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

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Abstract

Summary 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. Concurrently, 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. Lastly, the serine hydroxyl of the substrate rotates back toward D166 to form the Michaelis complex with the active site primed for phosphoryl transfer.

Original languageEnglish
Pages (from-to)2331-2340
Number of pages10
JournalStructure
Volume23
Issue number12
DOIs
StatePublished - Dec 1 2015

Funding

A.D. and W.T.H. were supported, and O.G. and P.L. were partly supported, by Laboratory Directed Research and Development funding from ORNL . P.L. was partly supported by an NIH/NIGMS -funded consortium (Grant 1R01GM071939-01 ) between ORNL and LBNL to develop computational tools for neutron protein crystallography. Use of the Advanced Photon Source was supported by the U.S. Department of Energy office of Basic Energy Sciences . We are grateful to the staff of the SBC-CAT of the 19-ID beamline for their support during data collection. This manuscript has been authored by UT-Battelle, LLC under Contract No. DE-AC05-00OR22725 for the U.S. Department of Energy. The United States Government retains copyright and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan ( http://energy.gov/downloads/doe-public-access-plan ).

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