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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 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.

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:
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
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY

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},
number = 3,
volume = 5,
place = {United States},
year = {2019},
month = {3}
}

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Figures / Tables:

Fig. 1 Fig. 1: Interweaving interactions of pPKS in the vicinity of the PKA-C active site. (A) Specific sites of the substrate’s direct interactions with PKA-C. The space-filled residue signifies the peptide’s hydrophobic P + 1 residue, Ile22. Here and in all the panels, pPKS is shown as a dark gray ribbon,more » ADP is colored with black carbon atoms, and the remaining colors are for the enzyme residues. This coloring scheme is kept for all figures throughout the manuscript. (B) P-site showing position of the D atom on pS21 and interactions with water molecules. The omit FOFC difference nuclear density map for the deuterium is shown as green mesh contoured at 3.5σ level. The 2FOFC nuclear density map contoured at 2.0σ level is shown in gray mesh. (C) The 2FOFC nuclear density map (gray mesh contoured at 2.0σ level) shows deprotonated Asp166, protonated Lys168, and the extent of the hydrogen-bonding network.« less

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