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

被引:69
作者
Bastidas, Adam C. [1 ]
Deal, Michael S. [2 ]
Steichen, Jon M. [2 ]
Guo, Yurong [2 ]
Wu, Jian [2 ]
Taylor, Susan S. [1 ,2 ,3 ]
机构
[1] Univ Calif San Diego, Dept Pharmacol, La Jolla, CA 92093 USA
[2] Univ Calif San Diego, Dept Chem & Biochem, La Jolla, CA 92093 USA
[3] Univ Calif San Diego, Howard Hughes Med Inst, La Jolla, CA 92093 USA
关键词
CAMP-DEPENDENT KINASE; CATALYTIC SUBUNIT; ACTIVE-SITE; ADENYLYL IMIDODIPHOSPHATE; PEPTIDE INHIBITOR; ESCHERICHIA-COLI; MECHANISM; ADENOSINE; HOLOENZYME; HYDROLYSIS;
D O I
10.1021/ja312237q
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
The catalytic (C) subunit of cAMP-dependent protein kinase (PICA) is a serine/threonine kinase responsible for most of the effects of cAMP signaling, and PICA 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 gamma-phosphate from the generally nonhydrolyzable analog of ATP, adenosine-5'-(beta,gamma-irnido)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 angstrom resolution shows two states of the protein with 55% displaying intact AMP-PNP and an unphosphorylated substrate and 45% displaying transfer of the gamma-phosphate of AMP-PNP onto the substrate peptide yielding AMP-PN and a phosphorylated substrate. Another structure refined to 2.15 angstrom 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.
引用
收藏
页码:4788 / 4798
页数:11
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