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 Asp 166 is 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.
Bibliographical noteFunding Information:
This research at ORNL’s High Flux Isotope Reactor (IMAGINE beamline) was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. We thank Institut Laue-Langevin (beamline LADI-III) for awarded neutron beamtime. The Office of Biological and Environmental Research supported research at Oak Ridge National Laboratory’s Center for Structural Molecular Biology (CSMB) involving protein deuteration, using facilities supported by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. This manuscript has been authored by UT-Battelle LLC under DOE contract no. DE-AC05-00OR22725. We thank Dr. A. Kornev (University of California at San Diego) for technical assistance with the manuscript preparation and critical analysis of the paper, and Y. Wang (University of Minnesota at Minneapolis) for help in retrieving the NMR data for the H/D exchange comparison. S.S.T. and G.V. were partly supported by NIH grants GM19301 and GM100310, respectively. O.G., S.S.T., and A.K. were partly supported by a UCOP grant. O.G. and A.K. were partly supported by the U.S. Department of Energy’s (DOE) Office of Basic Energy Sciences.