Protein Kinase A Catalytic Subunit Primed for Action: Time-Lapse Crystallography of Michaelis Complex Formation

Das, Amit; Gerlits, Oksana; Parks, Jerry M.; Langan, Paul; Kovalevsky, Andrey; Heller, William T.

doi:10.1016/j.str.2015.10.005

Title: Protein Kinase A Catalytic Subunit Primed for Action: Time-Lapse Crystallography of Michaelis Complex Formation

Abstract

The catalytic subunit of the cyclic AMP-dependent protein kinase A (PKAc) catalyzes the transfer of the γ-phosphate of bound Mg₂ATP to a serine or threonine residue of a protein substrate. Here, time-lapse X-ray crystallography was used to capture a series of complexes of PKAc with an oligopeptide substrate and unreacted Mg₂ATP, including the Michaelis complex, that reveal important geometric rearrangements in and near the active site preceding the phosphoryl transfer reaction. Contrary to the prevailing view, Mg²⁺ binds first to the M1 site as a complex with ATP and is followed by Mg²⁺ binding to the M2 site. Furthermore, the target serine hydroxyl of the peptide substrate rotates away from the active site toward the bulk solvent, which breaks the hydrogen bond with D166. In conclusion, the serine hydroxyl of the substrate rotates back toward D166 to form the Michaelis complex with the active site primed for phosphoryl transfer.

Authors:: Das, Amit; Gerlits, Oksana; Parks, Jerry M.; Langan, Paul; Kovalevsky, Andrey; Heller, William T.

Publication Date:: Tue Dec 01 00:00:00 EST 2015

Research Org.:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Org.:: USDOE Office of Science (SC), Basic Energy Sciences (BES)

OSTI Identifier:: 1334200

Alternate Identifier(s):: OSTI ID: 1235817; OSTI ID: 1250913

Grant/Contract Number:: AC05-00OR22725

Resource Type:: Published Article

Journal Name:: Structure

Additional Journal Information:: Journal Name: Structure Journal Volume: 23 Journal Issue: 12; Journal ID: ISSN 0969-2126

Publisher:: Elsevier

Country of Publication:: United Kingdom

Language:: English

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

Citation Formats


                    Das, Amit, Gerlits, Oksana, Parks, Jerry M., Langan, Paul, Kovalevsky, Andrey, and Heller, William T. Protein Kinase A Catalytic Subunit Primed for Action: Time-Lapse Crystallography of Michaelis Complex Formation.  United Kingdom: N. p., 2015. 
Web.  doi:10.1016/j.str.2015.10.005.

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                    Das, Amit, Gerlits, Oksana, Parks, Jerry M., Langan, Paul, Kovalevsky, Andrey, & Heller, William T. Protein Kinase A Catalytic Subunit Primed for Action: Time-Lapse Crystallography of Michaelis Complex Formation.  United Kingdom.  https://doi.org/10.1016/j.str.2015.10.005

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                    Das, Amit, Gerlits, Oksana, Parks, Jerry M., Langan, Paul, Kovalevsky, Andrey, and Heller, William T. Tue .  
"Protein Kinase A Catalytic Subunit Primed for Action: Time-Lapse Crystallography of Michaelis Complex Formation".  United Kingdom.  https://doi.org/10.1016/j.str.2015.10.005.

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@article{osti_1334200,

  title        = {Protein Kinase A Catalytic Subunit Primed for Action: Time-Lapse Crystallography of Michaelis Complex Formation},

  author       = {Das, Amit and Gerlits, Oksana and Parks, Jerry M. and Langan, Paul and Kovalevsky, Andrey and Heller, William T.},

  abstractNote = {The catalytic subunit of the cyclic AMP-dependent protein kinase A (PKAc) catalyzes the transfer of the γ-phosphate of bound Mg2ATP to a serine or threonine residue of a protein substrate. Here, time-lapse X-ray crystallography was used to capture a series of complexes of PKAc with an oligopeptide substrate and unreacted Mg2ATP, including the Michaelis complex, that reveal important geometric rearrangements in and near the active site preceding the phosphoryl transfer reaction. Contrary to the prevailing view, Mg2+ binds first to the M1 site as a complex with ATP and is followed by Mg2+ binding to the M2 site. Furthermore, the target serine hydroxyl of the peptide substrate rotates away from the active site toward the bulk solvent, which breaks the hydrogen bond with D166. In conclusion, the serine hydroxyl of the substrate rotates back toward D166 to form the Michaelis complex with the active site primed for phosphoryl transfer.},

  doi          = {10.1016/j.str.2015.10.005},

  journal      = {Structure},

  number       = 12,

  volume       = 23,

  place        = {United Kingdom},

  year         = {Tue Dec 01 00:00:00 EST 2015},

  month        = {Tue Dec 01 00:00:00 EST 2015}

}

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Works referencing / citing this record:

Globally correlated conformational entropy underlies positive and negative cooperativity in a kinase’s enzymatic cycle
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Assessing the performance of MM/PBSA and MM/GBSA methods. 9. Prediction reliability of binding affinities and binding poses for protein–peptide complexes
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Weng, Gaoqi; Wang, Ercheng; Chen, Fu
Physical Chemistry Chemical Physics, Vol. 21, Issue 19
DOI: 10.1039/c9cp01674k

Water-mediated conformational preselection mechanism in substrate binding cooperativity to protein kinase A
journal, March 2018

Setny, Piotr; Wiśniewska, Marta D.
Proceedings of the National Academy of Sciences, Vol. 115, Issue 15
DOI: 10.1073/pnas.1720024115

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Title: Protein Kinase A Catalytic Subunit Primed for Action: Time-Lapse Crystallography of Michaelis Complex Formation

Abstract

Citation Formats

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Assessing the performance of MM/PBSA and MM/GBSA methods. 9. Prediction reliability of binding affinities and binding poses for protein–peptide complexes journal, January 2019

Water-mediated conformational preselection mechanism in substrate binding cooperativity to protein kinase A journal, March 2018

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journal, February 2019

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journal, January 2019

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journal, March 2018