title: Phosphoryl transfer by protein kinase A is captured in a crystal lattice.
abstract: 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.
authors: Department of Pharmacology, University of California, San Diego, California 92093, USA.
authors: Adam C
authors: AC
authors: Bastidas
authors: Michael S
authors: MS
authors: Deal
authors: Jon M
authors: JM
authors: Steichen
authors: Yurong
authors: Y
authors: Guo
authors: Jian
authors: J
authors: Wu
authors: Susan S
authors: SS
authors: Taylor
mesh_terms: D000266:Adenylyl Imidodiphosphate / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D018360:Crystallography, X-Ray; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation
chemical_list: D010455:Peptides; D000266:Adenylyl Imidodiphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D008274:Magnesium