Cleavage of amyloid precursor protein (APP) by the intramembrane protease g-secretase is linked to Alzheimer’s disease (AD). We report an atomic structure of human g-secretase in complex with a transmembrane (TM) APP fragment at 2.6-angstrom resolution. The TM helix of APP closely interacts with five surrounding TMs of PS1 (the catalytic subunit of g-secretase). A hybrid b sheet, which is formed by a b strand from APP and two b strands from PS1, guides g-secretase to the scissile peptide bond of APP between its TM and b strand. Residues at the interface between PS1 and APP are heavily targeted by recurring mutations from AD patients. This structure, together with that of g-secretase bound to Notch, reveal contrasting features of substrate binding, which may be applied toward the design of substrate-specific inhibitors.

Rui Zhou;Guanghui Yang;Xuefei Guo;Qiang Zhou;Jianlin Lei;一公 施

Science

2019-2-15

γ-secretase, a membrane-embedded aspartate protease, catalyzes peptide bond hydrolysis of a large variety of type I integral membrane proteins exemplified by amyloid precursor protein (APP). Cleavage of APP leads to formation of β-amyloid plaque, which is a hallmark of Alzheimer's disease (AD). Over 200 AD-associated mutations are mapped to presenilin 1 (PS1), the catalytic component of γ-secretase. In the past three years, several cryo-electron microscopy (cryo-EM) structures of human γ-secretase have been determined at near atomic resolutions. Here we summarize the methods involved and discuss structural features of γ-secretase and the associated functional insights.

Guanghui Yang;Rui Zhou;一公 施

Current Opinion in Structural Biology

2017-10

Hemoglobin A (HbA), the oxygen delivery system in humans, comprises two α and two β subunits. Free α-hemoglobin (αHb) is unstable, and its precipitation contributes to the pathophysiology of β thalassemia. In erythrocytes, the α-hemoglobin stabilizing protein (AHSP) binds αHb and inhibits its precipitation. The crystal structure of AHSP bound to Fe(II)-αHb reveals that AHSP specifically recognizes the G and H helices of αHb through a hydrophobic interface that largely recapitulates the α₁-β₁ interface of hemoglobin. The AHSP-αHb interactions are extensive but suboptimal, explaining why β-hemoglobin can competitively displace AHSP to form HbA. Remarkably, the Fe(II)-heme group in AHSP bound αHb is coordinated by the distal but not the proximal histidine. Importantly, binding to AHSP facilitates the conversion of oxy-αHb to a deoxygenated, oxidized [Fe(III)], nonreactive form in which all six coordinate positions are occupied. These observations reveal the molecular mechanisms by which AHSP stabilizes free αHb.

Liang Feng;David A. Gell;Suiping Zhou;立川 谷;Yi Kong;Jianqing Li;Min Hu;Nieng Yan;Christopher Lee;Anne M. Rich;Robert S. Armstrong;Peter A. Lay;Andrew J. Gow;Mitchell J. Weiss;Joel P. MacKay;一公 施

Cell

2004-11-24

Smad proteins mediate transforming growth factor β signaling from the cell membrane to the nucleus. Upon phosphorylation by the activated receptor kinases, the receptor-regulated Smad, such as Smad2, forms a heterocomplex with the co-mediator Smad, Smad4. This heterocomplex is then translocated into the nucleus, where it associates with other transcription factors and regulates expression of ligand-responsive genes. The stoichiometry between receptor-regulated Smad and co-mediator Smad is important for understanding the molecular mechanisms of the signaling process. Using purified recombinant proteins, we demonstrate that Smad2 and Smad4 form a stable heterodimer and that the Smad4 activation domain is important for the formation of this complex. Many tumor-derived missense mutations disrupt the formation of this heterocomplex in in vitro interaction assays. Mapping these mutations onto the structures of Smad4 and Smad2 identifies a symmetric interface between these two Smad proteins. Importantly, two previous models on the formation of a heterocomplex are incompatible with our observations and other reported evidence.

Jia Wei Wu;Robert Fairman;Jack Penry;一公 施

Journal of Biological Chemistry

2001-6-8

Lijun Zhou;Yulin Zhou;Jing Hang;Ruixue Wan;Guifeng Lu;Chuangye Yan;一公 施

Cell Research

2014-4

Apoptosis is executed by a cascade of caspase activation. The autocatalytic activation of an initiator caspase, exemplified by caspase-9 in mammals or its ortholog, Dronc, in fruit flies, is facilitated by a multimeric adaptor complex known as the apoptosome. The underlying mechanism by which caspase-9 or Dronc is activated by the apoptosome remains unknown. Here we report the electron cryomicroscopic (cryo-EM) structure of the intact apoptosome from Drosophila melanogaster at 4.0 Å resolution. Analysis of the Drosophila apoptosome, which comprises 16 molecules of the Dark protein (Apaf-1 ortholog), reveals molecular determinants that support the assembly of the 2.5-MDa complex. In the absence of dATP or ATP, Dronc zymogen potently induces formation of the Dark apoptosome, within which Dronc is efficiently activated. At 4.1Å resolution, the cryo-EM structure of the Dark apoptosome bound to the caspase recruitment domain (CARD) of Dronc (Dronc-CARD) reveals two stacked rings of Dronc-CARD that are sandwiched between two octameric rings of the Dark protein. The specific interactions between Dronc-CARD and both the CARD and the WD40 repeats of a nearby Dark protomer are indispensable for Dronc activation. These findings reveal important mechanistic insights into the activation of initiator caspase by the apoptosome.

Yuxuan Pang;Xiao Chen Bai;Chuangye Yan;Qi Hao;Zheqin Chen;Jia Wei Wang;Sjors H.W. Scheres;一公 施

Genes and Development

2015

Enteropathogenic bacteria, exemplified by Escherichia coli, rely on acid-resistance systems (ARs) to survive the acidic environment of the stomach. AR3 consumes intracellular protons through decarboxylation of arginine (Arg) in the cytoplasm and exchange of the reaction product agmatine (Agm) with extracellular Arg. The latter process is mediated by the Arg:Agm antiporter AdiC, which is activated in response to acidic pH and remains fully active at pH 6.0 and below. Despite our knowledge of structural information, the molecular mechanism by which AdiC senses acidic pH remains completely unknown. Relying on alanine-scanning mutagenesis and an in vitro proteoliposome-based transport assay, we have identified Tyr74 as a critical pH sensor in AdiC. The AdiC variant Y74A exhibited robust transport activity at all pH values examined while maintaining stringent substrate specificity for Arg:Agm. Replacement of Tyr74 by Phe, but not by any other amino acid, led to the maintenance of pH-dependent substrate transport. These observations, in conjunction with structural information, identify a working model for pH-induced activation of AdiC in which a closed conformation is disrupted by cation-π interactions between proton and the aromatic side chain of Tyr74.

Sheng Wang;Renhong Yan;Xi Zhang;Qi Chu;一公 施

Proceedings of the National Academy of Sciences of the United States of America

2014-9-2

γ-Secretase is an intramembrane protease responsible for the generation of amyloid-β (Aβ) peptides. Aberrant accumulation of Aβ leads to the formation of amyloid plaques in the brain of patients with Alzheimer's disease. Nicastrin is the putative substrate-recruiting component of the γ-secretase complex. No atomic-resolution structure had been identified on γ-secretase or any of its four components, hindering mechanistic understanding of γ-secretase function. Here we report the crystal structure of nicas-trin from Dictyostelium purpureum at 1.95-Å resolution. The extracellular domain of nicastrin contains a large lobe and a small lobe. The large lobe of nicastrin, thought to be responsible for substrate recognition, associates with the small lobe through a hy-drophobic pivot at the center. The putative substrate-binding pocket is shielded from the small lobe by a lid, which blocks substrate entry. These structural features suggest a working model of nicastrin function. Analysis of nicastrin structure provides insights into the assembly and architecture of the γ-secretase complex.

Tian Xie;Chuangye Yan;Rui Zhou;Yanyu Zhao;Linfeng Sun;Guanghui Yang;Peilong Lu;Dan Ma;一公 施

Proceedings of the National Academy of Sciences of the United States of America

2014-9-16

Apaf-1 and cytochrome c coassemble in the presence of dATP to form the apoptosome. We have determined a structure of the apoptosome at 12.8 Å resolution by using electron cryomicroscopy and single-particle methods. We then docked appropriate crystal structures into the map to create an accurate domain model. Thus, we found that seven caspase recruitment domains (CARDs) form a central ring within the apoptosome. At a larger radius, seven copies of the nucleotide binding and oligomerization domain (NOD) associate laterally to form the hub, which encircles the CARD ring. Finally, an arm-like helical domain (HD2) links each NOD to a pair of β propellers, which bind a single cytochrome c. This model provides insights into the roles of dATP and cytochrome c in assembly. Our structure also reveals how a CARD ring and the central hub combine to create a platform for procaspase-9 activation.

Xinchao Yu;Devrim Acehan;Jean François Ménétret;Christopher R. Booth;Steven J. Ludtke;Stefan J. Riedl;一公 施;Xiaodong Wang;Christopher W. Akey

Structure

2005-11

Inhibitors of apoptosis proteins (IAPs) interact with caspases and inhibit their protease activity, whereas the IAP-inhibitory proteins Smac/DIABLO in mammals and Reaper, Hid, and Grim in flies relieve IAP-mediated inhibition [1-5] to induce cell death. Here we describe the functional characterization of the novel Drosophila cell death protein Sickle (Skl), which binds to IAPs and neutralizes their apoptotic inhibitory activity. Skl exhibits no sequence homology to Reaper, Hid, Grim, or Smac/DIABLO, except within the 4 residue N-terminal IAP binding motif. Skl interacts with Drosophila and mammalian IAPs and can promote caspase activation in the presence of IAPs. Consistent with these findings, expression of Skl in Drosophila and mammalian cell lines or in Drosophila embryos induces apoptosis. Skl can also synergize with Grim to induce cell death in the Drosophila eye imaginal disc. Based on biochemical and structural data, the N terminus of Skl, like that of the mammalian Smac/DIABLO, is absolutely required for its apoptotic and caspase-promoting activities and its ability to interact with IAPs. These findings point to conservation in the structure and function of the IAP-inhibitory proteins across species and suggest the existence of other family members.

Srinivasa M. Srinivasula;Pinaki Datta;Masatomo Kobayashi;Jia Wei Wu;Miki Fujioka;Ramesh Hegde;Zhi Jia Zhang;Rula Mukattash;Teresa Fernandes-Alnemri;一公 施;James B. Jaynes;Emad S. Alnemri

Current Biology

2002-1-22