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

doi:10.1016/bs.mie.2019.12.003

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 proceeding › Chapter › peer-review

3 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 language	English
Title of host publication	Neutron Crystallography in Structural Biology
Publisher	Academic Press Inc.
Pages	311-331
Number of pages	21
DOIs	https://doi.org/10.1016/bs.mie.2019.12.003
State	Published - 2020

Publication series

Name	Methods in Enzymology
Volume	634
ISSN (Print)	0076-6879
ISSN (Electronic)	1557-7988

Funding

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. 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.

Funders	Funder number
DOE BES
DOE Office of Biological and Environmental Research
Institut Laue Langevin
Office of Basic Energy Sciences
Scientific User Facilities Division
National Institutes of Health	GM100310
U.S. Department of Energy
National Institute of General Medical Sciences	R37GM019301
Basic Energy Sciences
Biological and Environmental Research	BES
Oak Ridge National Laboratory
Office of the President, University of California

Keywords

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

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10.1016/bs.mie.2019.12.003

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title = "Protein kinase A in the neutron beam: Insights for catalysis from directly observing protons",

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.",

keywords = "Enzyme mechanism, Hydrogen bond, Neutron crystallography, Phosphoryl transfer, Product complex, Protein kinase A, Proton transfer, Protonation state",

author = "Oksana Gerlits and Weiss, {Kevin L.} and Blakeley, {Matthew P.} and Gianluigi Veglia and Taylor, {Susan S.} and Andrey Kovalevsky",

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year = "2020",

doi = "10.1016/bs.mie.2019.12.003",

language = "English",

series = "Methods in Enzymology",

publisher = "Academic Press Inc.",

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T2 - Insights for catalysis from directly observing protons

AU - Gerlits, Oksana

AU - Weiss, Kevin L.

AU - Blakeley, Matthew P.

AU - Veglia, Gianluigi

AU - Taylor, Susan S.

AU - Kovalevsky, Andrey

PY - 2020

Y1 - 2020

N2 - 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.

AB - 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.

KW - Enzyme mechanism

KW - Hydrogen bond

KW - Neutron crystallography

KW - Phosphoryl transfer

KW - Product complex

KW - Protein kinase A

KW - Proton transfer

KW - Protonation state

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M3 - Chapter

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AN - SCOPUS:85078044275

T3 - Methods in Enzymology

SP - 311

EP - 331

BT - Neutron Crystallography in Structural Biology

PB - Academic Press Inc.

ER -

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

Abstract

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Keywords

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