Browsing by Author "Gu, Xinyu"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    A structural dynamics model for how CPEB3 binding to SUMO2 can regulate translational control in dendritic spines
    (Public Library of Science, 2022) Gu, Xinyu; Schafer, Nicholas P.; Bueno, Carlos; Lu, Wei; Wolynes, Peter G.; Center for Theoretical Biological Physics
    A prion-like RNA-binding protein, CPEB3, can regulate local translation in dendritic spines. CPEB3 monomers repress translation, whereas CPEB3 aggregates activate translation of its target mRNAs. However, the CPEB3 aggregates, as long-lasting prions, may raise the problem of unregulated translational activation. Here, we propose a computational model of the complex structure between CPEB3 RNA-binding domain (CPEB3-RBD) and small ubiquitin-like modifier protein 2 (SUMO2). Free energy calculations suggest that the allosteric effect of CPEB3-RBD/SUMO2 interaction can amplify the RNA-binding affinity of CPEB3. Combining with previous experimental observations on the SUMOylation mode of CPEB3, this model suggests an equilibrium shift of mRNA from binding to deSUMOylated CPEB3 aggregates to binding to SUMOylated CPEB3 monomers in basal synapses. This work shows how a burst of local translation in synapses can be silenced following a stimulation pulse, and explores the CPEB3/SUMO2 interplay underlying the structural change of synapses and the formation of long-term memories.
  • Thumbnail Image
    Item
    Mass Spectrometry of RNA-Binding Proteins during Liquid–Liquid Phase Separation Reveals Distinct Assembly Mechanisms and Droplet Architectures
    (American Chemical Society, 2023) Sahin, Cagla; Motso, Aikaterini; Gu, Xinyu; Feyrer, Hannes; Lama, Dilraj; Arndt, Tina; Rising, Anna; Gese, Genis Valentin; Hällberg, B. Martin; Marklund, Erik. G.; Schafer, Nicholas P.; Petzold, Katja; Teilum, Kaare; Wolynes, Peter G.; Landreh, Michael; Center for Theoretical Biological Physics
    Liquid–liquid phase separation (LLPS) of heterogeneous ribonucleoproteins (hnRNPs) drives the formation of membraneless organelles, but structural information about their assembled states is still lacking. Here, we address this challenge through a combination of protein engineering, native ion mobility mass spectrometry, and molecular dynamics simulations. We used an LLPS-compatible spider silk domain and pH changes to control the self-assembly of the hnRNPs FUS, TDP-43, and hCPEB3, which are implicated in neurodegeneration, cancer, and memory storage. By releasing the proteins inside the mass spectrometer from their native assemblies, we could monitor conformational changes associated with liquid–liquid phase separation. We find that FUS monomers undergo an unfolded-to-globular transition, whereas TDP-43 oligomerizes into partially disordered dimers and trimers. hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over LLPS. The divergent assembly mechanisms revealed by ion mobility mass spectrometry of soluble protein species that exist under LLPS conditions suggest structurally distinct complexes inside liquid droplets that may impact RNA processing and translation depending on biological context.
  • Thumbnail Image
    Item
    Multi-scale computational modeling of an RNA-binding prion, CPEB3, reveals its molecular mechanisms underlying the formation of long-term memory
    (2022-12-02) Gu, Xinyu; Wolynes, Peter G
    The growth and stabilization of dendritic spines is thought to be essential for strengthening the connections between neurons, and thereby memories. Actin cytoskeleton remodeling in spines is the basis of this growth and stabilization. CPEB proteins were first identified as a group of RNA-binding proteins regulating the translation of their target mRNAs, like actin mRNAs. Intriguingly, one isoform in CPEB family, CPEB3, has been recently reported as a functional prion that interacts with actin cytoskeleton. These observations make CPEB3 a seductively plausible candidate as a synaptic tag to strengthen the actin cytoskeleton and long-term memory by forming stable aggregates, and simultaneously regulate the local translation of synaptic proteins in spines. Numerous gene knockout experiments have been conducted about CPEB3 and its homologs to investigate CPEB3's function in memories. However, it is challenging for experimentalists to collect structural information of CPEB3 due to the conformational flexibility of prion-like proteins, and the picture of CPEB3's role in synaptic plasticity is still unclear at molecular level. In this thesis, to fill in the missing pieces in the molecular mechanisms of CPEB3, we utilized multi-scale computational modeling by conducting bioinformatic searches, setting up reaction-diffusion systems, and mainly by running molecular dynamics simulations using a coarse-grained protein force field - the Associative memory, Water-mediated, Structure and Energy Model (AWSEM). In the first part, we studied the interaction between actin and CPEB3 and proposed a molecular model for the complex structure of CPEB3 bound to an actin filament (F-actin). Our model gives insights into the molecular details of the F-actin/CPEB3 positive feedback loop underlying long-term memory which involves CPEB3's binding to F-actin, its aggregation triggered by F-actin, and its regulation by SUMOylation. The soluble CPEB3 monomers repress translation, whereas CPEB3 aggregates activate the translation of its target mRNAs. The CPEB3 aggregates, however, that act as long-lasting prions providing "conformational memory", may raise the problem of the consequent translational activation being unregulated. In the second part of the thesis, I propose a computational model of the complex structure between CPEB3 RNA-binding domain (CPEB3-RBD) and small ubiquitin-like modifier protein 2 (SUMO2). Free energy calculations suggest that the allosteric binding of CPEB3 with SUMO2 can confine the CPEB3-RBD to a conformation that favors RNA-binding, and thereby can amplify its RNA-binding affinity. Combining this model with previous experiments showing that CPEB3 monomers are SUMOylated in basal synapses but become deSUMOylated and start to aggregate upon stimulation, we suggest a way in which the translational control of CPEB3 can be switched back to a repressive mode after a stimulation pulse, through an RNA binding shift from binding to CPEB3 fibers to binding to SUMOylated CPEB3 monomers in basal synapses. In the last part, inspired by the specific geometry and polarity of the assembly of mRNAs and CPEB3 aggregates, a vectorial channeling mechanism is proposed to describe the local translational regulation by general mRNA/protein assemblies including functional prions and condensates. The analysis shows that the vectorial processive nature of translation can couple to transport via diffusion so as to repress or activate translation depending on the structure of the RNA protein assembly. We find that multiple factors including diffusivity changes and free energy biases in the assemblies can regulate the translation rate of mRNA by changing the balance between substrate recycling and competition between mRNAs.
  • Thumbnail Image
    Item
    OpenAWSEM with Open3SPN2: A fast, flexible, and accessible framework for large-scale coarse-grained biomolecular simulations
    (Public Library of Science, 2021) Lu, Wei; Bueno, Carlos; Schafer, Nicholas P.; Moller, Joshua; Jin, Shikai; Chen, Xun; Chen, Mingchen; Gu, Xinyu; Davtyan, Aram; Pablo, Juan J. de; Wolynes, Peter G.; Center for Theoretical Biological Physics
    We present OpenAWSEM and Open3SPN2, new cross-compatible implementations of coarse-grained models for protein (AWSEM) and DNA (3SPN2) molecular dynamics simulations within the OpenMM framework. These new implementations retain the chemical accuracy and intrinsic efficiency of the original models while adding GPU acceleration and the ease of forcefield modification provided by OpenMM’s Custom Forces software framework. By utilizing GPUs, we achieve around a 30-fold speedup in protein and protein-DNA simulations over the existing LAMMPS-based implementations running on a single CPU core. We showcase the benefits of OpenMM’s Custom Forces framework by devising and implementing two new potentials that allow us to address important aspects of protein folding and structure prediction and by testing the ability of the combined OpenAWSEM and Open3SPN2 to model protein-DNA binding. The first potential is used to describe the changes in effective interactions that occur as a protein becomes partially buried in a membrane. We also introduced an interaction to describe proteins with multiple disulfide bonds. Using simple pairwise disulfide bonding terms results in unphysical clustering of cysteine residues, posing a problem when simulating the folding of proteins with many cysteines. We now can computationally reproduce Anfinsen’s early Nobel prize winning experiments by using OpenMM’s Custom Forces framework to introduce a multi-body disulfide bonding term that prevents unphysical clustering. Our protein-DNA simulations show that the binding landscape is funneled towards structures that are quite similar to those found using experiments. In summary, this paper provides a simulation tool for the molecular biophysics community that is both easy to use and sufficiently efficient to simulate large proteins and large protein-DNA systems that are central to many cellular processes. These codes should facilitate the interplay between molecular simulations and cellular studies, which have been hampered by the large mismatch between the time and length scales accessible to molecular simulations and those relevant to cell biology.