Phosphoryl Transfer by Protein Kinase A Is Captured in a Crystal Lattice

The catalytic (C) subunit of cAMP-dependent
protein kinase (PKA)
is a serine/threonine kinase responsible for most of the effects of
cAMP signaling, and PKA serves as a prototype for the entire kinase
family. Despite multiple studies of PKA, the steps involved in phosphoryl
transfer, the roles of the catalytically essential magnesium ions,
and the processes that govern the rate-limiting step of ADP release
are unresolved. Here we identified conditions that yielded slow phosphoryl
transfer of the γ-phosphate from the generally nonhydrolyzable
analog of ATP, adenosine-5′-(β,γ-imido)­triphosphate
(AMP-PNP), onto a substrate peptide within protein crystals. By trapping
both products in the crystal lattice, we now have a complete resolution
profile of all the catalytic steps. One crystal structure refined
to 1.55 Å resolution shows two states of the protein with 55%
displaying intact AMP-PNP and an unphosphorylated substrate and 45%
displaying transfer of the γ-phosphate of AMP-PNP onto the substrate
peptide yielding AMP-PN and a phosphorylated substrate. Another structure
refined to 2.15 Å resolution displays complete phosphoryl transfer
to the substrate. These structures, in addition to trapping both products
in the crystal lattice, implicate one magnesium ion, previously termed
Mg2, as the more stably bound ion. Following phosphoryl transfer,
Mg2 recruits a water molecule to retain an octahedral coordination
geometry suggesting the strong binding character of this magnesium
ion, and Mg2 remains in the active site following complete phosphoryl
transfer while Mg1 is expelled. Loss of Mg1 may thus be an important
part of the rate-limiting step of ADP release.