA3 for LD binding and fluorescence cross-correlation spectroscopy demonstrates that PNPLA3 I148M greater associates with ABHD5 than WT PNPLA3. Moreover, the C-terminus of PNPLA3 is sufficient for functional targeting of PNPLAs to LD and the interaction with ABHD5. In addition, ABHD5 is a general binding partner of LD-bound PNPLAs. Finally, PNPLA3 I148M targeting to LD is required to promote steatosis in vitro and in the liver. Overall results suggest that PNPLA3 I148M is a gain of function mutation and that the interaction with ABHD5 on LD is required to promote liver steatosis.

cell biology↗

bioRxiv2023

Mechanistic insights into ligand dissociation from the SARS-CoV-2 spike glycoprotein

The COVID-19 pandemic, driven by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spurred an urgent need for effective therapeutic interventions. The spike glycoprotein of the SARS-CoV-2 is crucial for infiltrating host cells, rendering it a key candidate for drug development. By interacting with the human angiotensin-converting enzyme 2 (ACE2) receptor, the spike initiates the infection of SARS-CoV-2. Linoleate is known to bind the spike glycoprotein, subsequently reducing its interaction with ACE2. However, the detailed kinetics underlying the protein-ligand interaction remains unclear. In this study, we characterized the pathways of ligand dissociation and the conformational changes associated with the spike glycoprotein by using ligand Gaussian accelerated molecular dynamics (LiGaMD). Our simulations resulted in eight complete ligand dissociation trajectories, unveiling two distinct ligand unbinding pathways. The preference between these two pathways depends on the gate distance between two -helices in the receptor binding domain (RBD) and the position of the N-linked glycan at N343. Our study also highlights the essential contributions of K417, N121 glycan, and N165 glycan in ligand unbinding, which are equally crucial in enhancing spike-ACE2 binding. We suggest that the presence of the ligand influences the motions of these residues and glycans, consequently reducing accessibility for spike-ACE2 binding. These findings enhance our understanding of ligand dissociation from the spike glycoprotein and offer significant implications for drug design strategies in the battle against COVID-19.

biophysics↗