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Title: The human ACC2 CT-domain C-terminus is required for full functionality and has a novel twist

Abstract

The use of biophysical assays permitted the identification of a specific human ACC2 carboxyl transferase (CT) domain mutant that binds inhibitors and crystallizes in their presence. This mutant led to determination of the human ACC2 CT domain–CP-640186 complex crystal structure, which revealed differences in the inhibitor conformation from the yeast protein complex that are caused by differing residues in the binding pocket. Inhibition of acetyl-CoA carboxylase (ACC) may prevent lipid-induced insulin resistance and type 2 diabetes, making the enzyme an attractive pharmaceutical target. Although the enzyme is highly conserved amongst animals, only the yeast enzyme structure is available for rational drug design. The use of biophysical assays has permitted the identification of a specific C-terminal truncation of the 826-residue human ACC2 carboxyl transferase (CT) domain that is both functionally competent to bind inhibitors and crystallizes in their presence. This C-terminal truncation led to the determination of the human ACC2 CT domain–CP-640186 complex crystal structure, which revealed distinctions from the yeast-enzyme complex. The human ACC2 CT-domain C-terminus is comprised of three intertwined α-helices that extend outwards from the enzyme on the opposite side to the ligand-binding site. Differences in the observed inhibitor conformation between the yeast and human structures are causedmore » by differing residues in the binding pocket.« less

Authors:
 [1]; ;  [2];  [3];  [4];  [1];  [2];  [5];  [1]
  1. Department of Computational and Structural Chemistry, GlaxoSmithKline Inc., Five Moore Drive, Research Triangle Park, NC 27709 (United States)
  2. Department of Biochemical Reagents and Assay Development, GlaxoSmithKline Inc., Five Moore Drive, Research Triangle Park, NC 27709 (United States)
  3. Department of Chemistry in the Center for Excellence in Metabolic Pathways Drug Discovery, GlaxoSmithKline Inc., Five Moore Drive, Research Triangle Park, NC 27709 (United States)
  4. Institute for Genome Sciences and Policy and Department of Medicine, Division of Neurology, Duke University, Durham, NC 27708 (United States)
  5. Department of Physiology, UNC School of Medicine, University of North Carolina, Chapel Hill, NC 27515 (United States)
Publication Date:
OSTI Identifier:
22351182
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section D: Biological Crystallography; Journal Volume: 65; Journal Issue: Pt 5; Other Information: PMCID: PMC2725780; PMID: 19390150; PUBLISHER-ID: en5356; OAI: oai:pubmedcentral.nih.gov:2725780; Copyright (c) International Union of Crystallography 2009; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
Denmark
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CRYSTAL STRUCTURE; DESIGN; DRUGS; LIGANDS

Citation Formats

Madauss, Kevin P., Burkhart, William A., Consler, Thomas G., Cowan, David J., Gottschalk, William K., Miller, Aaron B., Short, Steven A., Tran, Thuy B., and Williams, Shawn P., E-mail: shawn.p.williams@gsk.com. The human ACC2 CT-domain C-terminus is required for full functionality and has a novel twist. Denmark: N. p., 2009. Web. doi:10.1107/S0907444909008014.
Madauss, Kevin P., Burkhart, William A., Consler, Thomas G., Cowan, David J., Gottschalk, William K., Miller, Aaron B., Short, Steven A., Tran, Thuy B., & Williams, Shawn P., E-mail: shawn.p.williams@gsk.com. The human ACC2 CT-domain C-terminus is required for full functionality and has a novel twist. Denmark. doi:10.1107/S0907444909008014.
Madauss, Kevin P., Burkhart, William A., Consler, Thomas G., Cowan, David J., Gottschalk, William K., Miller, Aaron B., Short, Steven A., Tran, Thuy B., and Williams, Shawn P., E-mail: shawn.p.williams@gsk.com. Fri . "The human ACC2 CT-domain C-terminus is required for full functionality and has a novel twist". Denmark. doi:10.1107/S0907444909008014.
@article{osti_22351182,
title = {The human ACC2 CT-domain C-terminus is required for full functionality and has a novel twist},
author = {Madauss, Kevin P. and Burkhart, William A. and Consler, Thomas G. and Cowan, David J. and Gottschalk, William K. and Miller, Aaron B. and Short, Steven A. and Tran, Thuy B. and Williams, Shawn P., E-mail: shawn.p.williams@gsk.com},
abstractNote = {The use of biophysical assays permitted the identification of a specific human ACC2 carboxyl transferase (CT) domain mutant that binds inhibitors and crystallizes in their presence. This mutant led to determination of the human ACC2 CT domain–CP-640186 complex crystal structure, which revealed differences in the inhibitor conformation from the yeast protein complex that are caused by differing residues in the binding pocket. Inhibition of acetyl-CoA carboxylase (ACC) may prevent lipid-induced insulin resistance and type 2 diabetes, making the enzyme an attractive pharmaceutical target. Although the enzyme is highly conserved amongst animals, only the yeast enzyme structure is available for rational drug design. The use of biophysical assays has permitted the identification of a specific C-terminal truncation of the 826-residue human ACC2 carboxyl transferase (CT) domain that is both functionally competent to bind inhibitors and crystallizes in their presence. This C-terminal truncation led to the determination of the human ACC2 CT domain–CP-640186 complex crystal structure, which revealed distinctions from the yeast-enzyme complex. The human ACC2 CT-domain C-terminus is comprised of three intertwined α-helices that extend outwards from the enzyme on the opposite side to the ligand-binding site. Differences in the observed inhibitor conformation between the yeast and human structures are caused by differing residues in the binding pocket.},
doi = {10.1107/S0907444909008014},
journal = {Acta Crystallographica. Section D: Biological Crystallography},
number = Pt 5,
volume = 65,
place = {Denmark},
year = {Fri May 01 00:00:00 EDT 2009},
month = {Fri May 01 00:00:00 EDT 2009}
}
  • Inhibition of acetyl-CoA carboxylase (ACC) may prevent lipid-induced insulin resistance and type 2 diabetes, making the enzyme an attractive pharmaceutical target. Although the enzyme is highly conserved amongst animals, only the yeast enzyme structure is available for rational drug design. The use of biophysical assays has permitted the identification of a specific C-terminal truncation of the 826-residue human ACC2 carboxyl transferase (CT) domain that is both functionally competent to bind inhibitors and crystallizes in their presence. This C-terminal truncation led to the determination of the human ACC2 CT domain-CP-640186 complex crystal structure, which revealed distinctions from the yeast-enzyme complex. Themore » human ACC2 CT-domain C-terminus is comprised of three intertwined -helices that extend outwards from the enzyme on the opposite side to the ligand-binding site. Differences in the observed inhibitor conformation between the yeast and human structures are caused by differing residues in the binding pocket.« less
  • The oncogenic potential of the high-risk human papillomavirus (HPV) relies on the expression of genes specifying the E7 and E6 proteins. To investigate further the variation in oligomeric structure that has been reported for different E7 proteins, an HPV-18 E7 cloned from a Hispanic woman with cervical intraepithelial neoplasia was purified to homogeneity most probably as a stable monomeric protein in aqueous solution. We determined that one zinc ion is present per HPV-18 E7 monomer by amino acid and inductively coupled plasma-atomic emission spectroscopy analysis. Intrinsic fluorescence and circular dichroism spectroscopic results indicate that the zinc ion is important formore » the correct folding and thermal stability of HPV-18 E7. Hydroxyl radical mediated protein footprinting coupled to mass spectrometry and other biochemical and biophysical data indicate that near the C-terminus, the four cysteines of the two Cys-X{sub 2}-Cys motifs that are coordinated to the zinc ion form a solvent inaccessible core. The N-terminal LXCXE pRb binding motif region is hydroxyl radical accessible and conformationally flexible. Both factors, the relative flexibility of the pRb binding motif at the N-terminus and the C-terminal metal-binding hydrophobic solvent-protected core, combine together and facilitate the biological functions of HPV-18 E7.« less
  • HMA2 is a Zn{sup 2+}-ATPase from Arabidopsis thaliana. It contributes to the maintenance of metal homeostasis in cells by driving Zn{sup 2+} efflux. Distinct from P1B-type ATPases, plant Zn{sup 2+}-ATPases have long C-terminal sequences rich in Cys and His. Removal of the 244 amino acid C terminus of HMA2 leads to a 43% reduction in enzyme turnover without significant effect on the Zn{sup 2+} K{sub 1/2} for enzyme activation. Characterization of the isolated HMA2 C terminus showed that this fragment binds three Zn{sup 2+} with high affinity (K{sub d} = 16 {+-} 3nM). Circular dichroism spectral analysis indicated the presencemore » of 8% {alpha}-helix, 45% {beta}-sheet, and 48% random coil in the C-terminal peptide with noticeable structural changes upon metal binding (8% {alpha}-helix, 39% {beta}-sheet, and 52% random coil). Zn K-edge XAS of Zn-C-MBD in the presence of one equivalent of Zn{sup 2+} shows that the average zinc complex formed is composed of three His and one Cys residues. Upon the addition of two extra Zn{sup 2+} ions per C-MBD, these appear coordinated primarily by His residues thus, suggesting that the three Zn{sup 2+} binding domains might not be identical. Modification of His residues with diethyl pyrocarbonate completely inhibited Zn{sup 2+} binding to the C terminus, pointing out the importance of His residues in Zn{sup 2+} coordination. In contrast, alkylation of Cys with iodoacetic acid did not prevent Zn{sup 2+} binding to the HMA2 C terminus. Zn K-edge XAS of the Cys-alkylated protein was consistent with (N/O){sub 4} coordination of the zinc site, with three of those ligands fitting for His residues. In summary, plant Zn{sup 2+}-ATPases contain novel metal binding domains in their cytoplasmic C terminus. Structurally distinct from the well characterized N-terminal metal binding domains present in most P{sub 1B}-type ATPases, they also appear to regulate enzyme turnover rate.« less
  • The amino acid sequence of the UL31 protein (UL31P) of equine herpesvirus 1 (EHV-1) has homology to that of the ICP8 of herpes simplex virus type 1 (HSV-1). Here we show that the UL31 gene is synergistically trans-activated by the IEP and the UL5P (EICP27). Detection of the UL31 RNA transcript and the UL31P in EHV-1-infected cells at 6 h post-infection (hpi) as well as metabolic inhibition assays indicated that UL31 is an early gene. The UL31P preferentially bound to single-stranded DNA over double-stranded DNA in gel shift assays. Subcellular localization of the green fluorescent protein (GFP)-UL31 fusion proteins revealedmore » that the C-terminal 32 amino acid residues of the UL31P are responsible for the nuclear localization. These findings may contribute to defining the role of the UL31P single-stranded DNA-binding protein in EHV-1 DNA replication.« less
  • The 37/67 kDa laminin receptor (LAMR) is a multifunctional protein, acting as an extracellular receptor, localizing to the nucleus, and playing roles in rRNA processing and ribosome assembly. LAMR is important for cell viability; however, it is unclear which of its functions are essential. We developed a silent mutant LAMR construct, resistant to siRNA, to rescue the phenotypic effects of knocking down endogenous LAMR, which include inhibition of protein synthesis, cell cycle arrest, and apoptosis. In addition, we generated a C-terminal-truncated silent mutant LAMR construct structurally homologous to the Archaeoglobus fulgidus S2 ribosomal protein and missing the C-terminal 75 residuesmore » of LAMR, which displays more sequence divergence. We found that HT1080 cells stably expressing either silent mutant LAMR construct still undergo arrest in the G{sub 1} phase of the cell cycle when treated with siRNA. However, the expression of full-length silent mutant LAMR rescues cell viability, whereas the expression of the C-terminal-truncated LAMR does not. Interestingly, we also found that both silent mutant constructs restore protein translation and localize to the nucleus. Our findings indicate that the ability of LAMR to regulate viability is associated with its C-terminal 75 residues. Furthermore, this function is distinct from its role in cell proliferation, independent of its ribosomal functions, and may be regulated by a nonnuclear localization.« less