Publications by authors named "Uhn Soo Cho"

30 Publications

  • Page 1 of 1

Dismantling and Rebuilding the Trisulfide Cofactor Demonstrates Its Essential Role in Human Sulfide Quinone Oxidoreductase.

J Am Chem Soc 2020 08 10;142(33):14295-14306. Epub 2020 Aug 10.

Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109, United States.

Sulfide quinone oxidoreductase (SQOR) catalyzes the first step in sulfide clearance, coupling HS oxidation to coenzyme Q reduction. Recent structures of human SQOR revealed a sulfur atom bridging the SQOR active site cysteines in a trisulfide configuration. Here, we assessed the importance of this cofactor using kinetic, crystallographic, and computational modeling approaches. Cyanolysis of SQOR proceeds via formation of an intense charge transfer complex that subsequently decays to eliminate thiocyanate. We captured a disulfanyl-methanimido thioate intermediate in the SQOR crystal structure, revealing how cyanolysis leads to reversible loss of SQOR activity that is restored in the presence of sulfide. Computational modeling and MD simulations revealed an ∼10-fold rate enhancement for nucleophilic addition of sulfide into the trisulfide versus a disulfide cofactor. The cysteine trisulfide in SQOR is thus critical for activity and provides a significant catalytic advantage over a cysteine disulfide.
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http://dx.doi.org/10.1021/jacs.0c06066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7442744PMC
August 2020

Insights on the regulation of the MLL/SET1 family histone methyltransferases.

Biochim Biophys Acta Gene Regul Mech 2020 07 15;1863(7):194561. Epub 2020 Apr 15.

Department of Pathology, University of Michigan, Ann Arbor, United States of America; Department of Biological Chemistry, University of Michigan, Ann Arbor, United States of America. Electronic address:

In eukaryotes, histone H3K4 methylation by the MLL/SET1 family histone methyltransferases is enriched at transcription regulatory elements including gene promoters and enhancers. The level of H3K4 methylation is highly correlated with transcription activation and is one of the most frequently used histone post-translational modifications to predict transcriptional outcome. Recently, it has been shown that rearrangement of the cellular landscape of H3K4 mono-methylation at distal enhancers precedes cell fate transition and is used for identification of novel regulatory elements for development and disease progression. Similarly, broad H3K4 tri-methylation regions have also been used to predict intrinsic tumor suppression properties of regulator regions in a variety of cellular models. Understanding the regulation for how H3K4 methylation is deposited and regulated is of paramount importance. In this review, we will discuss new findings on how the MLL/SET1 family enzymes are regulated on chromatin and their potential functional and regulatory implications. This article is part of a Special Issue entitled: The MLL family of proteins in normal development and disease edited by Thomas A Milne.
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http://dx.doi.org/10.1016/j.bbagrm.2020.194561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236755PMC
July 2020

An H3K9 methylation-dependent protein interaction regulates the non-enzymatic functions of a putative histone demethylase.

Elife 2020 03 20;9. Epub 2020 Mar 20.

Department of Biological Chemistry, University of Michigan, Ann Arbor, United States.

H3K9 methylation (H3K9me) specifies the establishment and maintenance of transcriptionally silent epigenetic states or heterochromatin. The enzymatic erasure of histone modifications is widely assumed to be the primary mechanism that reverses epigenetic silencing. Here, we reveal an inversion of this paradigm where a putative histone demethylase Epe1 in fission yeast, has a non-enzymatic function that opposes heterochromatin assembly. Mutations within the putative catalytic JmjC domain of Epe1 disrupt its interaction with Swi6 suggesting that this domain might have other functions besides enzymatic activity. The C-terminus of Epe1 directly interacts with Swi6, and H3K9 methylation stimulates this protein-protein interaction in vitro and in vivo. Expressing the Epe1 C-terminus is sufficient to disrupt heterochromatin by outcompeting the histone deacetylase, Clr3 from sites of heterochromatin formation. Our results underscore how histone modifying proteins that resemble enzymes have non-catalytic functions that regulate the assembly of epigenetic complexes in cells.
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http://dx.doi.org/10.7554/eLife.53155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192584PMC
March 2020

Publisher Correction: Cryo-EM structure of the human MLL1 core complex bound to the nucleosome.

Nat Commun 2020 02 27;11(1):1165. Epub 2020 Feb 27.

Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, 48109, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-14973-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046784PMC
February 2020

Cryo-EM structure of the human MLL1 core complex bound to the nucleosome.

Nat Commun 2019 12 5;10(1):5540. Epub 2019 Dec 5.

Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, 48109, USA.

Mixed lineage leukemia (MLL) family histone methyltransferases are enzymes that deposit histone H3 Lys4 (K4) mono-/di-/tri-methylation and regulate gene expression in mammals. Despite extensive structural and biochemical studies, the molecular mechanisms whereby the MLL complexes recognize histone H3K4 within nucleosome core particles (NCPs) remain unclear. Here we report the single-particle cryo-electron microscopy (cryo-EM) structure of the NCP-bound human MLL1 core complex. We show that the MLL1 core complex anchors to the NCP via the conserved RbBP5 and ASH2L, which interact extensively with nucleosomal DNA and the surface close to the N-terminal tail of histone H4. Concurrent interactions of RbBP5 and ASH2L with the NCP uniquely align the catalytic MLL1 domain at the nucleosome dyad, thereby facilitating symmetrical access to both H3K4 substrates within the NCP. Our study sheds light on how the MLL1 complex engages chromatin and how chromatin binding promotes MLL1 tri-methylation activity.
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http://dx.doi.org/10.1038/s41467-019-13550-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895043PMC
December 2019

Concurrent activation of growth factor and nutrient arms of mTORC1 induces oxidative liver injury.

Cell Discov 2019 19;5:60. Epub 2019 Nov 19.

1Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109 USA.

mTORC1 is a protein kinase important for metabolism and is regulated by growth factor and nutrient signaling pathways, mediated by the Rheb and Rag GTPases, respectively. Here we provide the first animal model in which both pathways were upregulated through concurrent mutations in their GTPase-activating proteins, and . Unlike former models that induced limited mTORC1 upregulation, hepatic deletion of both and (DKO) produced strong, synergistic activation of the mTORC1 pathway and provoked pronounced and widespread hepatocyte damage, leading to externally visible liver failure phenotypes, such as jaundice and systemic growth defects. The transcriptome profile of DKO was different from single knockout mutants but similar to those of diseased human livers with severe hepatitis and mouse livers challenged with oxidative stress-inducing chemicals. In addition, DKO liver cells exhibited prominent molecular pathologies associated with excessive endoplasmic reticulum (ER) stress, oxidative stress, DNA damage and inflammation. Although DKO liver pathologies were ameliorated by mTORC1 inhibition, ER stress suppression unexpectedly aggravated them, suggesting that ER stress signaling is not the major conduit of how hyperactive mTORC1 produces liver damage. Interestingly, superoxide scavengers N-acetylcysteine (NAC) and Tempol, chemicals that reduce oxidative stress, were able to recover liver phenotypes, indicating that mTORC1 hyperactivation induced liver damage mainly through oxidative stress pathways. Our study provides a new model of unregulated mTORC1 activation through concomitant upregulation of growth factor and nutrient signaling axes and shows that mTORC1 hyperactivation alone can provoke oxidative tissue injury.
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http://dx.doi.org/10.1038/s41421-019-0131-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868011PMC
November 2019

MMOD-induced structural changes of hydroxylase in soluble methane monooxygenase.

Sci Adv 2019 10 2;5(10):eaax0059. Epub 2019 Oct 2.

Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.

Soluble methane monooxygenase in methanotrophs converts methane to methanol under ambient conditions. The maximum catalytic activity of hydroxylase (MMOH) is achieved through the interplay of its regulatory protein (MMOB) and reductase. An additional auxiliary protein, MMOD, functions as an inhibitor of MMOH; however, its inhibitory mechanism remains unknown. Here, we report the crystal structure of the MMOH-MMOD complex from strain 5 (2.6 Å). Its structure illustrates that MMOD associates with the canyon region of MMOH where MMOB binds. Although MMOD and MMOB recognize the same binding site, each binding component triggers different conformational changes toward MMOH, which then respectively lead to the inhibition and activation of MMOH. Particularly, MMOD binding perturbs the di-iron geometry by inducing two major MMOH conformational changes, i.e., MMOH β subunit disorganization and subsequent His dissociation with Fe1 coordination. Furthermore, 1,6-hexanediol, a mimic of the products of sMMO, reveals the substrate access route.
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http://dx.doi.org/10.1126/sciadv.aax0059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6774732PMC
October 2019

A Catalytic Trisulfide in Human Sulfide Quinone Oxidoreductase Catalyzes Coenzyme A Persulfide Synthesis and Inhibits Butyrate Oxidation.

Cell Chem Biol 2019 11 4;26(11):1515-1525.e4. Epub 2019 Oct 4.

Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA. Electronic address:

Mitochondrial sulfide quinone oxidoreductase (SQR) catalyzes the oxidation of HS to glutathione persulfide with concomitant reduction of CoQ. We report herein that the promiscuous activity of human SQR supported the conversion of CoA to CoA-SSH (CoA-persulfide), a potent inhibitor of butyryl-CoA dehydrogenase, and revealed a molecular link between sulfide and butyrate metabolism, which are known to interact. Three different CoQ-bound crystal structures furnished insights into how diverse substrates access human SQR, and provided snapshots of the reaction coordinate. Unexpectedly, the active site cysteines in SQR are configured in a bridging trisulfide at the start and end of the catalytic cycle, and the presence of sulfane sulfur was confirmed biochemically. Importantly, our study leads to a mechanistic proposal for human SQR in which sulfide addition to the trisulfide cofactor eliminates Cys-SSH, forming an intense charge-transfer complex with flavin adenine dinucleotide, and Cys-SSH, which transfers sulfur to an external acceptor.
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http://dx.doi.org/10.1016/j.chembiol.2019.09.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906606PMC
November 2019

Structures of CENP-C cupin domains at regional centromeres reveal unique patterns of dimerization and recruitment functions for the inner pocket.

J Biol Chem 2019 09 31;294(38):14119-14134. Epub 2019 Jul 31.

Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109

The successful assembly and regulation of the kinetochore are critical for the equal and accurate segregation of genetic material during the cell cycle. CENP-C (centromere protein C), a conserved inner kinetochore component, has been broadly characterized as a scaffolding protein and is required for the recruitment of multiple kinetochore proteins to the centromere. At its C terminus, CENP-C harbors a conserved cupin domain that has an established role in protein dimerization. Although the crystal structure of the Mif2 cupin domain has been determined, centromeric organization and kinetochore composition vary greatly between (point centromere) and other eukaryotes (regional centromere). Therefore, whether the structural and functional role of the cupin domain is conserved throughout evolution requires investigation. Here, we report the crystal structures of the and CENP-C cupin domains at 2.52 and 1.81 Å resolutions, respectively. Although the central jelly roll architecture is conserved among the three determined CENP-C cupin domain structures, the cupin domains from organisms with regional centromeres contain additional structural features that aid in dimerization. Moreover, we found that the Cnp3 jelly roll fold harbors an inner binding pocket that is used to recruit the meiosis-specific protein Moa1. In summary, our results unveil the evolutionarily conserved and unique features of the CENP-C cupin domain and uncover the mechanism by which it functions as a recruitment factor.
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http://dx.doi.org/10.1074/jbc.RA119.008464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6755791PMC
September 2019

-3-Carboxypropyl-l-cysteine specifically inhibits cystathionine γ-lyase-dependent hydrogen sulfide synthesis.

J Biol Chem 2019 07 3;294(28):11011-11022. Epub 2019 Jun 3.

Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109 and. Electronic address:

Hydrogen sulfide (HS) is a gaseous signaling molecule, which modulates a wide range of mammalian physiological processes. Cystathionine γ-lyase (CSE) catalyzes HS synthesis and is a potential target for modulating HS levels under pathophysiological conditions. CSE is inhibited by propargylglycine (PPG), a widely used mechanism-based inhibitor. In this study, we report that inhibition of HS synthesis from cysteine, but not the canonical cystathionine cleavage reaction catalyzed by CSE , is sensitive to preincubation of the enzyme with PPG. In contrast, the efficacy of -3-carboxpropyl-l-cysteine (CPC) a new inhibitor described herein, was not dependent on the order of substrate/inhibitor addition. We observed that CPC inhibited the γ-elimination reaction of cystathionine and HS synthesis from cysteine by human CSE with values of 50 ± 3 and 180 ± 15 μm, respectively. We noted that CPC spared the other enzymes involved either directly (cystathionine β-synthase and mercaptopyruvate sulfurtransferase) or indirectly (cysteine aminotransferase) in HS biogenesis. CPC also targeted CSE in cultured cells, inhibiting transsulfuration flux by 80-90%, as monitored by the transfer of radiolabel from [S]methionine to GSH. The 2.5 Å resolution crystal structure of human CSE in complex with the CPC-derived aminoacrylate intermediate provided a structural framework for the molecular basis of its inhibitory effect. In summary, our study reveals a previously unknown confounding effect of PPG, widely used to inhibit CSE-dependent HS synthesis, and reports on an alternative inhibitor, CPC, which could be used as a scaffold to develop more potent HS biogenesis inhibitors.
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http://dx.doi.org/10.1074/jbc.RA119.009047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635441PMC
July 2019

Mis16 Switches Function from a Histone H4 Chaperone to a CENP-A-Specific Assembly Factor through Eic1 Interaction.

Structure 2018 07 24;26(7):960-971.e4. Epub 2018 May 24.

Department of Biological Chemistry, University of Michigan Medical School, 1150 W. Medical Center Drive, SPC 5606, Ann Arbor, MI 48109, USA; Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI 48109, USA. Electronic address:

The Mis18 complex, composed of Mis16, Eic1, and Mis18 in fission yeast, selectively deposits the centromere-specific histone H3 variant, CENP-A, at centromeres. How the intact Mis18 holo-complex oligomerizes and how Mis16, a well-known ubiquitous histone H4 chaperone, plays a centromere-specific role in the Mis18 holo-complex, remain unclear. Here, we report the stoichiometry of the intact Mis18 holo-complex as (Mis16):(Eic1):(Mis18) using analytical ultracentrifugation. We further determine the crystal structure of Schizosaccharomyces pombe Mis16 in complex with the C-terminal portion of Eic1 (Eic1-CT). Notably, Mis16 accommodates Eic1-CT through the binding pocket normally occupied by histone H4, indicating that Eic1 and H4 compete for the same binding site, providing a mechanism for Mis16 to switch its binding partner from histone H4 to Eic1. Thus, our analyses not only determine the stoichiometry of the intact Mis18 holo-complex but also uncover the molecular mechanism by which Mis16 plays a centromere-specific role through Eic1 association.
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http://dx.doi.org/10.1016/j.str.2018.04.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6031460PMC
July 2018

Integrative Structural Investigation on the Architecture of Human Importin4_Histone H3/H4_Asf1a Complex and Its Histone H3 Tail Binding.

J Mol Biol 2018 03 31;430(6):822-841. Epub 2018 Jan 31.

Department of Biological Sciences, Cancer Metastasis Control Center, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea. Electronic address:

Importin4 transports histone H3/H4 in complex with Asf1a to the nucleus for chromatin assembly. Importin4 recognizes the nuclear localization sequence located at the N-terminal tail of histones. Here, we analyzed the structures and interactions of human Importin4, histones and Asf1a by cross-linking mass spectrometry, X-ray crystallography, negative-stain electron microscopy, small-angle X-ray scattering and integrative modeling. The cross-linking mass spectrometry data showed that the C-terminal region of Importin4 was extensively cross-linked with the histone H3 tail. We determined the crystal structure of the C-terminal region of Importin4 bound to the histone H3 peptide, thus revealing that the acidic patch in Importin4 accommodates the histone H3 tail, and that histone H3 Lys14 contributes to the interaction with Importin4. In addition, we show that Asf1a modulates the binding of histone H3/H4 to Importin4. Furthermore, the molecular architecture of the Importin4_histone H3/H4_Asf1a complex was produced through an integrative modeling approach. Overall, this work provides structural insights into how Importin4 recognizes histones and their chaperone complex.
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http://dx.doi.org/10.1016/j.jmb.2018.01.015DOI Listing
March 2018

Structure-based nuclear import mechanism of histones H3 and H4 mediated by Kap123.

Elife 2017 10 16;6. Epub 2017 Oct 16.

Department of Biological Chemistry, University of Michigan Medical School, Michigan, United States.

Kap123, a major karyopherin protein of budding yeast, recognizes the nuclear localization signals (NLSs) of cytoplasmic histones H3 and H4 and translocates them into the nucleus during DNA replication. Mechanistic questions include H3- and H4-NLS redundancy toward Kap123 and the role of the conserved diacetylation of cytoplasmic H4 (K5ac and K12ac) in Kap123-mediated histone nuclear translocation. Here, we report crystal structures of full-length Kap123 alone and in complex with H3- and H4-NLSs. Structures reveal the unique feature of Kap123 that possesses two discrete lysine-binding pockets for NLS recognition. Structural comparison illustrates that H3- and H4-NLSs share at least one of two lysine-binding pockets, suggesting that H3- and H4-NLSs are mutually exclusive. Additionally, acetylation of key lysine residues at NLS, particularly H4-NLS diacetylation, weakens the interaction with Kap123. These data support that cytoplasmic histone H4 diacetylation weakens the Kap123-H4-NLS interaction thereby facilitating histone Kap123-H3-dependent H3:H4/Asf1 complex nuclear translocation.
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http://dx.doi.org/10.7554/eLife.30244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5677370PMC
October 2017

Structural and Mechanistic Insights into Hemoglobin-catalyzed Hydrogen Sulfide Oxidation and the Fate of Polysulfide Products.

J Biol Chem 2017 Mar 17;292(13):5584-5592. Epub 2017 Feb 17.

From the Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109 and

Hydrogen sulfide is a cardioprotective signaling molecule but is toxic at elevated concentrations. Red blood cells can synthesize HS but, lacking organelles, cannot dispose of HS via the mitochondrial sulfide oxidation pathway. We have recently shown that at high sulfide concentrations, ferric hemoglobin oxidizes HS to a mixture of thiosulfate and iron-bound polysulfides in which the latter species predominates. Here, we report the crystal structure of human hemoglobin containing low spin ferric sulfide, the first intermediate in heme-catalyzed sulfide oxidation. The structure provides molecular insights into why sulfide is susceptible to oxidation in human hemoglobin but is stabilized against it in HbI, a specialized sulfide-carrying hemoglobin from a mollusk adapted to life in a sulfide-rich environment. We have also captured a second sulfide bound at a postulated ligand entry/exit site in the α-subunit of hemoglobin, which, to the best of our knowledge, represents the first direct evidence for this site being used to access the heme iron. Hydrodisulfide, a postulated intermediate at the junction between thiosulfate and polysulfide formation, coordinates ferric hemoglobin and, in the presence of air, generated thiosulfate. At low sulfide/heme iron ratios, the product distribution between thiosulfate and iron-bound polysulfides was approximately equal. The iron-bound polysulfides were unstable at physiological glutathione concentrations and were reduced with concomitant formation of glutathione persulfide, glutathione disulfide, and HS. Hence, although polysulfides are unlikely to be stable in the reducing intracellular milieu, glutathione persulfide could serve as a persulfide donor for protein persulfidation, a posttranslational modification by which HS is postulated to signal.
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http://dx.doi.org/10.1074/jbc.M117.774943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5392699PMC
March 2017

Sestrin regulation of TORC1: Is Sestrin a leucine sensor?

Sci Signal 2016 06 7;9(431):re5. Epub 2016 Jun 7.

Departments of Pharmacology and Pathology, University of California, San Diego, School of Medicine, La Jolla, CA 92093-0723, USA.

Sestrins are highly conserved, stress-inducible proteins that inhibit target of rapamycin complex 1 (TORC1) signaling. After their transcriptional induction, both vertebrate and invertebrate Sestrins turn on the adenosine monophosphate (AMP)-activated protein kinase (AMPK), which activates the tuberous sclerosis complex (TSC), a key inhibitor of TORC1 activation. However, Sestrin overexpression, on occasion, can result in TORC1 inhibition even in AMPK-deficient cells. This effect has been attributed to Sestrin's ability to bind the TORC1-regulating GATOR2 protein complex, which was postulated to control trafficking of TORC1 to lysosomes. How the binding of Sestrins to GATOR2 is regulated and how it contributes to TORC1 inhibition are unknown. New findings suggest that the amino acid leucine specifically disrupts the association of Sestrin2 with GATOR2, thus explaining how leucine and related amino acids stimulate TORC1 activity. We discuss whether and how these findings fit what has already been learned about Sestrin-mediated TORC1 inhibition from genetic studies conducted in fruit flies and mammals.
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http://dx.doi.org/10.1126/scisignal.aaf2885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4902175PMC
June 2016

Biochemical Basis of Sestrin Physiological Activities.

Trends Biochem Sci 2016 07 10;41(7):621-632. Epub 2016 May 10.

Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA. Electronic address:

Excessive accumulation of reactive oxygen species (ROS) and chronic activation of mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) are well-characterized promoters of aging and age-associated degenerative pathologies. Sestrins, a family of highly conserved stress-inducible proteins, are important negative regulators of both ROS and mTORC1 signaling pathways; however, the mechanistic basis of how Sestrins suppress these pathways remains elusive. In the past couple of years, breakthrough discoveries about Sestrin signaling and its molecular nature have markedly increased our biochemical understanding of Sestrin function. These discoveries have also uncovered new potential therapeutic strategies that may eventually enable us to attenuate aging and age-associated diseases.
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http://dx.doi.org/10.1016/j.tibs.2016.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4930368PMC
July 2016

Janus-faced Sestrin2 controls ROS and mTOR signalling through two separate functional domains.

Nat Commun 2015 Nov 27;6:10025. Epub 2015 Nov 27.

Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.

Sestrins are stress-inducible metabolic regulators with two seemingly unrelated but physiologically important functions: reduction of reactive oxygen species (ROS) and inhibition of the mechanistic target of rapamycin complex 1 (mTORC1). How Sestrins fulfil this dual role has remained elusive so far. Here we report the crystal structure of human Sestrin2 (hSesn2), and show that hSesn2 is twofold pseudo-symmetric with two globular subdomains, which are structurally similar but functionally distinct from each other. While the N-terminal domain (Sesn-A) reduces alkylhydroperoxide radicals through its helix-turn-helix oxidoreductase motif, the C-terminal domain (Sesn-C) modified this motif to accommodate physical interaction with GATOR2 and subsequent inhibition of mTORC1. These findings clarify the molecular mechanism of how Sestrins can attenuate degenerative processes such as aging and diabetes by acting as a simultaneous inhibitor of ROS accumulation and mTORC1 activation.
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http://dx.doi.org/10.1038/ncomms10025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4674687PMC
November 2015

Mis16 Independently Recognizes Histone H4 and the CENP-ACnp1-Specific Chaperone Scm3sp.

J Mol Biol 2015 Oct 4;427(20):3230-3240. Epub 2015 Sep 4.

Department of Biological Chemistry, University of Michigan Medical School, 1150 West Medical Center Drive, SPC 5606, Ann Arbor, MI 48109, USA. Electronic address:

CENP-A is a centromere-specific histone H3 variant that is required for kinetochore assembly and accurate chromosome segregation. For it to function properly, CENP-A must be specifically localized to centromeres. In fission yeast, Scm3sp and the Mis18 complex, composed of Mis16, Eic1, and Mis18, function as a CENP-A(Cnp1)-specific chaperone and a recruiting factor, respectively, and together ensure accurate delivery of CENP-A(Cnp1) to centromeres. Although how Scm3sp specifically recognizes CENP-A(Cnp1) has been revealed recently, the recruiting mechanism of CENP-A(Cnp1) via the Mis18 complex remains unknown. In this study, we have determined crystal structures of Schizosaccharomyces japonicus Mis16 alone and in complex with the helix 1 of histone H4 (H4α1). Crystal structures followed by mutant analysis and affinity pull-downs have revealed that Mis16 recognizes both H4α1 and Scm3sp independently within the CENP-A(Cnp1)/H4:Scm3sp complex. This observation suggests that Mis16 gains CENP-A(Cnp1) specificity by recognizing both Scm3sp and histone H4. Our studies provide insights into the molecular mechanisms underlying specific recruitment of CENP-A(Cnp1)/H4:Scm3sp into centromeres.
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http://dx.doi.org/10.1016/j.jmb.2015.08.022DOI Listing
October 2015

Sestrin2 inhibits mTORC1 through modulation of GATOR complexes.

Sci Rep 2015 Mar 30;5:9502. Epub 2015 Mar 30.

Department of Molecular &Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA.

Sestrins are stress-inducible metabolic regulators that suppress a wide range of age- and obesity-associated pathologies, many of which are due to mTORC1 overactivation. Upon various stresses, the Sestrins inhibit mTORC1 activity through an indirect mechanism that is still unclear. GATORs are recently identified protein complexes that regulate the activity of RagB, a small GTPase essential for mTORC1 activation. GATOR1 is a GTPase activating protein (GAP) for RagB whereas GATOR2 functions as an inhibitor of GATOR1. However, how the GATORs are physiologically regulated is unknown. Here we show that Sestrin2 binds to GATOR2, and liberates GATOR1 from GATOR2-mediated inhibition. Released GATOR1 subsequently binds to and inactivates RagB, ultimately resulting in mTORC1 suppression. Consistent with this biochemical mechanism, genetic ablation of GATOR1 nullifies the mTORC1-inhibiting effect of Sestrin2 in both cell culture and Drosophila models. Collectively, we elucidate a new signaling cascade composed of Sestrin2-GATOR2-GATOR1-RagB that mediates stress-dependent suppression of mTORC1 activity.
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http://dx.doi.org/10.1038/srep09502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4377584PMC
March 2015

Control of substrate access to the active site in methane monooxygenase.

Nature 2013 Feb 10;494(7437):380-4. Epub 2013 Feb 10.

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

Methanotrophs consume methane as their major carbon source and have an essential role in the global carbon cycle by limiting escape of this greenhouse gas to the atmosphere. These bacteria oxidize methane to methanol by soluble and particulate methane monooxygenases (MMOs). Soluble MMO contains three protein components, a 251-kilodalton hydroxylase (MMOH), a 38.6-kilodalton reductase (MMOR), and a 15.9-kilodalton regulatory protein (MMOB), required to couple electron consumption with substrate hydroxylation at the catalytic diiron centre of MMOH. Until now, the role of MMOB has remained ambiguous owing to a lack of atomic-level information about the MMOH-MMOB (hereafter termed H-B) complex. Here we remedy this deficiency by providing a crystal structure of H-B, which reveals the manner by which MMOB controls the conformation of residues in MMOH crucial for substrate access to the active site. MMOB docks at the α(2)β(2) interface of α(2)β(2)γ(2) MMOH, and triggers simultaneous conformational changes in the α-subunit that modulate oxygen and methane access as well as proton delivery to the diiron centre. Without such careful control by MMOB of these substrate routes to the diiron active site, the enzyme operates as an NADH oxidase rather than a monooxygenase. Biological catalysis involving small substrates is often accomplished in nature by large proteins and protein complexes. The structure presented in this work provides an elegant example of this principle.
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http://dx.doi.org/10.1038/nature11880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3596810PMC
February 2013

Ndc10 is a platform for inner kinetochore assembly in budding yeast.

Nat Struct Mol Biol 2011 Dec 4;19(1):48-55. Epub 2011 Dec 4.

Jack and Eileen Connors Structural Biology Laboratory and Howard Hughes Medical Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.

Kinetochores link centromeric DNA to spindle microtubules and ensure faithful chromosome segregation during mitosis. In point-centromere yeasts, the CBF3 complex Skp1-Ctf13-(Cep3)(2)-(Ndc10)(2) recognizes a conserved centromeric DNA element through contacts made by Cep3 and Ndc10. We describe here the five-domain organization of Kluyveromyces lactis Ndc10 and the structure at 2.8 Å resolution of domains I-II (residues 1-402) bound to DNA. The structure resembles tyrosine DNA recombinases, although it lacks both endonuclease and ligase activities. Structural and biochemical data demonstrate that each subunit of the Ndc10 dimer binds a separate fragment of DNA, suggesting that Ndc10 stabilizes a DNA loop at the centromere. We describe in vitro association experiments showing that specific domains of Ndc10 interact with each of the known inner-kinetochore proteins or protein complexes in budding yeast. We propose that Ndc10 provides a central platform for inner-kinetochore assembly.
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http://dx.doi.org/10.1038/nsmb.2178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3252399PMC
December 2011

Recognition of the centromere-specific histone Cse4 by the chaperone Scm3.

Proc Natl Acad Sci U S A 2011 Jun 23;108(23):9367-71. Epub 2011 May 23.

Department of Biological Chemistry and Molecular Pharmacology and Howard Hughes Medical Institute, Jack and Eileen Connors Structural Biology Laboratory, Harvard Medical School, Boston, MA 02115, USA.

A specialized nucleosome is a component of all eukaryotic kinetochores. The core of this nucleosome contains a centromere-specific histone, CENP-A (the Cse4 gene product in budding yeast), instead of the usual H3. Assembly of a centromeric nucleosome depends on a specific chaperone, called Scm3 in yeast and HJURP in higher eukaryotes. We describe here the structure of a complex formed by an N-terminal fragment of Scm3 with the histone-fold domains of Cse4, and H4, all prepared as recombinant proteins derived from the budding yeast Kluyveromyces lactis. The contacts of Scm3 with Cse4 explain its selectivity for the centromere-specific histone; key residues at the interface are conserved in HJURP, indicating a common mechanism for centromeric-histone deposition. We also report the structure of a (Cse4 : H4)(2) heterotetramer; comparison with the structure of the Scm3:Cse4:H4 complex shows that tetramer formation and DNA-binding require displacement of Scm3 from the nucleosome core. The two structures together suggest that specific contacts between the chaperone and Cse4, rather than an altered overall structure of the nucleosome core, determine the selective presence of Cse4 at centromeres.
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http://dx.doi.org/10.1073/pnas.1106389108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3111289PMC
June 2011

Structure and function of the PP2A-shugoshin interaction.

Mol Cell 2009 Aug;35(4):426-41

Department of Biological Structure, University of Washington, Seattle, WA 98195, USA.

Accurate chromosome segregation during mitosis and meiosis depends on shugoshin proteins that prevent precocious dissociation of cohesin from centromeres. Shugoshins associate with PP2A, which is thought to dephosphorylate cohesin and thereby prevent cleavage by separase during meiosis I. A crystal structure of a complex between a fragment of human Sgo1 and an AB'C PP2A holoenzyme reveals that Sgo1 forms a homodimeric parallel coiled coil that docks simultaneously onto PP2A's C and B' subunits. Sgo1 homodimerization is a prerequisite for PP2A binding. While hSgo1 interacts only with the AB'C holoenzymes, its relative, Sgo2, interacts with all PP2A forms and may thus lead to dephosphorylation of distinct substrates. Mutant shugoshin proteins defective in the binding of PP2A cannot protect centromeric cohesin from separase during meiosis I or support the spindle assembly checkpoint in yeast. Finally, we provide evidence that PP2A's recruitment to chromosomes may be sufficient to protect cohesin from separase in mammalian oocytes.
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http://dx.doi.org/10.1016/j.molcel.2009.06.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2749713PMC
August 2009

Structural basis of PP2A inhibition by small t antigen.

PLoS Biol 2007 Aug;5(8):e202

Department of Biological Structure, University of Washington, Seattle, Washington, United States of America.

The SV40 small t antigen (ST) is a potent oncoprotein that perturbs the function of protein phosphatase 2A (PP2A). ST directly interacts with the PP2A scaffolding A subunit and alters PP2A activity by displacing regulatory B subunits from the A subunit. We have determined the crystal structure of full-length ST in complex with PP2A A subunit at 3.1 A resolution. ST consists of an N-terminal J domain and a C-terminal unique domain that contains two zinc-binding motifs. Both the J domain and second zinc-binding motif interact with the intra-HEAT-repeat loops of HEAT repeats 3-7 of the A subunit, which overlaps with the binding site of the PP2A B56 subunit. Intriguingly, the first zinc-binding motif is in a position that may allow it to directly interact with and inhibit the phosphatase activity of the PP2A catalytic C subunit. These observations provide a structural basis for understanding the oncogenic functions of ST.
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http://dx.doi.org/10.1371/journal.pbio.0050202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1945078PMC
August 2007

Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme.

Nature 2007 Jan 1;445(7123):53-7. Epub 2006 Nov 1.

Department of Biological Structure, University of Washington, Seattle, Washington 98195, USA.

Protein phosphatase 2A (PP2A) is a principal Ser/Thr phosphatase, the deregulation of which is associated with multiple human cancers, Alzheimer's disease and increased susceptibility to pathogen infections. How PP2A is structurally organized and functionally regulated remains unclear. Here we report the crystal structure of an AB'C heterotrimeric PP2A holoenzyme. The structure reveals that the HEAT repeats of the scaffold A subunit form a horseshoe-shaped fold, holding the catalytic C and regulatory B' subunits together on the same side. The regulatory B' subunit forms pseudo-HEAT repeats and interacts with the C subunit near the active site, thereby defining substrate specificity. The methylated carboxy-terminal tail of the C subunit interacts with a highly negatively charged region at the interface between A and B' subunits, suggesting that the C-terminal carboxyl methylation of the C subunit promotes B' subunit recruitment by neutralizing charge repulsion. Together, our structural results establish a crucial foundation for understanding PP2A assembly, substrate recruitment and regulation.
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http://dx.doi.org/10.1038/nature05351DOI Listing
January 2007

Crystal structure of a beta-catenin/BCL9/Tcf4 complex.

Mol Cell 2006 Oct;24(2):293-300

Department of Biological Structure, University of Washington, Seattle, Washington 98195, USA.

The canonical Wnt pathway plays critical roles in embryonic development, stem cell growth, and tumorigenesis. Stimulation of the Wnt pathway leads to the association of beta-catenin with Tcf and BCL9 in the nucleus, resulting in the transactivation of Wnt target genes. We have determined the crystal structure of a beta-catenin/BCL9/Tcf-4 triple complex at 2.6 A resolution. Our studies reveal that the beta-catenin binding site of BCL9 is distinct from that of most other beta-catenin partners and forms a good target for developing drugs that block canonical Wnt/beta-catenin signaling. The BCL9 beta-catenin binding domain (CBD) forms an alpha helix that binds to the first armadillo repeat of beta-catenin, which can be mutated to prevent beta-catenin binding to BCL9 without affecting cadherin or alpha-catenin binding. We also demonstrate that beta-catenin Y142 phosphorylation, which has been proposed to regulate BCL9-2 binding, does not directly affect the interaction of beta-catenin with either BCL9 or BCL9-2.
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http://dx.doi.org/10.1016/j.molcel.2006.09.001DOI Listing
October 2006

Metal bridges between the PhoQ sensor domain and the membrane regulate transmembrane signaling.

J Mol Biol 2006 Mar 27;356(5):1193-206. Epub 2005 Dec 27.

Department of Biological Structure, University of Washington, Seattle, WA 98195, USA.

Bacterial histidine kinases respond to environmental stimuli by transducing a signal from an extracytosolic sensor domain to a cytosolic catalytic domain. Among them, PhoQ promotes bacterial virulence and is tightly repressed by the divalent cations such as calcium and magnesium. We have determined the crystal structure of the PhoQ sensor domain from Salmonella typhimurium in the Ca2+-bound state, which reveals a highly negatively charged surface that is in close proximity to the inner membrane. This acidic surface binds at least three Ca2+, which mediate the PhoQ-membrane interaction. Mutagenesis analysis indicates that structural integrity at the membrane proximal region of the PhoQ sensor domain promotes metal-mediated repression. We propose that depletion or displacement of divalent cations leads to charge repulsion between PhoQ and the membrane, which initiates transmembrane signaling through a change in orientation between the PhoQ sensor domain and membrane. Therefore, both PhoQ and the membrane are required for extracytosolic sensing and transmembrane signaling.
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http://dx.doi.org/10.1016/j.jmb.2005.12.032DOI Listing
March 2006

Crystal structure of the Mycobacterium tuberculosis dUTPase: insights into the catalytic mechanism.

J Mol Biol 2004 Aug;341(2):503-17

UCLA-DOE Laboratory of Structural Biology and Molecular Medicine, 206 Boyer Hall, Box 951570, Los Angeles, CA 90095-1570, USA.

The structure of Mycobacterium tuberculosis dUTP nucleotidohydrolase (dUTPase) has been determined at 1.3 Angstrom resolution in complex with magnesium ion and the non-hydrolyzable substrate analog, alpha,beta-imido dUTP. dUTPase is an enzyme essential for depleting potentially toxic concentrations of dUTP in the cell. Given the importance of its biological role, it has been proposed that inhibiting M.tuberculosis dUTPase might be an effective means to treat tuberculosis infection in humans. The crystal structure presented here offers some insight into the potential for designing a specific inhibitor of the M.tuberculosis dUTPase enzyme. The structure also offers new insights into the mechanism of dUTP hydrolysis by providing an accurate representation of the enzyme-substrate complex in which both the metal ion and dUTP analog are included. The structure suggests that inclusion of a magnesium ion is important for stabilizing the position of the alpha-phosphorus for an in-line nucleophilic attack. In the absence of magnesium, the alpha-phosphate of dUTP can have either of the two positions which differ by 4.5 Angstrom. A transiently ordered C-terminal loop further assists catalysis by shielding the general base, Asp83, from solvent thus elevating its pK(a) so that it might in turn activate a tightly bound water molecule for nucleophilic attack. The metal ion coordinates alpha, beta, and gamma phosphate groups with tridentate geometry identical with that observed in the crystal structure of DNA polymerase beta complexed with magnesium and dNTP analog, revealing some common features in catalytic mechanism.
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http://dx.doi.org/10.1016/j.jmb.2004.06.028DOI Listing
August 2004

Tight-binding inhibition by alpha-naphthoflavone of human cytochrome P450 1A2.

Biochim Biophys Acta 2003 May;1648(1-2):195-202

Graduate School of Biotechnology, Korea University, 5-1 Anam dong, Sungbuk-gu, Seoul 136-701, South Korea.

Human cytochrome P450 (P450) enzymes exhibit remarkable diversity in their substrate specificities, participating in oxidation reactions of a wide range of xenobiotic drugs. Previously, we reported that alpha-naphthoflavone (ANF) is bound to the recombinant P450 1A2 tightly and stabilizes an overall enzyme conformation. The present study is designed to determine the type of P450 1A2 inhibition exerted by ANF, using two different substrates of P450 1A2, 7-ethoxycoumarin (EOC) and 7-ethoxyresorufin (EOR). ANF is generally known as a competitive inhibitor of the enzyme. However, in our tight-binding enzyme kinetics study, ANF acts as noncompetitive inhibitor in 7-ethoxycoumarin O-deethylation (ECOD) (K(i)=55.0 nM), but as competitive inhibitor in 7-ethoxyresorufin O-deethylation (EROD) (K(i)=1.4 nM). Based on homology modeling studies, ANF is positioned to bind to a hydrophobic cavity next to the active site where it may cause a direct effect on substrate binding. It is agreed with the predicted binding site of ANF in P450 3A4, in which ANF is rather known as a stimulating modulator. Our results suggest that ANF binds near the active site of P450 1A2 and exhibits differential inhibition mechanisms, possibly depending on the molecular structure of the substrate.
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http://dx.doi.org/10.1016/s1570-9639(03)00148-1DOI Listing
May 2003