Biology subjects

Zhan, R.

Publications and source records attributed to Zhan, R..

6 recordsLinked to original sources

bioRxiv2026

Zero-Shot Generation of Protein Conformational Ensembles Through AlphaFold Latent Flooding

Despite transformative advances in protein structure prediction, generating conformational ensembles directly from sequence in an efficient and accessible manner remains a central challenge. We introduce a heuristic importance sampling framework for the zero-shot prediction of functionally relevant, conformationally diverse structures, bypassing the need for extensive physics-based modeling or prior domain knowledge. Guided by an analysis of massive activations in AlphaFold (AF), we develop a latent flooding algorithm that enables adversarial exploration of the latent space while preserving local geometric integrity. We show that the AF latent flooding (AFLF) method robustly recovers experimental structural fluctuations, captures function-relevant conformational states, and reveals cryptic binding sites across diverse protein systems. These results suggest that AF latent features implicitly encode the biophysical principles of protein thermodynamics, which AFLF exploits. Finally, we show that AFLF is computationally accessible and interoperable, offering a test-time generalization of AF for investigating protein dynamics and accelerating structure-based ligand discovery.

biophysics↗

bioRxiv2026

RodZ acts through MreBCD to activate the elongasome in Escherichia coli

Most bacteria are surrounded by a peptidoglycan (PG) matrix that maintains cell shape and provides protection against turgor pressure. In many rod-shaped bacteria, synthesis of PG along the cell cylinder is organized by the elongasome, also called the Rod complex, which consists of six highly conserved proteins, including the actin-like MreB, the PG synthase complex RodA-PBP2 and three regulatory membrane proteins, MreC, MreD and RodZ. However, how these proteins interact with each other to form the elongasome and synthesize lateral PG remains elusive. In this study, by characterizing MreC mutations affecting elongasome activity in Escherichia coli, we provide evidence that MreC alternates between an active and an inactive state and these mutations affect MreC interaction with PBP2. We also find that RodZ interacts with MreC via a periplasmic region and disruption of this interaction compromises elongasome function. Additionally, we show that the cytoplasmic region of RodZ, which interacts with MreB, work synergistically with activating MreC mutations to promote rod shape formation. These results indicate that RodZ activates the elongasome by initiating two signaling cascades, one in the periplasm via MreCD, and another in the cytoplasm through MreB. This role of RodZ in regulating elongasome activity is analogous to that of FtsN in activating the divisome, suggesting that the mechanisms governing the activation of PG synthesis in cell elongation and division are similar in E. coli. ImportanceThe elongasome, or Rod complex, mediates lateral peptidoglycan synthesis during cell elongation in many rod-shaped bacteria. It consists of the cytoskeletal protein MreB, the peptidoglycan synthase RodA-PBP2, and three regulatory proteins MreCD and RodZ. Although it has been extensively studied, how its activity is controlled remains incompletely understood. Here, we reveal the roles of MreCD and RodZ in regulating elongasome activity in Escherichia coli. Our results indicate that RodZ triggers lateral peptidoglycan synthesis by interacting with MreB in the cytoplasm and MreCD in the periplasm. These interactions likely switch MreBCD to the active state such that they can stimulate the activity of the RodA-PBP2 complex. This regulatory mechanism resembles the activation mechanism of the divisome that mediates septal peptidoglycan synthesis.

microbiology↗

bioRxiv2025

Deciphering DEL Pocket Patterns Through Contrastive Learning

DNA-encoded libraries (DELs) facilitate high-throughput screening of trillions of molecules against protein targets through split-pool synthesis and DNA tagging. Despite their potential, only a few DEL-derived compounds have advanced to clinical trials or reached the market. A better understanding of the defining characteristics of target proteins, particularly those with binding pockets suitable for DEL screening, is critical to improving success rates. However, existing approaches remain limited in assessing pocket flexibility and functional similarity. Here, we present ErePOC, a pocket representation model based on contrastive learning with ESM-2 embeddings to address these challenges. ErePOC captures both structural and functional features of binding pockets, enabling identification of shared characteristics among DEL targets. By integrating analyses of low-dimensional physicochemical properties and high-dimensional ErePOC embeddings, we provide a comprehensive view of DEL target space. With 98% precision in downstream classification tasks, ErePOC demonstrates high performance in pocket representation, which is then applied to predict human proteins suitable for DEL screening, with enrichment uncovered across 18 functional categories. This work establishes a new framework for enhancing DEL-based drug discovery through more effective target selection and pocket similarity analysis.

biophysics↗

bioRxiv2025

Scrutinization on Docking Against Individually Generated Target Pockets for Each Ligand

The longstanding challenge of limited exploration in protein receptor conformational space continues to constrain the precision of molecular docking. Ensemble docking, which employs methods such as molecular dynamics simulations to generate multiple receptor conformations for docking, has improved accuracy but remains limited by incomplete sampling and an inability to fully account for ligand-induced fit. To address these limitations, we introduce the concept of individual docking, a novel approach that involves docking against receptor conformations generated individually for each ligand in the docking library. This approach has only very recently become feasible due to advances in protein structure prediction, in particular end-to-end protein-ligand complex prediction technologies. In this study, we performed individual docking on 27 targets from the DUD-E dataset, using a two-step protocol that integrates NeuralPLexer or AlphaFold3 for receptor conformation sampling, followed by physics-based docking. Our results reveal that individual docking with AlphaFold3 predictions yields more than a twofold improvement in enrichment factors compared to standard docking. Furthermore, individual docking recovers distinct sets of active ligands, thereby expanding the diversity of virtual screening hits. Detailed analyses of pocket and ligand conformations suggest several potential incompatibilities between deep learning-based and physics-based virtual screening tools.

bioinformatics↗

bioRxiv2021

Genetic analysis of the septal peptidoglycan synthase FtsWI complex supports a conserved activation mechanism for SEDS-bPBP complexes

SEDS family peptidoglycan (PG) glycosyltransferases RodA and FtsW require their cognate transpeptidases PBP2 and FtsI (class B penicillin binding proteins) to synthesize PG along the cell cylinder and at the septum, respectively. The activities of these SEDS-bPBPs complexes are tightly regulated to ensure proper cell elongation and division. In Escherichia coli FtsN switches FtsA and FtsQLB to the active forms that synergize to stimulate FtsWI, but the exact mechanism is not well understood. Previously, we isolated an activation mutation in ftsW (M269I) that allows cell division with reduced FtsN function. To try and understand the basis for activation we isolated additional substitutions at this position and found that only the original substitution produced an active mutant whereas drastic changes resulted in an inactive mutant. In another approach we isolated suppressors of an inactive FtsL mutant and obtained FtsWE289G and FtsIK211I and found they bypassed FtsN. Epistatic analysis of these mutations and others confirmed that the FtsN-triggered activation signal goes from FtsQLB to FtsI to FtsW. Mapping these mutations and others affecting the activities of FtsWI on the RodA-PBP2 structure revealed they are located at the interaction interface between the extracellular loop 4 (ECL4) of FtsW and the pedestal domain of FtsI (PBP3). This supports a model in which the interaction between the ECL4 of SEDS proteins and the pedestal domain of their cognate bPBPs plays a critical role in the activation mechanism. Author summaryBacterial cell division requires the synthesis of septal peptidoglycan by the widely conserved SEDS-bPBP protein complex FtsWI, but how the complex is activated during cell division is still poorly understood. Previous studies suggest that FtsN initiates a signaling cascade in the periplasm to activate FtsW. Here we isolated and characterized activated FtsW and FtsI mutants and confirmed that the signaling cascade for FtsW activation goes from FtsN to FtsQLB to FtsI and then to FtsW. The residues corresponding to mutations affecting FtsWI activation are clustered to a small region of the interaction interface between the pedestal domain of FtsI and the extracellular loop 4 of FtsW, suggesting that this interaction mediates activation of FtsW. This is strikingly similar to the proposed activation mechanism for the RodA-PBP2 complex, another SEDS-bPBP complex required for cell elongation. Thus, the two homologous SEDS-bPBP complexes are activated similarly by completely unrelated activators that modulate the interaction interface between the SEDS proteins and the bPBPs.

microbiology↗

bioRxiv2020

Structural and biochemical characterization of bifunctional XynA

Xylan and cellulose are the two major constituents in numerous types of lignocellulosic biomass, representing a promising resource for biofuels and other biobased industries. The efficient degradation of lignocellulose requires the synergistic actions of cellulase and xylanase. Thus, bifunctional enzyme incorporated xylanase/cellulase activity has attracted considerable attention since it has great cost savings potential. Recently, a novel GH10 family enzyme XynA identified from Bacillus sp. is found to degrade both cellulose and xylan. To understand its molecular catalytic mechanism, here we first solve the crystal structure of XynA at 2.3 [A]. XynA is characterized with a classic (/{beta})8 TIM-barrel fold (GH10 domain) flanked by the flexible N-terminal domain and C-terminal domain. Circular dichroism, protein thermal shift and enzyme activity assays reveal that conserved residues Glu182 and Glu280 are both important for catalytic activities of XynA, which is verified by the crystal structure of XynA with E182A/E280A double mutant. Molecular docking studies of XynA with xylohexaose and cellohexaose as well as site-directed mutagenesis and enzyme activity assay demonstrat that Gln250 and His252 are indispensible to cellulase and bifunctional activity, separately. These results elucidate the structural and biochemical features of XynA, providing clues for further modification of XynA for industrial application.

molecular biology↗