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
Miller, Michelle S; Schmidt-Kittler, Oleg; Bolduc, David M; Brower, Evan T; Chaves-Moreira, Daniele; Allaire, Marc; Kinzler, Kenneth W; Jennings, Ian G; Thompson, Philip E; Cole, Philip A; Amzel, L Mario; Vogelstein, Bert; Gabelli, Sandra B
Structural basis of nSH2 regulation and lipid binding in PI3Kα Journal Article
In: Oncotarget, vol. 5, no. 14, pp. 5198-208, 2014, ISSN: 1949-2553.
Abstract | Links | BibTeX | Tags: Amino Acid Sequence, Animals, Binding Sites, Boron Compounds, Models, Molecular, Molecular Sequence Data, Phosphatidylinositol 3-Kinases, Protein Binding, Protein Conformation, Sf9 Cells, Signal Transduction, Spodoptera, src Homology Domains
@article{1299980,
title = {Structural basis of nSH2 regulation and lipid binding in PI3Kα},
author = {Michelle S Miller and Oleg Schmidt-Kittler and David M Bolduc and Evan T Brower and Daniele Chaves-Moreira and Marc Allaire and Kenneth W Kinzler and Ian G Jennings and Philip E Thompson and Philip A Cole and L Mario Amzel and Bert Vogelstein and Sandra B Gabelli},
doi = {10.18632/oncotarget.2263},
issn = {1949-2553},
year = {2014},
date = {2014-07-01},
journal = {Oncotarget},
volume = {5},
number = {14},
pages = {5198-208},
abstract = {We report two crystal structures of the wild-type phosphatidylinositol 3-kinase α (PI3Kα) heterodimer refined to 2.9 Å and 3.4 Å resolution: the first as the free enzyme, the second in complex with the lipid substrate, diC4-PIP$_2$, respectively. The first structure shows key interactions of the N-terminal SH2 domain (nSH2) and iSH2 with the activation loop that suggest a mechanism by which the enzyme is inhibited in its basal state. In the second structure, the lipid substrate binds in a positively charged pocket adjacent to the ATP-binding site, bordered by the P-loop, the activation loop and the iSH2 domain. An additional lipid-binding site was identified at the interface of the ABD, iSH2 and kinase domains. The ability of PI3Kα to bind an additional PIP$_2$ molecule was confirmed in vitro by fluorescence quenching experiments. The crystal structures reveal key differences in the way the nSH2 domain interacts with wild-type p110α and with the oncogenic mutant p110αH1047R. Increased buried surface area and two unique salt-bridges observed only in the wild-type structure suggest tighter inhibition in the wild-type PI3Kα than in the oncogenic mutant. These differences may be partially responsible for the increased basal lipid kinase activity and increased membrane binding of the oncogenic mutant.},
keywords = {Amino Acid Sequence, Animals, Binding Sites, Boron Compounds, Models, Molecular, Molecular Sequence Data, Phosphatidylinositol 3-Kinases, Protein Binding, Protein Conformation, Sf9 Cells, Signal Transduction, Spodoptera, src Homology Domains},
pubstate = {published},
tppubtype = {article}
}
We report two crystal structures of the wild-type phosphatidylinositol 3-kinase α (PI3Kα) heterodimer refined to 2.9 Å and 3.4 Å resolution: the first as the free enzyme, the second in complex with the lipid substrate, diC4-PIP$_2$, respectively. The first structure shows key interactions of the N-terminal SH2 domain (nSH2) and iSH2 with the activation loop that suggest a mechanism by which the enzyme is inhibited in its basal state. In the second structure, the lipid substrate binds in a positively charged pocket adjacent to the ATP-binding site, bordered by the P-loop, the activation loop and the iSH2 domain. An additional lipid-binding site was identified at the interface of the ABD, iSH2 and kinase domains. The ability of PI3Kα to bind an additional PIP$_2$ molecule was confirmed in vitro by fluorescence quenching experiments. The crystal structures reveal key differences in the way the nSH2 domain interacts with wild-type p110α and with the oncogenic mutant p110αH1047R. Increased buried surface area and two unique salt-bridges observed only in the wild-type structure suggest tighter inhibition in the wild-type PI3Kα than in the oncogenic mutant. These differences may be partially responsible for the increased basal lipid kinase activity and increased membrane binding of the oncogenic mutant.
Maksimoska, Jasna; Segura-Peña, Dario; Cole, Philip A; Marmorstein, Ronen
Structure of the p300 histone acetyltransferase bound to acetyl-coenzyme A and its analogues Journal Article
In: Biochemistry, vol. 53, no. 21, pp. 3415-22, 2014, ISSN: 1520-4995.
Abstract | Links | BibTeX | Tags: Acetyl Coenzyme A, Catalytic Domain, Coenzyme A, Humans, Models, Molecular, p300-CBP Transcription Factors, Protein Binding, Protein Conformation
@article{1299982,
title = {Structure of the p300 histone acetyltransferase bound to acetyl-coenzyme A and its analogues},
author = {Jasna Maksimoska and Dario Segura-Peña and Philip A Cole and Ronen Marmorstein},
doi = {10.1021/bi500380f},
issn = {1520-4995},
year = {2014},
date = {2014-06-01},
journal = {Biochemistry},
volume = {53},
number = {21},
pages = {3415-22},
abstract = {The p300 and CBP transcriptional coactivator paralogs (p300/CBP) regulate a variety of different cellular pathways, in part, by acetylating histones and more than 70 non-histone protein substrates. Mutation, chromosomal translocation, or other aberrant activities of p300/CBP are linked to many different diseases, including cancer. Because of its pleiotropic biological roles and connection to disease, it is important to understand the mechanism of acetyl transfer by p300/CBP, in part so that inhibitors can be more rationally developed. Toward this goal, a structure of p300 bound to a Lys-CoA bisubstrate HAT inhibitor has been previously elucidated, and the enzyme’s catalytic mechanism has been investigated. Nonetheless, many questions underlying p300/CBP structure and mechanism remain. Here, we report a structural characterization of different reaction states in the p300 activity cycle. We present the structures of p300 in complex with an acetyl-CoA substrate, a CoA product, and an acetonyl-CoA inhibitor. A comparison of these structures with the previously reported p300/Lys-CoA complex demonstrates that the conformation of the enzyme active site depends on the interaction of the enzyme with the cofactor, and is not apparently influenced by protein substrate lysine binding. The p300/CoA crystals also contain two poly(ethylene glycol) moieties bound proximal to the cofactor binding site, implicating the path of protein substrate association. The structure of the p300/acetonyl-CoA complex explains the inhibitory and tight binding properties of the acetonyl-CoA toward p300. Together, these studies provide new insights into the molecular basis of acetylation by p300 and have implications for the rational development of new small molecule p300 inhibitors.},
keywords = {Acetyl Coenzyme A, Catalytic Domain, Coenzyme A, Humans, Models, Molecular, p300-CBP Transcription Factors, Protein Binding, Protein Conformation},
pubstate = {published},
tppubtype = {article}
}
The p300 and CBP transcriptional coactivator paralogs (p300/CBP) regulate a variety of different cellular pathways, in part, by acetylating histones and more than 70 non-histone protein substrates. Mutation, chromosomal translocation, or other aberrant activities of p300/CBP are linked to many different diseases, including cancer. Because of its pleiotropic biological roles and connection to disease, it is important to understand the mechanism of acetyl transfer by p300/CBP, in part so that inhibitors can be more rationally developed. Toward this goal, a structure of p300 bound to a Lys-CoA bisubstrate HAT inhibitor has been previously elucidated, and the enzyme’s catalytic mechanism has been investigated. Nonetheless, many questions underlying p300/CBP structure and mechanism remain. Here, we report a structural characterization of different reaction states in the p300 activity cycle. We present the structures of p300 in complex with an acetyl-CoA substrate, a CoA product, and an acetonyl-CoA inhibitor. A comparison of these structures with the previously reported p300/Lys-CoA complex demonstrates that the conformation of the enzyme active site depends on the interaction of the enzyme with the cofactor, and is not apparently influenced by protein substrate lysine binding. The p300/CoA crystals also contain two poly(ethylene glycol) moieties bound proximal to the cofactor binding site, implicating the path of protein substrate association. The structure of the p300/acetonyl-CoA complex explains the inhibitory and tight binding properties of the acetonyl-CoA toward p300. Together, these studies provide new insights into the molecular basis of acetylation by p300 and have implications for the rational development of new small molecule p300 inhibitors.
Chiang, Meng-Jung; Holbert, Marc A; Kalin, Jay H; Ahn, Young-Hoon; Giddens, John; Amin, Mohammed N; Taylor, Martin S; Collins, Samuel L; Chan-Li, Yee; Waickman, Adam; Hsiao, Po-Yuan; Bolduc, David; Leahy, Daniel J; Horton, Maureen R; Wang, Lai-Xi; Powell, Jonathan D; Cole, Philip A
An Fc domain protein-small molecule conjugate as an enhanced immunomodulator Journal Article
In: J Am Chem Soc, vol. 136, no. 9, pp. 3370-3, 2014, ISSN: 1520-5126.
Abstract | Links | BibTeX | Tags: Adenosine, Animals, CD4-Positive T-Lymphocytes, Immunoconjugates, Immunoglobulin Fc Fragments, Immunologic Factors, Mice, Models, Molecular, Phenethylamines, Protein Conformation
@article{1299984,
title = {An Fc domain protein-small molecule conjugate as an enhanced immunomodulator},
author = {Meng-Jung Chiang and Marc A Holbert and Jay H Kalin and Young-Hoon Ahn and John Giddens and Mohammed N Amin and Martin S Taylor and Samuel L Collins and Yee Chan-Li and Adam Waickman and Po-Yuan Hsiao and David Bolduc and Daniel J Leahy and Maureen R Horton and Lai-Xi Wang and Jonathan D Powell and Philip A Cole},
doi = {10.1021/ja5006674},
issn = {1520-5126},
year = {2014},
date = {2014-03-01},
journal = {J Am Chem Soc},
volume = {136},
number = {9},
pages = {3370-3},
abstract = {Proteins as well as small molecules have demonstrated success as therapeutic agents, but their pharmacologic properties sometimes fall short against particular drug targets. Although the adenosine 2a receptor (A(2A)R) has been identified as a promising target for immunotherapy, small molecule A(2A)R agonists have suffered from short pharmacokinetic half-lives and the potential for toxicity by modulating nonimmune pathways. To overcome these limitations, we have tethered the A(2A)R agonist CGS-21680 to the immunoglobulin Fc domain using expressed protein ligation with Sf9 cell secreted protein. The protein small molecule conjugate Fc-CGS retained potent Fc receptor and A(2A)R interactions and showed superior properties as a therapeutic for the treatment of a mouse model of autoimmune pneumonitis. This approach may provide a general strategy for optimizing small molecule therapeutics.},
keywords = {Adenosine, Animals, CD4-Positive T-Lymphocytes, Immunoconjugates, Immunoglobulin Fc Fragments, Immunologic Factors, Mice, Models, Molecular, Phenethylamines, Protein Conformation},
pubstate = {published},
tppubtype = {article}
}
Proteins as well as small molecules have demonstrated success as therapeutic agents, but their pharmacologic properties sometimes fall short against particular drug targets. Although the adenosine 2a receptor (A(2A)R) has been identified as a promising target for immunotherapy, small molecule A(2A)R agonists have suffered from short pharmacokinetic half-lives and the potential for toxicity by modulating nonimmune pathways. To overcome these limitations, we have tethered the A(2A)R agonist CGS-21680 to the immunoglobulin Fc domain using expressed protein ligation with Sf9 cell secreted protein. The protein small molecule conjugate Fc-CGS retained potent Fc receptor and A(2A)R interactions and showed superior properties as a therapeutic for the treatment of a mouse model of autoimmune pneumonitis. This approach may provide a general strategy for optimizing small molecule therapeutics.
Wang, Z.; Cole, P. A.
Catalytic mechanisms and regulation of protein kinases Journal Article
In: Methods Enzymol., vol. 548, pp. 1-21, 2014, ISSN: 1557-7988.
Abstract | Links | BibTeX | Tags: Adenosine Triphosphate, Animals, Biocatalysis, Humans, Models, Molecular, Mutation, Phosphorylation, Post-Translational, Protein Conformation, Protein Kinase Inhibitors, Protein Kinases, Protein Processing, Substrate Specificity
@article{1299974,
title = {Catalytic mechanisms and regulation of protein kinases},
author = {Z. Wang and P. A. Cole},
doi = {10.1016/B978-0-12-397918-6.00001-X},
issn = {1557-7988},
year = {2014},
date = {2014-00-00},
journal = {Methods Enzymol.},
volume = {548},
pages = {1-21},
abstract = {Protein kinases transfer a phosphoryl group from ATP onto target proteins and play a critical role in signal transduction and other cellular processes. Here, we review the kinase kinetic and chemical mechanisms and their application in understanding kinase structure and function. Aberrant kinase activity has been implicated in many human diseases, in particular cancer. We highlight applications of technologies and concepts derived from kinase mechanistic studies that have helped illuminate how kinases are regulated and contribute to pathophysiology.},
keywords = {Adenosine Triphosphate, Animals, Biocatalysis, Humans, Models, Molecular, Mutation, Phosphorylation, Post-Translational, Protein Conformation, Protein Kinase Inhibitors, Protein Kinases, Protein Processing, Substrate Specificity},
pubstate = {published},
tppubtype = {article}
}
Protein kinases transfer a phosphoryl group from ATP onto target proteins and play a critical role in signal transduction and other cellular processes. Here, we review the kinase kinetic and chemical mechanisms and their application in understanding kinase structure and function. Aberrant kinase activity has been implicated in many human diseases, in particular cancer. We highlight applications of technologies and concepts derived from kinase mechanistic studies that have helped illuminate how kinases are regulated and contribute to pathophysiology.