General Information

Database Accession: DI1100095

Name: cAMP-dependent protein kinase complexed with a phosphorylated PKI-alpha peptide

PDB ID: 4hpt PDB

Experimental method: X-ray (2.15 Å)

Source organism: Mus musculus

Proof of disorder: Inferred from homology

Primary publication of the structure:

Bastidas AC, Deal MS, Steichen JM, Guo Y, Wu J, Taylor SS
Phosphoryl Transfer by Protein Kinase A Is Captured in a Crystal Lattice.
(2013) J. Am. Chem. Soc. :

PMID: 23458248 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.

Function and Biology Annotations from the GeneOntology database. Only terms that fit at least two of the interacting proteins are shown.

Molecular function:

protein kinase binding Interacting selectively and non-covalently with a protein kinase, any enzyme that catalyzes the transfer of a phosphate group, usually from ATP, to a protein substrate. GeneOntology

protein kinase A binding Interacting selectively and non-covalently with any subunit of protein kinase A. GeneOntology

Biological process:

regulation of cellular process Any process that modulates the frequency, rate or extent of a cellular process, any of those that are carried out at the cellular level, but are not necessarily restricted to a single cell. For example, cell communication occurs among more than one cell, but occurs at the cellular level. GeneOntology

regulation of intracellular protein transport Any process that modulates the frequency, rate or extent of the directed movement of proteins within cells. GeneOntology

Cellular component:

nucleus A membrane-bounded organelle of eukaryotic cells in which chromosomes are housed and replicated. In most cells, the nucleus contains all of the cell's chromosomes except the organellar chromosomes, and is the site of RNA synthesis and processing. In some species, or in specialized cell types, RNA metabolism or DNA replication may be absent. GeneOntology

cytoplasm All of the contents of a cell excluding the plasma membrane and nucleus, but including other subcellular structures. GeneOntology

Structure Summary Structural annotations of the participating protein chains.

Entry contents: 2 distinct polypeptide molecules

Chains: I, E

Notes: No modifications of the original PDB file.

Chain I

Name: cAMP-dependent protein kinase inhibitor alpha Disordered Inferred from homology

Source organism: Mus musculus

Length: 20 residues

Sequence:Sequence according to PDB SEQRESTTYADFIASGRTGRRASIHD

The sequence contains the following modified/non-standard residues:

• phosphoserine (S) at position 22 (PDB position: 21)

UniProtKB AC: P63248 (positions: 6-25) UniProt Coverage: 26.3%

UniRef90 AC: UniRef90_P63248 (positions: 6-25) UniRef90

Chain E

Name: cAMP-dependent protein kinase catalytic subunit alpha Ordered

Source organism: Mus musculus

Length: 350 residues

Sequence:Sequence according to PDB SEQRESGNAAAAKKGSEQESVKEFLAKAKEDFLKKWETPSQNTAQLDQFDRIKTLGTGSFGRVMLVKHKESGNHYAMKILDKQKVVKLKQIEHTLNEKRILQAVNFPFLVKLEFSFKDNSNLYMVMEYVAGGEMFSHLRRIGRFSEPHARFYAAQIVLTFEYLHSLDLIYRDLKPENLLIDQQGYIQVTDFGFAKRVKGRTWTLCGTPEYLAPEIILSKGYNKAVDWWALGVLIYEMAAGYPPFFADQPIQIYEKIVSGKVRFPSHFSSDLKDLLRNLLQVDLTKRFGNLKNGVNDIKNHKWFATTDWIAIYQRKVEAPFIPKFKGPGDTSNFDDYEEEEIRVSINEKCGKEFTEF

UniProtKB AC: P05132 (positions: 2-351) UniProt Coverage: 99.7%

UniRef90 AC: UniRef90_P17612 (positions: 2-351) UniRef90

Evidence Evidence demonstrating that the participating proteins are unstructured prior to the interaction and their folding is coupled to binding.

Chain I: Disordered Inferred from homology

The corresponding region of a closely homologous protein has been shown to be disordered in the 1-76 region described in DisProt entry DP00015 (covers 100% of the sequence present in the structure).

Chain E: Ordered

The protein kinase domain involved in the interaction is known to adopt a stable structure in isolation (see Pfam domain PF00069). A solved monomeric structure of the domain is represented by PDB ID 4nts.

Related Structure(s) Structures from the PDB that contain the same number of proteins, and the proteins from the two structures show a sufficient degree of pairwise similarity, i.e. they belong to the same UniRef90 cluster (the full proteins exhibit at least 90% sequence identity) and convey roughly the same region to their respective interactions (the two regions from the two proteins share a minimum of 70% overlap).

There is 1 related structure in the Protein Data Bank:

• 4hpu

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