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Sharon Ruthstein - One of the best experts on this subject based on the ideXlab platform.

  • An EPR Study on the Interaction between the Cu(I) Metal Binding Domains of ATP7B and the Atox1 Metallochaperone.
    International Journal of Molecular Sciences, 2020
    Co-Authors: Michael Zaccak, Zena Qasem, Lada Gevorkyan-airapetov, Sharon Ruthstein
    Abstract:

    Copper’s essentiality and toxicity mean it requires a sophisticated regulation system for its acquisition, cellular distribution and excretion, which until now has remained elusive. Herein, we applied continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) spectroscopy in solution to resolve the copper trafficking mechanism in humans, by considering the route travelled by Cu(I) from the Metallochaperone Atox1 to the metal binding domains of ATP7B. Our study revealed that Cu(I) is most likely mediated by the binding of the Atox1 monomer to metal binding domain 1 (MBD1) and MBD4 of ATP7B in the final part of its extraction pathway, while the other MBDs mediate this interaction and participate in copper transfer between the various MBDs to the ATP7B membrane domain. This research also proposes that MBD1-3 and MBD4-6 act as two independent units.

  • The structural flexibility of the human copper chaperone Atox1: Insights from combined pulsed EPR studies and computations.
    Protein Science, 2017
    Co-Authors: Ariel R. Levy, Meital Turgeman, Lada Gevorkyan-aiapetov, Sharon Ruthstein
    Abstract:

    Metallochaperones are responsible for shuttling metal ions to target proteins. Thus, a Metallochaperone's structure must be sufficiently flexible both to hold onto its ion while traversing the cytoplasm and to transfer the ion to or from a partner protein. Here, we sought to shed light on the structure of Atox1, a Metallochaperone involved in the human copper regulation system. Atox1 shuttles copper ions from the main copper transporter, Ctr1, to the ATP7b transporter in the Golgi apparatus. Conventional biophysical tools such as X-ray or NMR cannot always target the various conformational states of Metallochaperones, owing to a requirement for crystallography or low sensitivity and resolution. Electron paramagnetic resonance (EPR) spectroscopy has recently emerged as a powerful tool for resolving biological reactions and mechanisms in solution. When coupled with computational methods, EPR with site-directed spin labeling and nanoscale distance measurements can provide structural information on a protein or protein complex in solution. We use these methods to show that Atox1 can accommodate at least four different conformations in the apo state (unbound to copper), and two different conformations in the holo state (bound to copper). We also demonstrate that the structure of Atox1 in the holo form is more compact than in the apo form. Our data provide insight regarding the structural mechanisms through which Atox1 can fulfill its dual role of copper binding and transfer.

  • Ctr1 Intracellular Loop Is Involved in the Copper Transfer Mechanism to the Atox1 Metallochaperone.
    The Journal of Physical Chemistry B, 2016
    Co-Authors: Ariel R. Levy, Matan Nissim, Netanel Mendelman, Jordan H. Chill, Sharon Ruthstein
    Abstract:

    Understanding the human copper cycle is essential to understand the role of metals in promoting neurological diseases and disorders. One of the cycles controlling the cellular concentration and distribution of copper involves the copper transporter, Ctr1; the Metallochaperone, Atox1; and the ATP7B transporter. It has been shown that the C-terminus of Ctr1, specifically the last three amino acids, HCH, is involved in both copper coordination and the transfer mechanism to Atox1. In contrast, the role of the intracellular loop of Ctr1, which is an additional intracellular segment of Ctr1, in facilitating the copper transfer mechanism has not been investigated yet. Here, we combine various biophysical methods to explore the interaction between this Ctr1 segment and Metallochaperone Atox1 and clearly demonstrate that the Ctr1 intracellular loop (1) can coordinate Cu(I) via interactions with the side chains of one histidine and two methionine residues and (2) closely interacts with the Atox1 Metallochaperone. Our findings are another important step in elucidating the mechanistic details of the eukaryotic copper cycle.

  • Ctr1 Intracellular Loop Is Involved in the Copper Transfer Mechanism to the Atox1 Metallochaperone
    2016
    Co-Authors: Ariel R. Levy, Netanel Mendelman, Matan Nissim, Jordan H. Chill, Sharon Ruthstein
    Abstract:

    Understanding the human copper cycle is essential to understand the role of metals in promoting neurological diseases and disorders. One of the cycles controlling the cellular concentration and distribution of copper involves the copper transporter, Ctr1; the Metallochaperone, Atox1; and the ATP7B transporter. It has been shown that the C-terminus of Ctr1, specifically the last three amino acids, HCH, is involved in both copper coordination and the transfer mechanism to Atox1. In contrast, the role of the intracellular loop of Ctr1, which is an additional intracellular segment of Ctr1, in facilitating the copper transfer mechanism has not been investigated yet. Here, we combine various biophysical methods to explore the interaction between this Ctr1 segment and Metallochaperone Atox1 and clearly demonstrate that the Ctr1 intracellular loop (1) can coordinate Cu­(I) via interactions with the side chains of one histidine and two methionine residues and (2) closely interacts with the Atox1 Metallochaperone. Our findings are another important step in elucidating the mechanistic details of the eukaryotic copper cycle

Robert P. Hausinger - One of the best experts on this subject based on the ideXlab platform.

  • mutational and computational evidence that a nickel transfer tunnel in ured is used for activation of klebsiella aerogenes urease
    Biochemistry, 2015
    Co-Authors: Mark A Farrugia, Beibei Wang, Michael Feig, Robert P. Hausinger
    Abstract:

    Nickel-containing urease from Klebsiella aerogenes requires four accessory proteins for proper active site metalation. The Metallochaperone UreE delivers nickel to UreG, a GTPase that forms a UreD/UreF/UreG complex, which binds to urease apoprotein via UreD. Prior in silico analysis of the homologous, structurally characterized UreH/UreF/UreG complex from Helicobacter pylori identified a water tunnel originating at a likely nickel-binding motif in UreG, passing through UreF, and exiting UreH, suggestive of a role for the channel in providing the metal to urease apoprotein for its activation; however, no experimental support was reported for the significance of this tunnel. Here, specific variants were designed to disrupt a comparable 34.6 A predicted internal tunnel, alternative channels, and surface sites for UreD. Cells producing a set of tunnel-disrupting variants of UreD exhibited greatly reduced urease specific activities, whereas other mutants had no appreciable effect on activity. Affinity pull-dow...

  • interplay of metal ions and urease
    Metallomics, 2009
    Co-Authors: Eric L Carter, Scott B. Mulrooney, Nicholas Flugga, Jodi L Boer, Robert P. Hausinger
    Abstract:

    Urease, the first enzyme to be crystallized, contains a dinuclear nickel metallocenter that catalyzes the decomposition of urea to produce ammonia, a reaction of great agricultural and medical importance. Several mechanisms of urease catalysis have been proposed on the basis of enzyme crystal structures, model complexes, and computational efforts, but the precise steps in catalysis and the requirement of nickel versus other metals remain unclear. Purified bacterial urease is partially activated via incubation with carbon dioxide plus nickel ions; however, in vitro activation also has been achieved with manganese and cobalt. In vivo activation of most ureases requires accessory proteins that function as nickel Metallochaperones and GTP-dependent molecular chaperones or play other roles in the maturation process. In addition, some microorganisms control their levels of urease by metal ion-dependent regulatory mechanisms.

  • crystal structure of klebsiella aerogenes uree a nickel binding Metallochaperone for urease activation
    Journal of Biological Chemistry, 2001
    Co-Authors: Hyun Kyu Song, Scott B. Mulrooney, Robert Huber, Robert P. Hausinger
    Abstract:

    Abstract UreE is proposed to be a Metallochaperone that delivers nickel ions to urease during activation of this bacterial virulence factor. Wild-type Klebsiella aerogenes UreE binds approximately six nickel ions per homodimer, whereas H144*UreE (a functional C-terminal truncated variant) was previously reported to bind two. We determined the structure of H144*UreE by multi-wavelength anomalous diffraction and refined it to 1.5 A resolution. The present structure reveals an Hsp40-like peptide-binding domain, an Atx1-like metal-binding domain, and a flexible C terminus. Three metal-binding sites per dimer, defined by structural analysis of Cu-H144*UreE, are on the opposite face of the Atx1-like domain than observed in the copper Metallochaperone. One metal bridges the two subunits via the pair of His-96 residues, whereas the other two sites involve metal coordination by His-110 and His-112 within each subunit. In contrast to the copper Metallochaperone mechanism involving thiol ligand exchanges between structurally similar chaperones and target proteins, we propose that the Hsp40-like module interacts with urease apoprotein and/or other urease accessory proteins, while the Atx1-like domain delivers histidyl-bound nickel to the urease active site.

  • identification of metal binding residues in the klebsiella aerogenes urease nickel Metallochaperone uree
    Biochemistry, 1999
    Co-Authors: Gerard J Colpas, Timothy G Brayman, Lijune Ming, Robert P. Hausinger
    Abstract:

    The urease accessory protein encoded by ureE from Klebsiella aerogenes is proposed to bind intracellular Ni(II) for transfer to urease apoprotein. While native UreE possesses a histidine-rich region at its carboxyl terminus that binds several equivalents of Ni, the Ni-binding sites associated with urease activation are internal to the protein as shown by studies involving truncated H144*UreE [Brayman and Hausinger (1996) J. Bacteriol. 178, 5410−5416]. Nine potential Ni-binding residues (five His, two Cys, one Asp, and one Tyr) within H144*UreE were independently substituted by mutagenesis to determine their roles in metal binding and urease activation. In vivo effects of these substitutions on urease activity were measured in Escherichia coli strains containing the K. aerogenes urease gene cluster with the mutated ureE genes. Several mutational changes led to reductions in specific activity, with substitution of His96 producing urease activity below the level obtained from a ureE deletion mutant. The meta...

Deborah B. Zamble - One of the best experts on this subject based on the ideXlab platform.

  • Bimodal Nickel-Binding Site on Escherichia coli [NiFe]-Hydrogenase Metallochaperone HypA.
    Inorganic Chemistry, 2019
    Co-Authors: Michael J. Lacasse, Kelly L. Summers, Mozhgan Khorasani-motlagh, Graham N. George, Deborah B. Zamble
    Abstract:

    [NiFe]-hydrogenase enzymes catalyze the reversible oxidation of hydrogen at a bimetallic cluster and are used by bacteria and archaea for anaerobic growth and pathogenesis. Maturation of the [NiFe]...

  • bimodal nickel binding site on escherichia coli nife hydrogenase Metallochaperone hypa
    Inorganic Chemistry, 2019
    Co-Authors: Michael J. Lacasse, Kelly L. Summers, Graham N. George, Mozhgan Khorasanimotlagh, Deborah B. Zamble
    Abstract:

    [NiFe]-hydrogenase enzymes catalyze the reversible oxidation of hydrogen at a bimetallic cluster and are used by bacteria and archaea for anaerobic growth and pathogenesis. Maturation of the [NiFe]-hydrogenase requires several accessory proteins to assemble and insert the components of the active site. The penultimate maturation step is the delivery of nickel to a primed hydrogenase enzyme precursor protein, a process that is accomplished by two nickel Metallochaperones, the accessory protein HypA and the GTPase HypB. Recent work demonstrated that nickel is rapidly transferred to HypA from GDP-loaded HypB within the context of a protein complex in a nickel selective and unidirectional process. To investigate the mechanism of metal transfer, we examined the allosteric effects of nucleotide cofactors and partner proteins on the nickel environments of HypA and HypB by using a combination of biochemical, microbiological, computational, and spectroscopic techniques. We observed that loading HypB with either GDP or a nonhydrolyzable GTP analogue resulted in a similar nickel environment. In addition, interaction with a mutant version of HypA with disrupted nickel binding, H2Q-HypA, does not induce substantial changes to the HypB G-domain nickel site. Instead, the results demonstrate that HypB modifies the acceptor site of HypA. Analysis of a peptide maquette derived from the N-terminus of HypA revealed that nickel is predominately coordinated by atoms from the N-terminal Met-His motif. Furthermore, HypA is capable of two nickel-binding modes at the N-terminus, a HypB-induced mode and a binding mode that mirrors the peptide maquette. Collectively, these results reveal that HypB brings about changes in the nickel coordination of HypA, providing a mechanism for the HypB-dependent control of the acquisition and release of nickel by HypA.

Thomas V Ohalloran - One of the best experts on this subject based on the ideXlab platform.

  • tetrathiomolybdate inhibits copper trafficking proteins through metal cluster formation
    Science, 2010
    Co-Authors: Hamsell M Alvarez, Chandler D Robinson, Monica A Canalizohernandez, Rebecca G Marvin, Rebekah A Kelly, Alfonso Mondragon, James E Pennerhahn, Thomas V Ohalloran
    Abstract:

    Tetrathiomolybdate (TM) is an orally active agent for treatment of disorders of copper metabolism. Here we describe how TM inhibits proteins that regulate copper physiology. Crystallographic results reveal that the surprising stability of the drug complex with the Metallochaperone Atx1 arises from formation of a sulfur-bridged copper-molybdenum cluster reminiscent of those found in molybdenum and iron sulfur proteins. Spectroscopic studies indicate that this cluster is stable in solution and corresponds to physiological clusters isolated from TM-treated Wilson’s disease animal models. Finally, mechanistic studies show that the drug-Metallochaperone inhibits metal transfer functions between copper-trafficking proteins. The results are consistent with a model wherein TM can directly and reversibly down-regulate copper delivery to secreted metalloenzymes and suggest that proteins involved in metal regulation might be fruitful drug targets.

  • a place for thioether chemistry in cellular copper ion recognition and trafficking
    Nature Chemical Biology, 2008
    Co-Authors: Anna V Davis, Thomas V Ohalloran
    Abstract:

    Over the last decade, cysteine thiolate ligands have been shown to be critical to the Cu(I) (cuprous) binding chemistry of many cytosolic Metallochaperone and metalloregulatory proteins involved in copper physiology. More recently, the thioether group of methionine has begun to emerge as an important Cu(I) ligand for trafficking proteins in more oxidizing cellular environments.

  • transition metal speciation in the cell insights from the chemistry of metal ion receptors
    Science, 2003
    Co-Authors: Lydia Finney, Thomas V Ohalloran
    Abstract:

    The essential transition metal ions are avidly accumulated by cells, yet they have two faces: They are put to use as required cofactors, but they also can catalyze cytotoxic reactions. Several families of proteins are emerging that control the activity of intracellular metal ions and help confine them to vital roles. These include integral transmembrane transporters, metalloregulatory sensors, and diffusible cytoplasmic Metallochaperone proteins that protect and guide metal ions to targets. It is becoming clear that many of these proteins use atypical coordination chemistry to accomplish their unique goals. The different coordination numbers, types of coordinating residues, and solvent accessibilities of these sites are providing insight into the inorganic chemistry of the cytoplasm.

  • structural basis for copper transfer by the Metallochaperone for the menkes wilson disease proteins
    Nature Structural & Molecular Biology, 2000
    Co-Authors: A K Wernimont, David L Huffman, Audrey L Lamb, Thomas V Ohalloran, Amy C Rosenzweig
    Abstract:

    The Hah1 Metallochaperone protein is implicated in copper delivery to the Menkes and Wilson disease proteins. Hah1 and the N-termini of its target proteins belong to a family of metal binding domains characterized by a conserved MT/HCXXC sequence motif. The crystal structure of Hah1 has been determined in the presence of Cu(I), Hg(II), and Cd(II). The 1.8 A resolution structure of CuHah1 reveals a copper ion coordinated by Cys residues from two adjacent Hah1 molecules. The CuHah1 crystal structure is the first of a copper chaperone bound to copper and provides structural support for direct metal ion exchange between conserved MT/HCXXC motifs in two domains. The structures of HgHah1 and CdHah1, determined to 1.75 A resolution, also reveal metal ion coordination by two MT/HCXXC motifs. An extended hydrogen bonding network, unique to the complex of two Hah1 molecules, stabilizes the metal binding sites and suggests specific roles for several conserved residues. Taken together, the structures provide models for intermediates in metal ion transfer and suggest a detailed molecular mechanism for protein recognition and metal ion exchange between MT/HCXXC containing domains.

  • energetics of copper trafficking between the atx1 Metallochaperone and the intracellular copper transporter ccc2
    Journal of Biological Chemistry, 2000
    Co-Authors: David L Huffman, Thomas V Ohalloran
    Abstract:

    Abstract The Atx1 Metallochaperone protein is a cytoplasmic Cu(I) receptor that functions in intracellular copper trafficking pathways in plants, microbes, and humans. A key physiological partner of the Saccharomyces cerevisiae Atx1 is Ccc2, a cation transporting P-type ATPase located in secretory vesicles. Here, we show that Atx1 donates its metal ion cargo to the first N-terminal Atx1-like domain of Ccc2 in a direct and reversible manner. The thermodynamic gradient for metal transfer is shallow (K exchange = 1.4 ± 0.2), establishing that vectorial delivery of copper by Atx1 is not based on a higher copper affinity of the target domain. Instead, Atx1 allows rapid metal transfer to its partner. This equilibrium is unaffected by a 50-fold excess of the Cu(I) competitor, glutathione, indicating that Atx1 also protects Cu(I) from nonspecific reactions. Mechanistically, we propose that a low activation barrier for transfer between partners results from complementary electrostatic forces that ultimately orient the metal-binding loops of Atx1 and Ccc2 for formation of copper-bridged intermediates. These thermodynamic and kinetic considerations suggest that copper trafficking proteins overcome the extraordinary copper chelation capacity of the eukaryotic cytoplasm by catalyzing the rate of copper transfer between physiological partners. In this sense, Metallochaperones work like enzymes, carefully tailoring energetic barriers along specific reaction pathways but not others.

Darren R Carpizo - One of the best experts on this subject based on the ideXlab platform.

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    2016
    Co-Authors: Aaron J. Wolfe, David J Augeri, David S Kimball, John A. Gilleran, Eric C. Olson, Thomas J. Emge, Liviu Movileanu, Darren R Carpizo
    Abstract:

    Synthetic Metallochaperone ZMC1 rescues mutant p53 conformation by transporting zinc into cells as an ionophor

  • abstract 3833 restoration of wildtype structure and function of mutant p53 by thiosemicarbozones using a novel zinc Metallochaperone based mechanism
    Cancer Research, 2016
    Co-Authors: Adam R Blanden, Ashley T Tsang, Saif Zaman, John Gelleran, David J Augeri, David S Kimball, Stewart N Loh, Darren R Carpizo
    Abstract:

    NSC319726 (ZMC1) is a small molecule that reactivates mutant p53 by restoration of WT structure and function to the most common p53 missense mutant (p53-R175H). We identified that ZMC1 functions as a zinc-Metallochaperone, providing an optimal concentration of zinc to facilitate proper folding of p53 protein, and increasing cellular reactive oxygen species to transactivate the newly conformed p53-R175H (via post-translational modifications). ZMC1 was identified from an in silico screen of the NCI anti-cancer drug screen along with two other thiosemicarbazones (TSCs), NSC319725 and NSC328784. We investigated these TSCs to determine if they could reactivate mutant p53 using a zinc Metallochaperone mechanism. We found that indeed these compounds could reactivate mutant p53 by functioning as zinc Metallochaperones. In distinction, Triapine the only TSC in clinical development, does not function as a zinc Metallochaperone and is not a mutant p53 reactivator. Citation Format: Xin Yu, Adam R. Blanden, Ashley T. Tsang, Saif Zaman, John Gelleran, David Augeri, S. David Kimball, Stewart N. Loh, Darren R. Carpizo. Restoration of wildtype structure and function of mutant p53 by thiosemicarbozones using a novel zinc Metallochaperone based mechanism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3833.

  • small molecule restoration of wildtype structure and function of mutant p53 using a novel zinc Metallochaperone based mechanism
    Oncotarget, 2014
    Co-Authors: Adam R Blanden, David J Augeri, David S Kimball, Stewart N Loh, Sumana Narayanan, Lalithapriya Jayakumar, David Lubin, Darren R Carpizo
    Abstract:

    // Xin Yu 1,2,* , Adam R. Blanden 3,* , Sumana Narayanan 2 , Lalithapriya Jayakumar 2 , David Lubin 3 , David Augeri 4 , S. David Kimball 4 , Stewart N. Loh 3 and Darren R. Carpizo 1,2 1 Rutgers Cancer Institute of New Jersey, New Jersey 2 Department of Surgery, Rutgers Robert Wood Johnson Medical School, New Brunswick, New Jersey 3 Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 4 Department of Medicinal Chemistry, Rutgers Ernest Mario School of Pharmacy, Piscataway, New Jersey * These authors contributed equally to this work Correspondence: Stewart N. Loh, email: // Darren R. Carpizo, email: // Keywords : mutant p53 reactivation, zinc-Metallochaperone, mutant p53 targeted drug, thiosemicarbazone, reactive oxygen species (ROS) Received : July 29, 2014 Accepted : September 02, 2014 Published : September 03, 2014 Abstract NSC319726 (ZMC1) is a small molecule that reactivates mutant p53 by restoration of WT structure/function to the most common p53 missense mutant (p53-R175H). We investigated the mechanism by which ZMC1 reactivates p53-R175H and provide evidence that ZMC1: 1) restores WT structure by functioning as a zinc-Metallochaperone, providing an optimal concentration of zinc to facilitate proper folding; and 2) increases cellular reactive oxygen species that transactivate the newly conformed p53-R175H (via post-translational modifications), inducing an apoptotic program. We not only demonstrate that this zinc Metallochaperone function is possessed by other zinc-binding small molecules, but that it can reactivate other p53 mutants with impaired zinc binding. This represents a novel mechanism for an anti-cancer drug and a new pathway to drug mutant p53. Significance: We have elucidated a novel mechanism to restore wild-type structure/function to mutant p53 using small molecules functioning as zinc-Metallochaperones. The pharmacologic delivery of a metal ion to restore proper folding of a mutant protein is unique to medicinal chemistry and represents a new pathway to drug mutant p53.