Bio

Honors & Awards


  • The Presidential Award, Chinese Academy of Sciences (2011)
  • Stanford CVI Travel Award, Stanford Cardiovascular Institute (2015)
  • Postdoctoral Fellowship, American Heart Association (01/01/2014-12/31/2015)

Professional Education


  • Ph.D., University of Science and Technology of China, Structural Biology (2011)

Stanford Advisors


Research & Scholarship

Current Research and Scholarly Interests


Structural and Functional Studies of beta-2 Adrenergic Receptor and Signalling Protein Complexes

Publications

All Publications


  • Highly Branched Pentasaccharide-Bearing Amphiphiles for Membrane Protein Studies. Journal of the American Chemical Society Ehsan, M., Du, Y., Scull, N. J., Tikhonova, E., Tarrasch, J., Mortensen, J. S., Loland, C. J., Skiniotis, G., Guan, L., Byrne, B., Kobilka, B. K., Chae, P. S. 2016; 138 (11): 3789-3796

    Abstract

    Detergents are essential tools for membrane protein manipulation. Micelles formed by detergent molecules have the ability to encapsulate the hydrophobic domains of membrane proteins. The resulting protein-detergent complexes (PDCs) are compatible with the polar environments of aqueous media, making structural and functional analysis feasible. Although a number of novel agents have been developed to overcome the limitations of conventional detergents, most have traditional head groups such as glucoside or maltoside. In this study, we introduce a class of amphiphiles, the PSA/Es with a novel highly branched pentasaccharide hydrophilic group. The PSA/Es conferred markedly increased stability to a diverse range of membrane proteins compared to conventional detergents, indicating a positive role for the new hydrophilic group in maintaining the native protein integrity. In addition, PDCs formed by PSA/Es were smaller and more suitable for electron microscopic analysis than those formed by DDM, indicating that the new agents have significant potential for the structure-function studies of membrane proteins.

    View details for DOI 10.1021/jacs.5b13233

    View details for PubMedID 26966956

  • The α-Arrestin ARRDC3 Regulates the Endosomal Residence Time and Intracellular Signaling of the β2-Adrenergic Receptor. The Journal of biological chemistry Tian, X., Irannejad, R., Bowman, S. L., Du, Y., Puthenveedu, M. A., von Zastrow, M., Benovic, J. L. 2016

    Abstract

    Arrestin domain-containing protein 3 (ARRDC3) is a member of the mammalian α-arrestin family, which is predicted to share similar tertiary structure with visual-/β-arrestins and also contains C-terminal PPxY motifs that mediate interaction with E3 ubiquitin ligases. Recently, ARRDC3 has been proposed to play a role in regulating the trafficking of G protein-coupled receptors, although mechanistic insight into this process is lacking. Here we focused on characterizing the role of ARRDC3 in regulating the trafficking of the β2-adrenergic receptor (β2AR). We find that ARRDC3 primarily localizes to EEA1-positive early endosomes and directly interacts with the β2AR in a ligand-independent manner. While ARRDC3 has no effect on β2AR endocytosis or degradation, it negatively regulates β2AR entry into SNX27-occupied endosomal tubules. This results in delayed recycling of the receptor and a concomitant increase in β2AR-dependent endosomal signaling. Thus, ARRDC3 functions as a switch to modulate the endosomal residence time and subsequent intracellular signaling of the β2AR.

    View details for DOI 10.1074/jbc.M116.716589

    View details for PubMedID 27226565

  • Accessible Mannitol-Based Amphiphiles (MNAs) for Membrane Protein Solubilisation and Stabilisation. Chemistry (Weinheim an der Bergstrasse, Germany) Hussain, H., Du, Y., Scull, N. J., Mortensen, J. S., Tarrasch, J., Bae, H. E., Loland, C. J., Byrne, B., Kobilka, B. K., Chae, P. S. 2016

    Abstract

    Integral membrane proteins are amphipathic molecules crucial for all cellular life. The structural study of these macromolecules starts with protein extraction from the native membranes, followed by purification and crystallisation. Detergents are essential tools for these processes, but detergent-solubilised membrane proteins often denature and aggregate, resulting in loss of both structure and function. In this study, a novel class of agents, designated mannitol-based amphiphiles (MNAs), were prepared and characterised for their ability to solubilise and stabilise membrane proteins. Some of MNAs conferred enhanced stability to four membrane proteins including a G protein-coupled receptor (GPCR), the β2 adrenergic receptor (β2 AR), compared to both n-dodecyl-d-maltoside (DDM) and the other MNAs. These agents were also better than DDM for electron microscopy analysis of the β2 AR. The ease of preparation together with the enhanced membrane protein stabilisation efficacy demonstrates the value of these agents for future membrane protein research.

    View details for DOI 10.1002/chem.201600533

    View details for PubMedID 27072057

  • Allosteric coupling from G protein to the agonist-binding pocket in GPCRs. Nature DeVree, B. T., Mahoney, J. P., Vélez-Ruiz, G. A., Rasmussen, S. G., Kuszak, A. J., Edwald, E., Fung, J. J., Manglik, A., Masureel, M., Du, Y., Matt, R. A., Pardon, E., Steyaert, J., Kobilka, B. K., Sunahara, R. K. 2016; 535 (7610): 182-186

    Abstract

    G-protein-coupled receptors (GPCRs) remain the primary conduit by which cells detect environmental stimuli and communicate with each other. Upon activation by extracellular agonists, these seven-transmembrane-domain-containing receptors interact with heterotrimeric G proteins to regulate downstream second messenger and/or protein kinase cascades. Crystallographic evidence from a prototypic GPCR, the β2-adrenergic receptor (β2AR), in complex with its cognate G protein, Gs, has provided a model for how agonist binding promotes conformational changes that propagate through the GPCR and into the nucleotide-binding pocket of the G protein α-subunit to catalyse GDP release, the key step required for GTP binding and activation of G proteins. The structure also offers hints about how G-protein binding may, in turn, allosterically influence ligand binding. Here we provide functional evidence that G-protein coupling to the β2AR stabilizes a 'closed' receptor conformation characterized by restricted access to and egress from the hormone-binding site. Surprisingly, the effects of G protein on the hormone-binding site can be observed in the absence of a bound agonist, where G-protein coupling driven by basal receptor activity impedes the association of agonists, partial agonists, antagonists and inverse agonists. The ability of bound ligands to dissociate from the receptor is also hindered, providing a structural explanation for the G-protein-mediated enhancement of agonist affinity, which has been observed for many GPCR-G-protein pairs. Our data also indicate that, in contrast to agonist binding alone, coupling of a G protein in the absence of an agonist stabilizes large structural changes in a GPCR. The effects of nucleotide-free G protein on ligand-binding kinetics are shared by other members of the superfamily of GPCRs, suggesting that a common mechanism may underlie G-protein-mediated enhancement of agonist affinity.

    View details for DOI 10.1038/nature18324

    View details for PubMedID 27362234

  • β-arrestin-biased signaling through the β2-adrenergic receptor promotes cardiomyocyte contraction. Proceedings of the National Academy of Sciences of the United States of America Carr, R., Schilling, J., Song, J., Carter, R. L., Du, Y., Yoo, S. M., Traynham, C. J., Koch, W. J., Cheung, J. Y., Tilley, D. G., Benovic, J. L. 2016

    Abstract

    β-adrenergic receptors (βARs) are critical regulators of acute cardiovascular physiology. In response to elevated catecholamine stimulation during development of congestive heart failure (CHF), chronic activation of Gs-dependent β1AR and Gi-dependent β2AR pathways leads to enhanced cardiomyocyte death, reduced β1AR expression, and decreased inotropic reserve. β-blockers act to block excessive catecholamine stimulation of βARs to decrease cellular apoptotic signaling and normalize β1AR expression and inotropy. Whereas these actions reduce cardiac remodeling and mortality outcomes, the effects are not sustained. Converse to G-protein-dependent signaling, β-arrestin-dependent signaling promotes cardiomyocyte survival. Given that β2AR expression is unaltered in CHF, a β-arrestin-biased agonist that operates through the β2AR represents a potentially useful therapeutic approach. Carvedilol, a currently prescribed nonselective β-blocker, has been classified as a β-arrestin-biased agonist that can inhibit basal signaling from βARs and also stimulate cell survival signaling pathways. To understand the relative contribution of β-arrestin bias to the efficacy of select β-blockers, a specific β-arrestin-biased pepducin for the β2AR, intracellular loop (ICL)1-9, was used to decouple β-arrestin-biased signaling from occupation of the orthosteric ligand-binding pocket. With similar efficacy to carvedilol, ICL1-9 was able to promote β2AR phosphorylation, β-arrestin recruitment, β2AR internalization, and β-arrestin-biased signaling. Interestingly, ICL1-9 was also able to induce β2AR- and β-arrestin-dependent and Ca(2+)-independent contractility in primary adult murine cardiomyocytes, whereas carvedilol had no efficacy. Thus, ICL1-9 is an effective tool to access a pharmacological profile stimulating cardioprotective signaling and inotropic effects through the β2AR and serves as a model for the next generation of cardiovascular drug development.

    View details for DOI 10.1073/pnas.1606267113

    View details for PubMedID 27354517

  • Novel Xylene-Linked Maltoside Amphiphiles (XMAs) for Membrane Protein Stabilisation CHEMISTRY-A EUROPEAN JOURNAL Cho, K. H., Du, Y., Scull, N. J., Hariharan, P., Gotfryd, K., Loland, C. J., Guan, L., Byrne, B., Kobilka, B. K., Chae, P. S. 2015; 21 (28): 10008-10013

    Abstract

    Membrane proteins are key functional players in biological systems. These biomacromolecules contain both hydrophilic and hydrophobic regions and thus amphipathic molecules are necessary to extract membrane proteins from their native lipid environments and stabilise them in aqueous solutions. Conventional detergents are commonly used for membrane protein manipulation, but membrane proteins surrounded by these agents often undergo denaturation and aggregation. In this study, a novel class of maltoside-bearing amphiphiles, with a xylene linker in the central region, designated xylene-linked maltoside amphiphiles (XMAs) was developed. When these novel agents were evaluated with a number of membrane proteins, it was found that XMA-4 and XMA-5 have particularly favourable efficacy with respect to membrane protein stabilisation, indicating that these agents hold significant potential for membrane protein structural study.

    View details for DOI 10.1002/chem.201501083

    View details for Web of Science ID 000357026700011

    View details for PubMedID 26013293

  • Effective Application of Bicelles for Conformational Analysis of G Protein-Coupled Receptors by Hydrogen/Deuterium Exchange Mass Spectrometry JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY Nguyen Minh Duc, N. M., Du, Y., Thorsen, T. S., Lee, S. Y., Zhang, C., Kato, H., Kobilka, B. K., Chung, K. Y. 2015; 26 (5): 808-817

    Abstract

    G protein-coupled receptors (GPCRs) have important roles in physiology and pathology, and 40% of drugs currently on the market target GPCRs for the treatment of various diseases. Because of their therapeutic importance, the structural mechanism of GPCR signaling is of great interest in the field of drug discovery. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for analyzing ligand binding sites, the protein-protein interaction interface, and conformational changes of proteins. However, its application to GPCRs has been limited for various reasons, including the hydrophobic nature of GPCRs and the use of detergents in their preparation. In the present study, we tested the application of bicelles as a means of solubilizing GPCRs for HDX-MS studies. GPCRs (e.g., β2-adrenergic receptor [β2AR], μ-opioid receptor, and protease-activated receptor 1) solubilized in bicelles produced better sequence coverage (greater than 90%) than GPCRs solubilized in n-dodecyl-β-D-maltopyranoside (DDM), suggesting that bicelles are a more effective method of solubilization for HDX-MS studies. The HDX-MS profile of β2AR in bicelles showed that transmembrane domains (TMs) undergo lower deuterium uptake than intracellular or extracellular regions, which is consistent with the fact that the TMs are highly ordered and embedded in bicelles. The overall HDX-MS profiles of β2AR solubilized in bicelles and in DDM were similar except for intracellular loop 3. Interestingly, we detected EX1 kinetics, an important phenomenon in protein dynamics, at the C-terminus of TM6 in β2AR. In conclusion, we suggest the application of bicelles as a useful method for solubilizing GPCRs for conformational analysis by HDX-MS.

    View details for DOI 10.1007/s13361-015-1083-4

    View details for Web of Science ID 000352876100014

  • Development and Characterization of Pepducins as G(s)-biased Allosteric Agonists JOURNAL OF BIOLOGICAL CHEMISTRY Carr, R., Du, Y., Quoyer, J., Panettieri, R. A., Janz, J. M., Bouvier, M., Kobilka, B. K., Benovic, J. L. 2014; 289 (52): 35668-35684

    Abstract

    The β2-adrenergic receptor (β2AR) is a prototypical G protein-coupled receptor that mediates many hormonal responses, including cardiovascular and pulmonary function. β-Agonists used to combat hypercontractility in airway smooth muscle stimulate β2AR-dependent cAMP production that ultimately promotes airway relaxation. Chronic stimulation of the β2AR by long acting β-agonists used in the treatment of asthma can promote attenuated responsiveness to agonists and an increased frequency of fatal asthmatic attacks. β2AR desensitization to β-agonists is primarily mediated by G protein-coupled receptor kinases and β-arrestins that attenuate receptor-Gs coupling and promote β2AR internalization and degradation. A biased agonist that can selectively stimulate Gs signaling without promoting receptor interaction with G protein-coupled receptor kinases and β-arrestins should serve as an advantageous asthma therapeutic. To identify such molecules, we screened ∼50 lipidated peptides derived from the intracellular loops of the β2AR, known as pepducins. This screen revealed two classes of Gs-biased pepducins, receptor-independent and receptor-dependent, as well as several β-arrestin-biased pepducins. The receptor-independent Gs-biased pepducins operate by directly stimulating G protein activation. In contrast, receptor-dependent Gs-biased pepducins appear to stabilize a Gs-biased conformation of the β2AR that couples to Gs but does not undergo G protein-coupled receptor kinase-mediated phosphorylation or β-arrestin-mediated internalization. Functional studies in primary human airway smooth muscle cells demonstrate that Gs-biased pepducins are not subject to conventional desensitization and thus may be good candidates for the development of next generation asthma therapeutics. Our study reports the first Gs-biased activator of the β2AR and provides valuable tools for the study of β2AR function.

    View details for DOI 10.1074/jbc.M114.618819

    View details for Web of Science ID 000346897900001

  • Expression profiling reveals an unexpected growth-stimulating effect of surplus iron on the yeast Saccharomyces cerevisiae MOLECULES AND CELLS Du, Y., Cheng, W., Li, W. 2012; 34 (2): 127-32
  • Structures of the substrate-binding protein provide insights into the multiple compatible solute binding specificities of the Bacillus subtilis ABC transporter OpuC BIOCHEMICAL JOURNAL Du, Y., Shi, W., He, Y., Yang, Y., Zhou, C., Chen, Y. 2011; 436: 283-289

    Abstract

    The compatible solute ABC (ATP-binding cassette) transporters are indispensable for acquiring a variety of compatible solutes under osmotic stress in Bacillus subtilis. The substrate-binding protein OpuCC (Opu is osmoprotectant uptake) of the ABC transporter OpuC can recognize a broad spectrum of compatible solutes, compared with its 70% sequence-identical paralogue OpuBC that can solely bind choline. To explore the structural basis of this difference of substrate specificity, we determined crystal structures of OpuCC in the apo-form and in complex with carnitine, glycine betaine, choline and ectoine respectively. OpuCC is composed of two α/β/α globular sandwich domains linked by two hinge regions, with a substrate-binding pocket located at the interdomain cleft. Upon substrate binding, the two domains shift towards each other to trap the substrate. Comparative structural analysis revealed a plastic pocket that fits various compatible solutes, which attributes themultiple-substrate binding property to OpuCC. This plasticity is a gain-of-function via a single-residue mutation of Thr⁹⁴ in OpuCC compared with Asp⁹⁶ in OpuBC.

    View details for DOI 10.1042/BJ20102097

    View details for Web of Science ID 000291413200009

    View details for PubMedID 21366542

  • Crystal structure of the mucin-binding domain of Spr1345 from Streptococcus pneumoniae JOURNAL OF STRUCTURAL BIOLOGY Du, Y., He, Y., Zhang, Z., Yang, Y., Shi, W., Frolet, C., Di Guilmi, A., Vernet, T., Zhou, C., Chen, Y. 2011; 174 (1): 252-257

    Abstract

    The surface protein Spr1345 from Streptococcus pneumoniae R6 is a 22-kDa mucin-binding protein (MucBP) involved in adherence and colonization of the human lung and respiratory tract. It is composed of a mucin-binding domain (MucBD) and a proline-rich domain (PRD) followed by an LPxTG motif, which is recognized and cleaved by sortase, resulting in a mature form of 171 residues (MF171) that is anchored to the cell wall. We found that the MucBD alone possesses comparable in vitro mucin-binding affinity to the mature form, and can be specifically enriched at the surface of human lung carcinoma A549 cells. Using single-wavelength anomalous dispersion (SAD) phasing method with the iodine signals, we solved the crystal structure of the MucBD at 2.0Å resolution, the first structure of MucBDs from pathogenic bacteria. The overall structure adopts an immunoglobulin-like fold with an elongated rod-like shape, composed of six anti-parallel β-strands and a long loop. Structural comparison suggested that the conserved C-terminal moiety may participate in the recognition of mucins. These findings provided structural insights into host-pathogen interaction mediated by mucins, which might be useful for designing novel vaccines and antibiotic drugs against human diseases caused by pneumococci.

    View details for DOI 10.1016/j.jsb.2010.10.016

    View details for Web of Science ID 000288640100030

    View details for PubMedID 21055474

  • Crystal structures of the apo and GDP-bound forms of a cupin-like protein BbDUF985 from Branchiostoma belcheri tsingtauense PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS Du, Y., He, Y., Gaowa, S., Zhang, X., Chen, Y., Zhang, S., Zhou, C. 2010; 78 (12): 2714-2719

    View details for DOI 10.1002/prot.22771

    View details for Web of Science ID 000280822000013

    View details for PubMedID 20589641

  • Crystal structure of Saccharomyces cerevisiae glutamine synthetase Gln1 suggests a nanotube-like supramolecular assembly PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS He, Y., Gui, L., Liu, Y., Du, Y., Zhou, Y., Li, P., Zhou, C. 2009; 76 (1): 249-254

    View details for DOI 10.1002/prot.22403

    View details for Web of Science ID 000266370100021

    View details for PubMedID 19322816