Biology subjects

Wu, C.-G.

Publications and source records attributed to Wu, C.-G..

6 recordsLinked to original sources

bioRxiv2026

CryoSHIELDs: tunable amphipathic peptide additives for cryo-EM

While cryogenic electron microscopy (cryo-EM) has transformed structural biology, sample preparation remains a major determinant of success. Specifically, the air-water interface (AWI) can damage complexes, impose preferred orientation, and reduce particle density. Here, we introduce cryoSHIELDs, a chemically diverse library of peptide additives designed to passivate the AWI and improve cryo-EM grid preparation. Across 24 protein systems spanning GPCRs, ABC and SLC transporters, soluble and Fab-containing complexes, and enzymes, cryoSHIELDs increased the success rate of structure determination. Individual cryoSHIELDs improved particle density, angular sampling, complex stability, and, for routine targets, map quality and resolution. This toolkit enabled the structural characterization of important targets, including the ABCC6 transporter, revealing unique TMD0-mediated stabilization, and DAMGO-bound -opioid receptor-Gi complex using substantially lower protein concentration. CryoSHIELDs provide a simple, tunable, and broadly applicable platform for more reliable cryo-EM in discovery biology and structure-based drug development.

biophysics↗

bioRxiv2026

Progressive Loosening of a Dual Autoinhibitory Interface Activates PP2A-B56δ

Protein phosphatase 2A containing the B56{delta} regulatory subunit (PP2A-B56{delta}) is a critical signaling enzyme whose dysregulation is associated with cancer, neurodegenerative disorders, and Jordan's syndrome, a severe intellectual disability disorder caused by mutations in B56{delta}. Unlike other PP2A holoenzymes, PP2A-B56{delta} is regulated through a unique dual autoinhibition mechanism in which the N- and C-arms occlude the catalytic site while a substrate-mimicking short linear motif (SLiM) blocks the substrate-binding pocket. Although disease-associated mutations have been shown to alter enzyme activity, the molecular mechanism underlying activation of PP2A-B56{delta} and the effects of pathogenic mutations remain poorly understood. Here, we combined cryo-electron microscopy (cryo-EM), enhanced-sampling molecular dynamics (MD) simulations, Markov state model (MSM) construction, and transition-state analysis using Transition State identification via Dispersion and vAriational principle Regularized neural networks (TS-DAR) to characterize the conformational landscape of the disease variant E198K. Our cryo-EM analysis identified two distinct structures of E198K: an inactive closed-form with the N/C-arms resolved and an active loose-form in which the N/C-arms become highly flexible and could not be fully resolved. These structures therefore established that activation is governed by conformational changes of the N/C-arms but did not reveal the underlying mechanism. Starting from the inactive closed-form, we generated over 1,600 trajectories with an average length of 1,260 ns combined for E198K and wild-type (WT) PP2A-B56{delta}. TS-DAR identified four metastable states and two major activation pathways connecting inactive and active conformations. We found that activation occurs through progressive loosening of the N/C-arm interface while maintaining the overall holoenzyme architecture, rather than a complete opening of the interface. This mechanism exposes both the catalytic site and substrate-binding pocket. Comparison of E198K and WT revealed that the disease-associated mutation shifts the conformational equilibrium toward active states while leaving the transition-state ensemble largely unchanged. Mechanistically, E198K disrupts a salt-bridge network and weakens interactions between the internal loop and the C-arm that normally stabilize active-site occlusion. The resulting increase in C-arm mobility promotes active-site exposure and explains the elevated catalytic activity of the mutant. Together, these findings establish a previously uncharacterized activation mechanism for PP2A-B56{delta} and provide an atomic-level explanation for how the pathogenic E198K mutation allosterically promotes holoenzyme activation.

biophysics↗

bioRxiv2026

Library docking for Cannabinoid-2 Receptor ligands

Cannabinoid receptors are therapeutically promising GPCRs that are also interesting test systems for structure-based methods, which have targeted them previously. Here we used the CB2 receptor as a template to explore several topical questions in library docking. Whereas an earlier campaign against the CB1 receptor led to potent but relatively non-selective ligands, here we found that targeting interactions with polar, orthosteric site residues led to subtype-selective ligands. Docking hit rate and especially hit affinity improved in moving from a 7 million to a 2.6 billion molecule library. Similar to earlier studies, docking against active and inactive states of the receptor did not reliably bias toward the discovery of agonists or inverse agonists. Cryo-EM structures of two of the new agonists, each in a different chemotype, superposed well on the docking predictions. Correspondingly, structure-based optimization led to 10- to 140-fold improvements within three different series, also consistent with well-behaved ligand families. Hit rates with a fully enumerated 2.6 billion molecule library resembled those of an implied 11 billion molecule library from a building-block method, consistent with the latters ability to explore this space, though higher affinities were discovered from the fully enumerated set. Overall, eight diverse families of ligands, with potencies <100 nM and mostly unrelated to previously known ligands were found. Implications for future studies are considered.

biochemistry↗

bioRxiv2023

Structure-based discovery of positive allosteric modulators for the calcium sensing receptor

Drugs acting as positive allosteric modulators (PAMs) to enhance the activation of the calcium sensing receptor (CaSR) and to suppress parathyroid hormone (PTH) secretion can treat hyperparathyroidism but suffer from side effects including hypocalcemia and arrhythmias. Seeking new CaSR modulators, we docked libraries of 2.7 million and 1.2 billion molecules against transforming pockets in the active-state receptor dimer structure. Consistent with simulations suggesting that docking improves with library size, billion-molecule docking found new PAMs with a hit rate that was 2.7-fold higher than the million-molecule library and with hits up to 37-fold more potent. Structure-based optimization of ligands from both campaigns led to nanomolar leads, one of which was advanced to animal testing. This PAM displays 100-fold the potency of the standard of care, cinacalcet, in ex vivo organ assays, and reduces serum PTH levels in mice by up to 80% without the hypocalcemia typical of CaSR drugs. Cryo-EM structures with the new PAMs show that they induce residue rearrangements in the binding pockets and promote CaSR dimer conformations that are closer to the G-protein coupled state compared to established drugs. These findings highlight the promise of large library docking for therapeutic leads, especially when combined with experimental structure determination and mechanism. One sentence summaryStructure-based virtual screening uncovers novel CaSR allosteric modulators with enhanced efficacy and less side effects.

biophysics↗

bioRxiv2023

Extended regulation interface coupled to the allosteric network and disease mutations in the PP2A-B56 delta holoenzyme

An increasing number of mutations associated with devastating human diseases are diagnosed by whole-genome/exon sequencing. Recurrent de novo missense mutations have been discovered in B56{delta} (encoded by PPP2R5D), a regulatory subunit of protein phosphatase 2A (PP2A), that cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Single-particle cryo-EM structures show that the PP2A-B56{delta} holoenzyme possesses closed latent and open active forms. In the closed form, the long, disordered arms of B56{delta} termini fold against each other and the holoenzyme core, establishing dual autoinhibition of the phosphatase active site and the substrate-binding protein groove. The resulting interface spans over 190 [A] and harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is close to an allosteric network responsive to activation phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations perturb the activation phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the wild variant.

biochemistry↗

bioRxiv2022

Coupling to short linear motifs creates versatile PME-1 activities in PP2A holoenzyme demethylation and inhibition

Protein phosphatase 2A (PP2A) holoenzymes target broad substrates by recognizing short motifs via regulatory subunits. PP2A methylesterase 1 (PME-1) is a cancer-promoting enzyme and undergoes methylesterase activation upon binding to the PP2A core enzyme. Here we showed that PME-1 readily demethylates different families of PP2A holoenzymes and blocks substrate recognition in vitro. The high-resolution cryo-EM structure of a PP2A-B56 holoenzyme-PME-1 complex reveals that PME-1 disordered regions, including a substrate-mimicking motif, tether to the B56 regulatory subunit at remote sites. They occupy the holoenzyme substrate-binding groove and allow large structural shifts in both holoenzyme and PME-1 to enable multi-partite contacts at structured cores to activate the methylesterase. B56-interface mutations selectively block PME-1 activity toward PP2A-B56 holoenzymes and affect the methylation of a fraction of total cellular PP2A. The B56-interface mutations allow us to uncover B56-specific PME-1 functions in p53 signaling. Our studies reveal multiple mechanisms of PME-1 in suppressing holoenzyme functions and versatile PME-1 activities derived from coupling substrate-mimicking motifs to dynamic structured cores.

biochemistry↗