Person: Mao, Youdong
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Mao
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Youdong
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Mao, Youdong
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Publication Evaluation of the contribution of the transmembrane region to the ectodomain conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein(BioMed Central, 2017) Nguyen, Hanh; Madani, Navid; Ding, Haitao; Elder, Emerald; Princiotto, Amy; Gu, Christopher; Darby, Patrice; Alin, James; Herschhorn, Alon; Kappes, John C.; Mao, Youdong; Sodroski, JosephBackground: The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env), a Type 1 transmembrane protein, assembles into a trimeric spike complex that mediates virus entry into host cells. The high potential energy of the metastable, unliganded Env trimer is maintained by multiple non-covalent contacts among the gp120 exterior and gp41 transmembrane Env subunits. Structural studies suggest that the gp41 transmembrane region forms a left-handed coiled coil that contributes to the Env trimer interprotomer contacts. Here we evaluate the contribution of the gp41 transmembrane region to the folding and stability of Env trimers. Methods: Multiple polar/charged amino acid residues, which hypothetically disrupt the stop-transfer signal, were introduced in the proposed lipid-interactive face of the transmembrane coiled coil, allowing release of soluble cleavage-negative Envs containing the modified transmembrane region (TMmod). We also examined effects of cleavage, the cytoplasmic tail and a C-terminal fibritin trimerization (FT) motif on oligomerization, antigenicity and functionality of soluble and membrane-bound Envs. Results: The introduction of polar/charged amino acids into the transmembrane region resulted in the secretion of soluble Envs from the cell. However, these TMmod Envs primarily formed dimers. By contrast, control cleavage-negative sgp140 Envs lacking the transmembrane region formed soluble trimers, dimers and monomers. TMmod and sgp140 trimers were stabilized by the addition of a C-terminal FT sequence, but still exhibited carbohydrate and antigenic signatures of a flexible ectodomain structure. On the other hand, detergent-solubilized cleaved and uncleaved Envs isolated from the membranes of expressing cells exhibited "tighter” ectodomain structures, based on carbohydrate modifications. These trimers were found to be unstable in detergent solutions, but could be stabilized by the addition of a C-terminal FT moiety. The C-terminal FT domain decreased Env cleavage and syncytium-forming ability by approximately three-fold; alteration of the FT trimerization interface restored Env cleavage and syncytium formation to near-wild-type levels. Conclusion: The modified transmembrane region was not conducive to trimerization of soluble Envs. However, for HIV-1 Env ectodomains that are minimally modified, membrane-anchored Envs exhibit the most native structures and can be stabilized by appropriately positioned FT domains.Publication Molecular architecture of the uncleaved HIV-1 envelope glycoprotein trimer(BioMed Central, 2013) Mao, Youdong; Castillo-Menendez, Luis; Wang, Liping; Gu, Christopher; Herschhorn, Alon; Désormeaux, Anik; Finzi, Andres; Xiang, Shi-Hua; Sodroski, JosephPublication A Twin-Cysteine Motif in the V2 Region of gp120 Is Associated with SIV Envelope Trimer Stabilization(Public Library of Science, 2013) Bohl, Christopher; Bowder, Dane; Thompson, Jesse; Abrahamyan, Levon; Gonzalez-Ramirez, Sandra; Mao, Youdong; Sodroski, Joseph; Wood, Charles; Xiang, Shi-huaThe V1 and V2 variable regions of the primate immunodeficiency viruses contribute to the trimer association domain of the gp120 exterior envelope glycoprotein. A pair of V2 cysteine residues at 183 and 191 (“twin cysteines”) is present in several simian immunodeficiency viruses, human immunodeficiency virus type 2 (HIV-2) and some SIVcpz lineages, but not in HIV-1. To examine the role of this potentially disulfide-bonded twin-cysteine motif, the cysteine residues in the SIVmac239 envelope glycoproteins were individually and pairwise substituted by alanine residues. All of the twin-cysteine mutants exhibited decreases in gp120 association with the Env trimer, membrane-fusing activity, and ability to support virus entry. Thus, the twin-cysteine motif plays a role in Env trimer stabilization in SIV and may do so in HIV-2 and some SIVcpz as well. This implies that HIV-1 lost the twin-cysteines, and may have relatively unstable Env trimers compared to SIV and HIV-2.Publication Molecular basis of caspase-1 polymerization and its inhibition by a novel capping mechanism(2016) Lu, Alvin; Li, Yang; Schmidt, Florian I.; Yin, Qian; Chen, Shuobing; Fu, Tian-Min; Tong, Alexander B.; Ploegh, Hidde L.; Mao, Youdong; Wu, HaoInflammasomes are cytosolic caspase-1 activation complexes that sense intrinsic and extrinsic danger signals to trigger inflammatory responses and pyroptotic cell death. Homotypic interactions by Pyrin domains (PYD) and caspase recruitment domains (CARD) in inflammasome component proteins mediate oligomerization into filamentous assemblies. Several cytosolic proteins consisting of only the interaction domains exert inhibitory effects on inflammasome assembly. In this study, we determined the structure of human caspase-1CARD filament by cryo-electron microscopy and investigated the biophysical properties of two caspase-1-like CARD-only proteins, human inhibitor of CARD (INCA or CARD17) and ICEBERG (or CARD18). Our results reveal the surprising finding that INCA caps caspase-1 filament, thereby exerting potent inhibition with low nanomolar Ki on caspase-1CARD polymerization in vitro and inflammasome activation in cells. While caspase-1CARD uses six complementary surfaces of three types for filament assembly, INCA is defective in two of the six interfaces to terminate caspase-1 filament.Publication Unsupervised Cryo-EM Data Clustering through Adaptively Constrained K-Means Algorithm(Public Library of Science, 2016) Xu, Yaofang; Wu, Jiayi; Yin, Chang-Cheng; Mao, YoudongIn single-particle cryo-electron microscopy (cryo-EM), K-means clustering algorithm is widely used in unsupervised 2D classification of projection images of biological macromolecules. 3D ab initio reconstruction requires accurate unsupervised classification in order to separate molecular projections of distinct orientations. Due to background noise in single-particle images and uncertainty of molecular orientations, traditional K-means clustering algorithm may classify images into wrong classes and produce classes with a large variation in membership. Overcoming these limitations requires further development on clustering algorithms for cryo-EM data analysis. We propose a novel unsupervised data clustering method building upon the traditional K-means algorithm. By introducing an adaptive constraint term in the objective function, our algorithm not only avoids a large variation in class sizes but also produces more accurate data clustering. Applications of this approach to both simulated and experimental cryo-EM data demonstrate that our algorithm is a significantly improved alterative to the traditional K-means algorithm in single-particle cryo-EM analysis.Publication Subunit organization of the membrane-bound HIV-1 envelope glycoprotein trimer(2012) Mao, Youdong; Wang, Liping; Gu, Christopher; Herschhorn, Alon; Xiang, Shi-Hua; Haim, Hillel; Yang, Xinzhen; Sodroski, JosephThe trimeric human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) spike is a molecular machine that mediates virus entry into host cells and is the sole target for virus-neutralizing antibodies. The mature Env spike results from cleavage of a trimeric gp160 precursor into three gp120 and three gp41 subunits. Here we describe an ~11-Å cryo-EM structure of the trimeric HIV-1 Env precursor in its unliganded state. The three gp120 and three gp41 subunits form a cage-like structure with an interior void surrounding the trimer axis. Interprotomer contacts are limited to the gp41 transmembrane region, the torus-like gp41 ectodomain, and a gp120 trimer association domain composed of the V1/V2 and V3 variable regions. The cage-like architecture, which is unique among characterized viral envelope proteins, restricts antibody access, reflecting requirements imposed by HIV-1 persistence in the host.Publication Cryo-EM structures and dynamics of substrate-engaged human 26S proteasome(Springer Nature, 2018-11-12) Dong, Yuanchen; Zhang, Shuwen; Wu, Zhaolong; Li, Xuemei; Wang, Wei; Zhu, Yanan; Stoilova-McPhie, Svetla; Lu, Ying; Finley, Daniel; Mao, YoudongThe proteasome is an ATP-dependent, 2.5-megadalton molecular machine that is responsible for selective protein degradation in eukaryotic cells. Here we present cryo-electron microscopy structures of the substrate-engaged human proteasome in seven conformational states at 2.8–3.6 Å resolution, captured during breakdown of a polyubiquitylated protein. These structures illuminate a spatiotemporal continuum of dynamic substrate–proteasome interactions from ubiquitin recognition to substrate translocation, during which ATP hydrolysis sequentially navigates through all six ATPases. There are three principal modes of coordinated hydrolysis, featuring hydrolytic events in two oppositely positioned ATPases, in two adjacent ATPases and in one ATPase at a time. These hydrolytic modes regulate deubiquitylation, initiation of translocation and processive unfolding of substrates, respectively. Hydrolysis of ATP powers a hinge-like motion in each ATPase that regulates its substrate interaction. Synchronization of ATP binding, ADP release and ATP hydrolysis in three adjacent ATPases drives rigid-body rotations of substrate-bound ATPases that are propagated unidirectionally in the ATPase ring and unfold the substrate.