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Title: Zooming in on protons: Neutron structure of protein kinase A trapped in a product complex

Abstract

The question vis-à-vis the chemistry of phosphoryl group transfer catalyzed by protein kinases remains a major challenge. The neutron diffraction structure of the catalytic subunit of cAMP-dependent protein kinase (PKA-C) provides a more complete chemical portrait of key proton interactions at the active site. By using a high-affinity protein kinase substrate (PKS) peptide, we captured the reaction products, dephosphorylated nucleotide [adenosine diphosphate (ADP)] and phosphorylated PKS (pPKS), bound at the active site. In the complex, the phosphoryl group of the peptide is protonated, whereas the carboxyl group of the catalytic Asp 166is not. Our structure, including conserved waters, shows how the peptide links the distal parts of the cleft together, creating a network that engages the entire molecule. By comparing slow-exchanging backbone amides to those determined by the NMR analysis of PKA-C with ADP and inhibitor peptide (PKI), we identified exchangeable amides that likely distinguish catalytic and inhibited states.

Authors:
ORCiD logo [1]; ORCiD logo [2]; ORCiD logo [3];  [4]; ORCiD logo [5]; ORCiD logo [2]
  1. Univ. of Tennessee, Knoxville, TN (United States). Bredesen Center
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Neutron Scattering Division
  3. Inst. Laue-Langevin, Grenoble (France). Large Scale Structures Group
  4. Univ. of Minnesota, Minneapolis, MN (United States). Dept. of Chemistry; Univ. of Minnesota, Minneapolis, MN (United States). Dept. of Biochemistry, Molecular Biology, and Biophysics
  5. Univ. of California, San Diego, CA (United States). Dept. of Pharmacology, and Dept. of Chemistry and Biochemistry
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1506775
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Science Advances
Additional Journal Information:
Journal Volume: 5; Journal Issue: 3; Journal ID: ISSN 2375-2548
Publisher:
AAAS
Country of Publication:
United States
Language:
English

Citation Formats

Gerlits, Oksana, Weiss, Kevin L., Blakeley, Matthew P., Veglia, Gianluigi, Taylor, Susan S., and Kovalevsky, Andrey. Zooming in on protons: Neutron structure of protein kinase A trapped in a product complex. United States: N. p., 2019. Web. doi:10.1126/sciadv.aav0482.
Gerlits, Oksana, Weiss, Kevin L., Blakeley, Matthew P., Veglia, Gianluigi, Taylor, Susan S., & Kovalevsky, Andrey. Zooming in on protons: Neutron structure of protein kinase A trapped in a product complex. United States. doi:10.1126/sciadv.aav0482.
Gerlits, Oksana, Weiss, Kevin L., Blakeley, Matthew P., Veglia, Gianluigi, Taylor, Susan S., and Kovalevsky, Andrey. Wed . "Zooming in on protons: Neutron structure of protein kinase A trapped in a product complex". United States. doi:10.1126/sciadv.aav0482. https://www.osti.gov/servlets/purl/1506775.
@article{osti_1506775,
title = {Zooming in on protons: Neutron structure of protein kinase A trapped in a product complex},
author = {Gerlits, Oksana and Weiss, Kevin L. and Blakeley, Matthew P. and Veglia, Gianluigi and Taylor, Susan S. and Kovalevsky, Andrey},
abstractNote = {The question vis-à-vis the chemistry of phosphoryl group transfer catalyzed by protein kinases remains a major challenge. The neutron diffraction structure of the catalytic subunit of cAMP-dependent protein kinase (PKA-C) provides a more complete chemical portrait of key proton interactions at the active site. By using a high-affinity protein kinase substrate (PKS) peptide, we captured the reaction products, dephosphorylated nucleotide [adenosine diphosphate (ADP)] and phosphorylated PKS (pPKS), bound at the active site. In the complex, the phosphoryl group of the peptide is protonated, whereas the carboxyl group of the catalytic Asp166is not. Our structure, including conserved waters, shows how the peptide links the distal parts of the cleft together, creating a network that engages the entire molecule. By comparing slow-exchanging backbone amides to those determined by the NMR analysis of PKA-C with ADP and inhibitor peptide (PKI), we identified exchangeable amides that likely distinguish catalytic and inhibited states.},
doi = {10.1126/sciadv.aav0482},
journal = {Science Advances},
issn = {2375-2548},
number = 3,
volume = 5,
place = {United States},
year = {2019},
month = {3}
}

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Works referenced in this record:

A short history of SHELX
journal, December 2007

  • Sheldrick, George M.
  • Acta Crystallographica Section A Foundations of Crystallography, Vol. 64, Issue 1, p. 112-122
  • DOI: 10.1107/S0108767307043930

The CCP4 suite programs for protein crystallography
journal, September 1994