4HPT

Crystal structure of the catalytic subunit of cAMP-dependent protein kinase displaying complete phosphoryl transfer of AMP-PNP onto a substrate peptide

Summary for 4HPT

• Entry DOI: 10.2210/pdb4hpt/pdb
• Related: 1ATP 1JBP 1JLU 1L3R 4DFX 4DG0 4DH1 4hpu
• Descriptor: cAMP-dependent protein kinase catalytic subunit alpha, cAMP-dependent protein kinase inhibitor alpha, PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER, ... (5 entities in total)
• Functional Keywords: protein kinase, phosphotransferase, regulatory subunits, pki, magnesium, phosphorylation, transferase-transferase inhibitor complex, transferase/transferase inhibitor
• Biological source: Mus musculus (mouse); More
• Cellular location: Cytoplasm . Isoform 2: Cell projection, cilium, flagellum : P05132
• Total number of polymer chains: 2
• Total formula weight: 43491.49

Authors

Bastidas, A.C.,Steichen, J.M.,Wu, J.,Taylor, S.S. (deposition date: 2012-10-24, release date: 2013-03-20, Last modification date: 2017-11-15)

Primary citation

Bastidas, A.C.,Deal, M.S.,Steichen, J.M.,Guo, Y.,Wu, J.,Taylor, S.S.
Phosphoryl transfer by protein kinase a is captured in a crystal lattice.
J.Am.Chem.Soc., 135:4788-4798, 2013

PubMed 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.

PubMed: 23458248
DOI: 10.1021/ja312237q

Experimental method

X-RAY DIFFRACTION (2.15 Å)