Professional Education

  • Doctor of Philosophy, University of Wisconsin Madison (2020)
  • Master of Science, National Taiwan University (2014)
  • Ph.D., University of Wisconsin-Madison, Biophysics (2020)
  • M.S., National Taiwan University, Biochemistry (2014)
  • B.S., National Yang-Ming University, Biology (2012)

Stanford Advisors

All Publications

  • Allosteric modulation and G-protein selectivity of the Ca2+-sensing receptor. Nature He, F., Wu, C. G., Gao, Y., Rahman, S. N., Zaoralová, M., Papasergi-Scott, M. M., Gu, T. J., Robertson, M. J., Seven, A. B., Li, L., Mathiesen, J. M., Skiniotis, G. 2024


    The calcium-sensing receptor (CaSR) is a family C G-protein-coupled receptor1 (GPCR) that has a central role in regulating systemic calcium homeostasis2,3. Here we use cryo-electron microscopy and functional assays to investigate the activation of human CaSR embedded in lipid nanodiscs and its coupling to functional Gi versus Gq proteins in the presence and absence of the calcimimetic drug cinacalcet. High-resolution structures show that both Gi and Gq drive additional conformational changes in the activated CaSR dimer to stabilize a more extensive asymmetric interface of the seven-transmembrane domain (7TM) that involves key protein-lipid interactions. Selective Gi and Gq coupling by the receptor is achieved through substantial rearrangements of intracellular loop 2 and the C terminus, which contribute differentially towards the binding of the two G-protein subtypes, resulting in distinct CaSR-G-protein interfaces. The structures also reveal that natural polyamines target multiple sites on CaSR to enhance receptor activation by zipping negatively charged regions between two protomers. Furthermore, we find that the amino acid L-tryptophan, a well-known ligand of CaSR extracellular domains, occupies the 7TM bundle of the G-protein-coupled protomer at the same location as cinacalcet and other allosteric modulators. Together, these results provide a framework for G-protein activation and selectivity by CaSR, as well as its allosteric modulation by endogenous and exogenous ligands.

    View details for DOI 10.1038/s41586-024-07055-2

    View details for PubMedID 38326620

    View details for PubMedCentralID 5264458

  • B56δ long-disordered arms form a dynamic PP2A regulation interface coupled with global allostery and Jordan's syndrome mutations. Proceedings of the National Academy of Sciences of the United States of America Wu, C. G., Balakrishnan, V. K., Merrill, R. A., Parihar, P. S., Konovolov, K., Chen, Y. C., Xu, Z., Wei, H., Sundaresan, R., Cui, Q., Wadzinski, B. E., Swingle, M. R., Musiyenko, A., Chung, W. K., Honkanen, R. E., Suzuki, A., Huang, X., Strack, S., Xing, Y. 2024; 121 (1): e2310727120


    Intrinsically disordered regions (IDR) and short linear motifs (SLiMs) play pivotal roles in the intricate signaling networks governed by phosphatases and kinases. B56δ (encoded by PPP2R5D) is a regulatory subunit of protein phosphatase 2A (PP2A) with long IDRs that harbor a substrate-mimicking SLiM and multiple phosphorylation sites. De novo missense mutations in PPP2R5D cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Our single-particle cryo-EM structures of the PP2A-B56δ holoenzyme reveal that the long, disordered arms at the B56δ termini fold against each other and the holoenzyme core. This architecture suppresses both the phosphatase active site and the substrate-binding protein groove, thereby stabilizing the enzyme in a closed latent form with dual autoinhibition. The resulting interface spans over 190 Å and harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is coupled to an allosteric network responsive to phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations increase the holoenzyme activity and perturb the phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the normal variant.

    View details for DOI 10.1073/pnas.2310727120

    View details for PubMedID 38150499

    View details for PubMedCentralID PMC10769853

  • Disease mutations and phosphorylation alter the allosteric pathways involved in autoinhibition of protein phosphatase 2A. The Journal of chemical physics Konovalov, K. A., Wu, C. G., Qiu, Y., Balakrishnan, V. K., Parihar, P. S., O'Connor, M. S., Xing, Y., Huang, X. 2023; 158 (21)


    Mutations in protein phosphatase 2A (PP2A) are connected to intellectual disability and cancer. It has been hypothesized that these mutations might disrupt the autoinhibition and phosphorylation-induced activation of PP2A. Since they are located far from both the active and substrate binding sites, it is unclear how they exert their effect. We performed allosteric pathway analysis based on molecular dynamics simulations and combined it with biochemical experiments to investigate the autoinhibition of PP2A. In the wild type (WT), the C-arm of the regulatory subunit B56δ obstructs the active and substrate binding sites exerting a dual autoinhibition effect. We find that the disease mutant, E198K, severely weakens the allosteric pathways that stabilize the C-arm in the WT. Instead, the strongest allosteric pathways in E198K take a different route that promotes exposure of the substrate binding site. To facilitate the allosteric pathway analysis, we introduce a path clustering algorithm for lumping pathways into channels. We reveal remarkable similarities between the allosteric channels of E198K and those in phosphorylation-activated WT, suggesting that the autoinhibition can be alleviated through a conserved mechanism. In contrast, we find that another disease mutant, E200K, which is in spatial proximity of E198, does not repartition the allosteric pathways leading to the substrate binding site; however, it may still induce exposure of the active site. This finding agrees with our biochemical data, allowing us to predict the activity of PP2A with the phosphorylated B56δ and provide insight into how disease mutations in spatial proximity alter the enzymatic activity in surprisingly different mechanisms.

    View details for DOI 10.1063/5.0150272

    View details for PubMedID 37260014

    View details for PubMedCentralID PMC10238128

  • Glucose dissociates DDX21 dimers to regulate mRNA splicing and tissue differentiation. Cell Miao, W., Porter, D. F., Lopez-Pajares, V., Siprashvili, Z., Meyers, R. M., Bai, Y., Nguyen, D. T., Ko, L. A., Zarnegar, B. J., Ferguson, I. D., Mills, M. M., Jilly-Rehak, C. E., Wu, C., Yang, Y., Meyers, J. M., Hong, A. W., Reynolds, D. L., Ramanathan, M., Tao, S., Jiang, S., Flynn, R. A., Wang, Y., Nolan, G. P., Khavari, P. A. 2023; 186 (1): 80


    Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.

    View details for DOI 10.1016/j.cell.2022.12.004

    View details for PubMedID 36608661

  • Coupling to short linear motifs creates versatile PME-1 activities in PP2A holoenzyme demethylation and inhibition ELIFE Li, Y., Balakrishnan, V., Rowse, M., Wu, C., Bravos, A., Yadav, V. K., Ivarsson, Y., Strack, S., Novikova, I., Xing, Y. 2022; 11


    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 cryoelectron microscopy 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 multipartite 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.

    View details for DOI 10.7554/eLife.79736

    View details for Web of Science ID 000868454200001

    View details for PubMedID 35924897

    View details for PubMedCentralID PMC9398451

  • Small-molecule inhibitors that disrupt the MTDH-SND1 complex suppress breast cancer progression and metastasis NATURE CANCER Shen, M., Wei, Y., Kim, H., Wan, L., Jiang, Y., Hang, X., Raba, M., Remiszewski, S., Rowicki, M., Wu, C., Wu, S., Zhang, L., Lu, X., Yuan, M., Smith, H. A., Zheng, A., Bertino, J., Jin, J. F., Xing, Y., Shao, Z., Kang, Y. 2021
  • Roles of constitutive and signal-dependent protein phosphatase 2A docking motifs in burst attenuation of the cyclic AMP response element-binding protein JOURNAL OF BIOLOGICAL CHEMISTRY Kim, S., Wu, C., Jia, W., Xing, Y., Tibbetts, R. S. 2021; 297 (1): 100908


    The cAMP response element-binding protein (CREB) is an important regulator of cell growth, metabolism, and synaptic plasticity. CREB is activated through phosphorylation of an evolutionarily conserved Ser residue (S133) within its intrinsically disordered kinase-inducible domain (KID). Phosphorylation of S133 in response to cAMP, Ca2+, and other stimuli triggers an association of the KID with the KID-interacting (KIX) domain of the CREB-binding protein (CBP), a histone acetyl transferase (HAT) that promotes transcriptional activation. Here we addressed the mechanisms of CREB attenuation following bursts in CREB phosphorylation. We show that phosphorylation of S133 is reversed by protein phosphatase 2A (PP2A), which is recruited to CREB through its B56 regulatory subunits. We found that a B56-binding site located at the carboxyl-terminal boundary of the KID (BS2) mediates high-affinity B56 binding, while a second binding site (BS1) located near the amino terminus of the KID mediates low affinity binding enhanced by phosphorylation of adjacent casein kinase (CK) phosphosites. Mutations that diminished B56 binding to BS2 elevated both basal and stimulus-induced phosphorylation of S133, increased CBP interaction with CREB, and potentiated the expression of CREB-dependent reporter genes. Cells from mice harboring a homozygous CrebE153D mutation that disrupts BS2 exhibited increased S133 phosphorylation stoichiometry and elevated transcriptional bursts to cAMP. These findings provide insights into substrate targeting by PP2A holoenzymes and establish a new mechanism of CREB attenuation that has implications for understanding CREB signaling in cell growth, metabolism, synaptic plasticity, and other physiologic contexts.

    View details for DOI 10.1016/j.jbc.2021.100908

    View details for Web of Science ID 000678068400095

    View details for PubMedID 34171357

    View details for PubMedCentralID PMC8294589

  • Methylation-regulated decommissioning of multimeric PP2A complexes NATURE COMMUNICATIONS Wu, C., Zheng, A., Jiang, L., Rowse, M., Stanevich, V., Chen, H., Li, Y., Satyshur, K. A., Johnson, B., Gu, T., Liu, Z., Xing, Y. 2017; 8: 2272


    Dynamic assembly/disassembly of signaling complexes are crucial for cellular functions. Specialized latency and activation chaperones control the biogenesis of protein phosphatase 2A (PP2A) holoenzymes that contain a common scaffold and catalytic subunits and a variable regulatory subunit. Here we show that the butterfly-shaped TIPRL (TOR signaling pathway regulator) makes highly integrative multibranching contacts with the PP2A catalytic subunit, selective for the unmethylated tail and perturbing/inactivating the phosphatase active site. TIPRL also makes unusual wobble contacts with the scaffold subunit, allowing TIPRL, but not the overlapping regulatory subunits, to tolerate disease-associated PP2A mutations, resulting in reduced holoenzyme assembly and enhanced inactivation of mutant PP2A. Strikingly, TIPRL and the latency chaperone, α4, coordinate to disassemble active holoenzymes into latent PP2A, strictly controlled by methylation. Our study reveals a mechanism for methylation-responsive inactivation and holoenzyme disassembly, illustrating the complexity of regulation/signaling, dynamic complex disassembly, and disease mutations in cancer and intellectual disability.

    View details for DOI 10.1038/s41467-017-02405-3

    View details for Web of Science ID 000418570600017

    View details for PubMedID 29273778

    View details for PubMedCentralID PMC5741625

  • PP2A-B ' holoenzyme substrate recognition, regulation and role in cytokinesis CELL DISCOVERY Wu, C., Chen, H., Guo, F., Yadav, V. K., Mcilwain, S. J., Rowse, M., Choudhary, A., Lin, Z., Li, Y., Gu, T., Zheng, A., Xu, Q., Lee, W., Resch, E., Johnson, B., Day, J., Ge, Y., Ong, I. M., Burkard, M. E., Ivarsson, Y., Xing, Y. 2017; 3: 17027


    Protein phosphatase 2A (PP2A) is a major Ser/Thr phosphatase; it forms diverse heterotrimeric holoenzymes that counteract kinase actions. Using a peptidome that tiles the disordered regions of the human proteome, we identified proteins containing [LMFI]xx[ILV]xEx motifs that serve as interaction sites for B'-family PP2A regulatory subunits and holoenzymes. The B'-binding motifs have important roles in substrate recognition and in competitive inhibition of substrate binding. With more than 100 novel ligands identified, we confirmed that the recently identified LxxIxEx B'α-binding motifs serve as common binding sites for B' subunits with minor variations, and that S/T phosphorylation or D/E residues at positions 2, 7, 8 and 9 of the motifs reinforce interactions. Hundreds of proteins in the human proteome harbor intrinsic or phosphorylation-responsive B'-interaction motifs, and localize at distinct cellular organelles, such as midbody, predicting kinase-facilitated recruitment of PP2A-B' holoenzymes for tight spatiotemporal control of phosphorylation at mitosis and cytokinesis. Moroever, Polo-like kinase 1-mediated phosphorylation of Cyk4/RACGAP1, a centralspindlin component at the midbody, facilitates binding of both RhoA guanine nucleotide exchange factor (epithelial cell transforming sequence 2 (Ect2)) and PP2A-B' that in turn dephosphorylates Cyk4 and disrupts Ect2 binding. This feedback signaling loop precisely controls RhoA activation and specifies a restricted region for cleavage furrow ingression. Our results provide a framework for further investigation of diverse signaling circuits formed by PP2A-B' holoenzymes in various cellular processes.

    View details for DOI 10.1038/celldisc.2017.27

    View details for Web of Science ID 000414925200001

    View details for PubMedID 28884018

    View details for PubMedCentralID PMC5586252

  • Structure of a Highly Active Cephalopod S-crystallin Mutant: New Molecular Evidence for Evolution from an Active Enzyme into Lens-Refractive Protein SCIENTIFIC REPORTS Tan, W., Cheng, S., Liu, Y., Wu, C., Lin, M., Chen, C., Lin, C., Chou, C. 2016; 6: 31176


    Crystallins are found widely in animal lenses and have important functions due to their refractive properties. In the coleoid cephalopods, a lens with a graded refractive index provides good vision and is required for survival. Cephalopod S-crystallin is thought to have evolved from glutathione S-transferase (GST) with various homologs differentially expressed in the lens. However, there is no direct structural information that helps to delineate the mechanisms by which S-crystallin could have evolved. Here we report the structural and biochemical characterization of novel S-crystallin-glutathione complex. The 2.35-Å crystal structure of a S-crystallin mutant from Octopus vulgaris reveals an active-site architecture that is different from that of GST. S-crystallin has a preference for glutathione binding, although almost lost its GST enzymatic activity. We've also identified four historical mutations that are able to produce a "GST-like" S-crystallin that has regained activity. This protein recapitulates the evolution of S-crystallin from GST. Protein stability studies suggest that S-crystallin is stabilized by glutathione binding to prevent its aggregation; this contrasts with GST-σ, which do not possess this protection. We suggest that a tradeoff between enzyme activity and the stability of the lens protein might have been one of the major driving force behind lens evolution.

    View details for DOI 10.1038/srep31176

    View details for Web of Science ID 000392101500001

    View details for PubMedID 27499004

    View details for PubMedCentralID PMC4976375

  • Mechanism for controlling the monomer-dimer conversion of SARS coronavirus main protease ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY Wu, C., Cheng, S., Chen, S., Li, J., Fang, Y., Chen, Y., Chou, C. 2013; 69: 747-755


    The Severe acute respiratory syndrome coronavirus (SARS-CoV) main protease (M(pro)) cleaves two virion polyproteins (pp1a and pp1ab); this essential process represents an attractive target for the development of anti-SARS drugs. The functional unit of M(pro) is a homodimer and each subunit contains a His41/Cys145 catalytic dyad. Large amounts of biochemical and structural information are available on M(pro); nevertheless, the mechanism by which monomeric M(pro) is converted into a dimer during maturation still remains poorly understood. Previous studies have suggested that a C-terminal residue, Arg298, interacts with Ser123 of the other monomer in the dimer, and mutation of Arg298 results in a monomeric structure with a collapsed substrate-binding pocket. Interestingly, the R298A mutant of M(pro) shows a reversible substrate-induced dimerization that is essential for catalysis. Here, the conformational change that occurs during substrate-induced dimerization is delineated by X-ray crystallography. A dimer with a mutual orientation of the monomers that differs from that of the wild-type protease is present in the asymmetric unit. The presence of a complete substrate-binding pocket and oxyanion hole in both protomers suggests that they are both catalytically active, while the two domain IIIs show minor reorganization. This structural information offers valuable insights into the molecular mechanism associated with substrate-induced dimerization and has important implications with respect to the maturation of the enzyme.

    View details for DOI 10.1107/S0907444913001315

    View details for Web of Science ID 000318240200008

    View details for PubMedID 23633583

    View details for PubMedCentralID PMC7161611