Protein kinase A in the neutron beam: Insights for catalysis from directly observing protons

Oksana Gerlits, Kevin L. Weiss, Matthew P. Blakeley, Gianluigi Veglia, Susan S. Taylor, Andrey Kovalevsky

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Scopus citations

Abstract

Protein kinases transmit chemical signals by phosphorylating substrate proteins, thus regulating a multitude of cellular processes. cAMP-dependent protein kinase (PKA), a prototypical enzyme for the whole kinase family, has been the focus of research for several decades, however, the details of the chemical mechanism of phosphoryl group transfer have remained unknown. We used neutron crystallography to map key proton sites and hydrogen bonding interactions in the PKA catalytic subunit (PKAc) in a product complex containing adenosine diphosphate (ADP) and the phosphorylated high affinity protein kinase substrate (pPKS) peptide. To improve neutron diffraction, we deuterated PKAc allowing us to use very small crystals. In the product complex, the phosphoryl group of pPKS is protonated whereas the catalytic Asp166 is not. H/D exchange analysis of the main-chain amides and comparison with the NMR analysis of PKAc with inhibitor peptide complex revealed exchangeable amides that may distinguish the catalytic and inhibited states.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages311-331
Number of pages21
DOIs
StatePublished - 2020

Publication series

NameMethods in Enzymology
Volume634
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Bibliographical note

Funding Information:
The neutron diffraction experiments were performed on IMAGINE beamline at Oak Ridge National Laboratory's High Flux Isotope Reactor sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy (DOE), and on beamline LADI-III at the Institut Laue Langevin. Protein deuteration, purification and crystallization were carried out at ORNL's Center for Structural Molecular Biology (CSMB) supported by the DOE Office of Biological and Environmental Research (BER), using facilities supported by the Scientific User Facilities Division, Office of Basic Energy Sciences (BES). This work was supported by the NIH grants GM19301 and GM100310, by a UCOP grant, and by the DOE BES.

Funding Information:
The neutron diffraction experiments were performed on IMAGINE beamline at Oak Ridge National Laboratory's High Flux Isotope Reactor sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy (DOE), and on beamline LADI-III at the Institut Laue Langevin. Protein deuteration, purification and crystallization were carried out at ORNL's Center for Structural Molecular Biology (CSMB) supported by the DOE Office of Biological and Environmental Research (BER), using facilities supported by the Scientific User Facilities Division, Office of Basic Energy Sciences (BES). This work was supported by the NIH grants GM19301 and GM100310, by a UCOP grant, and by the DOE BES.

Keywords

  • Enzyme mechanism
  • Hydrogen bond
  • Neutron crystallography
  • Phosphoryl transfer
  • Product complex
  • Protein kinase A
  • Proton transfer
  • Protonation state

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