2021
Dempsey, D. R.; Viennet, T.; Iwase, R.; Park, E. Y.; Henriquez, S.; Chen, Z.; Jeliazkov, J. R.; Palanski, B. A.; Phan, K. L.; Coote, P.; Gray, J. J.; Eck, M. J.; Gabelli, S. B.; Arthanari, H.; Cole, P. A.
The structural basis of PTEN regulation by multi-site phosphorylation Journal Article
In: Nat. Struct. Mol. Biol., vol. 28, no. 10, pp. 858-868, 2021, ISSN: 1545-9985.
Abstract | Links | BibTeX | Tags: Animals, Ciona intestinalis, Crystallography, Fluorescence Polarization, Humans, Magnetic Resonance Spectroscopy, Molecular Docking Simulation, Phosphorylation, PTEN Phosphohydrolase, X-Ray
@article{1624365,
title = {The structural basis of PTEN regulation by multi-site phosphorylation},
author = {D. R. Dempsey and T. Viennet and R. Iwase and E. Y. Park and S. Henriquez and Z. Chen and J. R. Jeliazkov and B. A. Palanski and K. L. Phan and P. Coote and J. J. Gray and M. J. Eck and S. B. Gabelli and H. Arthanari and P. A. Cole},
doi = {10.1038/s41594-021-00668-5},
issn = {1545-9985},
year = {2021},
date = {2021-10-28},
urldate = {2021-10-28},
journal = {Nat. Struct. Mol. Biol.},
volume = {28},
number = {10},
pages = {858-868},
abstract = {Phosphatase and tensin homolog (PTEN) is a phosphatidylinositol-3,4,5-triphosphate (PIP3) phospholipid phosphatase that is commonly mutated or silenced in cancer. PTEN’s catalytic activity, cellular membrane localization and stability are orchestrated by a cluster of C-terminal phosphorylation (phospho-C-tail) events on Ser380, Thr382, Thr383 and Ser385, but the molecular details of this multi-faceted regulation have remained uncertain. Here we use a combination of protein semisynthesis, biochemical analysis, NMR, X-ray crystallography and computational simulations on human PTEN and its sea squirt homolog, VSP, to obtain a detailed picture of how the phospho-C-tail forms a belt around the C2 and phosphatase domains of PTEN. We also visualize a previously proposed dynamic N-terminal α-helix and show that it is key for PTEN catalysis but disordered upon phospho-C-tail interaction. This structural model provides a comprehensive framework for how C-tail phosphorylation can impact PTEN’s cellular functions.},
keywords = {Animals, Ciona intestinalis, Crystallography, Fluorescence Polarization, Humans, Magnetic Resonance Spectroscopy, Molecular Docking Simulation, Phosphorylation, PTEN Phosphohydrolase, X-Ray},
pubstate = {published},
tppubtype = {article}
}
Phosphatase and tensin homolog (PTEN) is a phosphatidylinositol-3,4,5-triphosphate (PIP3) phospholipid phosphatase that is commonly mutated or silenced in cancer. PTEN’s catalytic activity, cellular membrane localization and stability are orchestrated by a cluster of C-terminal phosphorylation (phospho-C-tail) events on Ser380, Thr382, Thr383 and Ser385, but the molecular details of this multi-faceted regulation have remained uncertain. Here we use a combination of protein semisynthesis, biochemical analysis, NMR, X-ray crystallography and computational simulations on human PTEN and its sea squirt homolog, VSP, to obtain a detailed picture of how the phospho-C-tail forms a belt around the C2 and phosphatase domains of PTEN. We also visualize a previously proposed dynamic N-terminal α-helix and show that it is key for PTEN catalysis but disordered upon phospho-C-tail interaction. This structural model provides a comprehensive framework for how C-tail phosphorylation can impact PTEN’s cellular functions.
2019
Miller, Michelle S; Maheshwari, Sweta; Shi, Wuxian; Gao, Yuan; Chu, Nam; Soares, Alexei S; Cole, Philip A; Amzel, L Mario; Fuchs, Martin R; Jakoncic, Jean; Gabelli, Sandra B
Getting the Most Out of Your Crystals: Data Collection at the New High-Flux, Microfocus MX Beamlines at NSLS-II Journal Article
In: Molecules, vol. 24, no. 3, 2019, ISSN: 1420-3049.
Abstract | Links | BibTeX | Tags: Crystallography, Models, Molecular, Phosphatidylinositol 3-Kinases, Protein Conformation, Proteins, Pyrophosphatases, X-Ray
@article{1457440,
title = {Getting the Most Out of Your Crystals: Data Collection at the New High-Flux, Microfocus MX Beamlines at NSLS-II},
author = {Michelle S Miller and Sweta Maheshwari and Wuxian Shi and Yuan Gao and Nam Chu and Alexei S Soares and Philip A Cole and L Mario Amzel and Martin R Fuchs and Jean Jakoncic and Sandra B Gabelli},
doi = {10.3390/molecules24030496},
issn = {1420-3049},
year = {2019},
date = {2019-01-01},
journal = {Molecules},
volume = {24},
number = {3},
abstract = {Advances in synchrotron technology are changing the landscape of macromolecular crystallography. The two recently opened beamlines at NSLS-II-AMX and FMX-deliver high-flux microfocus beams that open new possibilities for crystallographic data collection. They are equipped with state-of-the-art experimental stations and automation to allow data collection on previously intractable crystals. Optimized data collection strategies allow users to tailor crystal positioning to optimally distribute the X-ray dose over its volume. Vector data collection allows the user to define a linear trajectory along a well diffracting volume of the crystal and perform rotational data collection while moving along the vector. This is particularly well suited to long, thin crystals. We describe vector data collection of three proteins-Akt1, PI3Kα, and CDP-Chase-to demonstrate its application and utility. For smaller crystals, we describe two methods for multicrystal data collection in a single loop, either manually selecting multiple centers (using H108A-PHM as an example), or "raster-collect", a more automated approach for a larger number of crystals (using CDP-Chase as an example).},
keywords = {Crystallography, Models, Molecular, Phosphatidylinositol 3-Kinases, Protein Conformation, Proteins, Pyrophosphatases, X-Ray},
pubstate = {published},
tppubtype = {article}
}
Advances in synchrotron technology are changing the landscape of macromolecular crystallography. The two recently opened beamlines at NSLS-II-AMX and FMX-deliver high-flux microfocus beams that open new possibilities for crystallographic data collection. They are equipped with state-of-the-art experimental stations and automation to allow data collection on previously intractable crystals. Optimized data collection strategies allow users to tailor crystal positioning to optimally distribute the X-ray dose over its volume. Vector data collection allows the user to define a linear trajectory along a well diffracting volume of the crystal and perform rotational data collection while moving along the vector. This is particularly well suited to long, thin crystals. We describe vector data collection of three proteins-Akt1, PI3Kα, and CDP-Chase-to demonstrate its application and utility. For smaller crystals, we describe two methods for multicrystal data collection in a single loop, either manually selecting multiple centers (using H108A-PHM as an example), or "raster-collect", a more automated approach for a larger number of crystals (using CDP-Chase as an example).
2016
Zucconi, B. E.; Luef, B.; Xu, W.; Henry, R. A.; Nodelman, I. M.; Bowman, G. D.; Andrews, A. J; Cole, P. A.
Modulation of p300/CBP Acetylation of Nucleosomes by Bromodomain Ligand I-CBP112 Journal Article
In: Biochemistry, vol. 55, no. 27, pp. 3727-34, 2016, ISSN: 1520-4995.
Abstract | Links | BibTeX | Tags: Acetylation, Bromine Compounds, Cell Proliferation, Crystallography, Cultured, E1A-Associated p300 Protein, Histones, Humans, Leukemia, Male, Models, Molecular, Mutagenesis, Nucleosomes, p300-CBP Transcription Factors, Prostatic Neoplasms, Protein Binding, Protein Conformation, Site-Directed, Tumor Cells, X-Ray
@article{1299963,
title = {Modulation of p300/CBP Acetylation of Nucleosomes by Bromodomain Ligand I-CBP112},
author = {B. E. Zucconi and B. Luef and W. Xu and R. A. Henry and I. M. Nodelman and G. D. Bowman and A. J Andrews and P. A. Cole},
doi = {10.1021/acs.biochem.6b00480},
issn = {1520-4995},
year = {2016},
date = {2016-00-00},
journal = {Biochemistry},
volume = {55},
number = {27},
pages = {3727-34},
abstract = {The histone acetyltransferase (HAT) enzymes p300 and CBP are closely related paralogs that serve as transcriptional coactivators and have been found to be dysregulated in cancer and other diseases. p300/CBP is a multidomain protein and possesses a highly conserved bromodomain that has been shown to bind acetylated Lys residues in both proteins and various small molecules, including I-CBP112 and CBP30. Here we show that the ligand I-CBP112 can stimulate nucleosome acetylation up to 3-fold while CBP30 does not. Activation of p300/CBP by I-CBP112 is not observed with the isolated histone H3 substrate but requires a nucleosome substrate. I-CBP112 does not impact nucleosome acetylation by the isolated p300 HAT domain, and the effects of I-CBP112 on p300/CBP can be neutralized by CBP30, suggesting that I-CBP112 likely allosterically activates p300/CBP through bromodomain interactions. Using mass spectrometry and Western blots, we have found that I-CBP112 particularly stimulates acetylation of Lys18 of histone H3 (H3K18) in nucleosomes, an established in vivo site of p300/CBP. In addition, we show that I-CBP112 enhances H3K18 acetylation in acute leukemia and prostate cancer cells in a concentration range commensurate with its antiproliferative effects. Our findings extend the known pharmacology of bromodomain ligands in the regulation of p300/CBP and suggest a novel approach to modulating histone acetylation in cancer.},
keywords = {Acetylation, Bromine Compounds, Cell Proliferation, Crystallography, Cultured, E1A-Associated p300 Protein, Histones, Humans, Leukemia, Male, Models, Molecular, Mutagenesis, Nucleosomes, p300-CBP Transcription Factors, Prostatic Neoplasms, Protein Binding, Protein Conformation, Site-Directed, Tumor Cells, X-Ray},
pubstate = {published},
tppubtype = {article}
}
The histone acetyltransferase (HAT) enzymes p300 and CBP are closely related paralogs that serve as transcriptional coactivators and have been found to be dysregulated in cancer and other diseases. p300/CBP is a multidomain protein and possesses a highly conserved bromodomain that has been shown to bind acetylated Lys residues in both proteins and various small molecules, including I-CBP112 and CBP30. Here we show that the ligand I-CBP112 can stimulate nucleosome acetylation up to 3-fold while CBP30 does not. Activation of p300/CBP by I-CBP112 is not observed with the isolated histone H3 substrate but requires a nucleosome substrate. I-CBP112 does not impact nucleosome acetylation by the isolated p300 HAT domain, and the effects of I-CBP112 on p300/CBP can be neutralized by CBP30, suggesting that I-CBP112 likely allosterically activates p300/CBP through bromodomain interactions. Using mass spectrometry and Western blots, we have found that I-CBP112 particularly stimulates acetylation of Lys18 of histone H3 (H3K18) in nucleosomes, an established in vivo site of p300/CBP. In addition, we show that I-CBP112 enhances H3K18 acetylation in acute leukemia and prostate cancer cells in a concentration range commensurate with its antiproliferative effects. Our findings extend the known pharmacology of bromodomain ligands in the regulation of p300/CBP and suggest a novel approach to modulating histone acetylation in cancer.
2014
Wang, Yun; Kavran, Jennifer M; Chen, Zan; Karukurichi, Kannan R; Leahy, Daniel J; Cole, Philip A
Regulation of S-adenosylhomocysteine hydrolase by lysine acetylation Journal Article
In: J Biol Chem, vol. 289, no. 45, pp. 31361-72, 2014, ISSN: 1083-351X.
Abstract | Links | BibTeX | Tags: Acetylation, Adenosylhomocysteinase, Amino Acid, Amino Acid Sequence, Catalysis, Crystallography, Humans, Hydrogen Bonding, Lysine, Methylation, Models, Molecular, Molecular Sequence Data, Mutagenesis, NAD, Plasmids, Post-Translational, Protein Binding, Protein Processing, Protein Structure, Recombinant Proteins, Sequence Homology, Site-Directed, Structure-Activity Relationship, Tertiary, X-Ray
@article{1299977,
title = {Regulation of S-adenosylhomocysteine hydrolase by lysine acetylation},
author = {Yun Wang and Jennifer M Kavran and Zan Chen and Kannan R Karukurichi and Daniel J Leahy and Philip A Cole},
doi = {10.1074/jbc.M114.597153},
issn = {1083-351X},
year = {2014},
date = {2014-11-01},
journal = {J Biol Chem},
volume = {289},
number = {45},
pages = {31361-72},
abstract = {S-Adenosylhomocysteine hydrolase (SAHH) is an NAD(+)-dependent tetrameric enzyme that catalyzes the breakdown of S-adenosylhomocysteine to adenosine and homocysteine and is important in cell growth and the regulation of gene expression. Loss of SAHH function can result in global inhibition of cellular methyltransferase enzymes because of high levels of S-adenosylhomocysteine. Prior proteomics studies have identified two SAHH acetylation sites at Lys(401) and Lys(408) but the impact of these post-translational modifications has not yet been determined. Here we use expressed protein ligation to produce semisynthetic SAHH acetylated at Lys(401) and Lys(408) and show that modification of either position negatively impacts the catalytic activity of SAHH. X-ray crystal structures of 408-acetylated SAHH and dually acetylated SAHH have been determined and reveal perturbations in the C-terminal hydrogen bonding patterns, a region of the protein important for NAD(+) binding. These crystal structures along with mutagenesis data suggest that such hydrogen bond perturbations are responsible for SAHH catalytic inhibition by acetylation. These results suggest how increased acetylation of SAHH may globally influence cellular methylation patterns.},
keywords = {Acetylation, Adenosylhomocysteinase, Amino Acid, Amino Acid Sequence, Catalysis, Crystallography, Humans, Hydrogen Bonding, Lysine, Methylation, Models, Molecular, Molecular Sequence Data, Mutagenesis, NAD, Plasmids, Post-Translational, Protein Binding, Protein Processing, Protein Structure, Recombinant Proteins, Sequence Homology, Site-Directed, Structure-Activity Relationship, Tertiary, X-Ray},
pubstate = {published},
tppubtype = {article}
}
S-Adenosylhomocysteine hydrolase (SAHH) is an NAD(+)-dependent tetrameric enzyme that catalyzes the breakdown of S-adenosylhomocysteine to adenosine and homocysteine and is important in cell growth and the regulation of gene expression. Loss of SAHH function can result in global inhibition of cellular methyltransferase enzymes because of high levels of S-adenosylhomocysteine. Prior proteomics studies have identified two SAHH acetylation sites at Lys(401) and Lys(408) but the impact of these post-translational modifications has not yet been determined. Here we use expressed protein ligation to produce semisynthetic SAHH acetylated at Lys(401) and Lys(408) and show that modification of either position negatively impacts the catalytic activity of SAHH. X-ray crystal structures of 408-acetylated SAHH and dually acetylated SAHH have been determined and reveal perturbations in the C-terminal hydrogen bonding patterns, a region of the protein important for NAD(+) binding. These crystal structures along with mutagenesis data suggest that such hydrogen bond perturbations are responsible for SAHH catalytic inhibition by acetylation. These results suggest how increased acetylation of SAHH may globally influence cellular methylation patterns.