title: A transition path ensemble study reveals a linchpin role for Mg(2+) during rate-limiting ADP release from protein kinase A.
abstract: Protein kinases are key regulators of diverse signaling networks critical for growth and development. Protein kinase A (PKA) is an important kinase prototype that phosphorylates protein targets at Ser and Thr residues by converting ATP to ADP. Mg(2+) ions play a crucial role in regulating phosphoryl transfer and can limit overall enzyme turnover by affecting ADP release. However, the mechanism by which Mg(2+) participates in ADP release is poorly understood. Here we use a novel transition path ensemble technique, the harmonic Fourier beads method, to explore the atomic and energetic details of the Mg(2+)-dependent ADP binding and release. Our studies demonstrate that adenine-driven ADP binding to PKA creates three ion-binding sites at the ADP/PKA interface that are absent otherwise. Two of these sites bind the previously characterized Mg(2+) ions, whereas the third site binds a monovalent cation with high affinity. This third site can bind the P-3 residue of substrate proteins and may serve as a reporter of the active site occupation. Binding of Mg(2+) ions restricts mobility of the Gly-rich loop that closes over the active site. We find that simultaneous release of ADP with Mg(2+) ions from the active site is unfeasible. Thus, we conclude that Mg(2+) ions act as a linchpin and that at least one ion must be removed prior to pyrophosphate-driven ADP release. The results of the present study enhance understanding of Mg(2+)-dependent association of nucleotides with protein kinases.
authors.affiliation: Howard Hughes Medical Institute, University of California San Diego,La Jolla, California 92093-0365, USA. ikhavru@mccammon.ucsd.edu
authors.forename: Ilja V
authors.initials: IV
authors.lastname: Khavrutskii
authors.forename: Barry
authors.initials: B
authors.lastname: Grant
authors.forename: Susan S
authors.initials: SS
authors.lastname: Taylor
authors.forename: J Andrew
authors.initials: JA
authors.lastname: McCammon
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000465:Algorithms; D001665:Binding Sites; D002413:Cations, Divalent; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D005998:Glycine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D013912:Threonine / Q000737:chemistry / Q000378:metabolism
chemical_list: D002413:Cations, Divalent; D013912:Threonine; D012694:Serine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D008274:Magnesium; D005998:Glycine