PDBsum entry 3idc

Transferase

PDB id: 3idc

Contents

Protein chains

343 a.a. *

160 a.a. *

Ligands

ANP

Metals

_MN ×2

Waters ×43

* Residue conservation analysis

PDB id:

3idc

Name:

Transferase

Title:

Crystal structure of (102-265)riib:c holoenzyme of camp-dependent protein kinase

Structure:

Camp-dependent protein kinase catalytic subunit alpha. Chain: a. Fragment: isoform 1 (c-alpha-1): unp residues 2-351. Synonym: pka c-alpha. Engineered: yes. Other_details: (102-265)riib:c holoenzyme of camp-dependent protein kinase. Camp-dependent protein kinase type ii-beta regulatory subunit.

Source:

Mus musculus. Mouse. Organism_taxid: 10090. Gene: pkaca, prkaca. Expressed in: escherichia coli. Expression_system_taxid: 562. Rattus norvegicus. Rat. Organism_taxid: 10116.

Resolution:

2.70Å R-factor: 0.241 R-free: 0.319

Authors:

S.H.J.Brown,J.Wu,C.Kim,K.Alberto,S.S.Taylor

Key ref:

S.H.Brown et al. (2009). Novel isoform-specific interfaces revealed by PKA RIIbeta holoenzyme structures. J Mol Biol, 393, 1070-1082. PubMed id: 19748511 DOI: 10.1016/j.jmb.2009.09.014

Date:

20-Jul-09 Release date: 29-Sep-09

Protein chain

P05132  (KAPCA_MOUSE) -  cAMP-dependent protein kinase catalytic subunit alpha from Mus musculus

Seq: 351 a.a.

Struc: 343 a.a.*

Protein chain

P12369  (KAP3_RAT) -  cAMP-dependent protein kinase type II-beta regulatory subunit from Rattus norvegicus

Seq: 416 a.a.

Struc: 160 a.a.

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: Chain A: E.C.2.7.11.11 - cAMP-dependent protein kinase.
• Reaction:
  1. L-seryl-[protein] + ATP = O-phospho-L-seryl-[protein] + ADP + H⁺
  2. L-threonyl-[protein] + ATP = O-phospho-L-threonyl-[protein] + ADP + H⁺

L-seryl-[protein] + ATP = O-phospho-L-seryl-[protein] (Bound ligand (Het Group name = ANP) matches with 81.25% similarity) + ADP + H(+)

L-threonyl-[protein] + ATP = O-phospho-L-threonyl-[protein] (Bound ligand (Het Group name = ANP) matches with 81.25% similarity) + ADP + H(+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1016/j.jmb.2009.09.014

J Mol Biol 393:1070-1082 (2009)

PubMed id: 19748511

Novel isoform-specific interfaces revealed by PKA RIIbeta holoenzyme structures.

S.H.Brown, J.Wu, C.Kim, K.Alberto, S.S.Taylor.

ABSTRACT

The cAMP-dependent protein kinase catalytic (C) subunit is inhibited by two classes of functionally nonredundant regulatory (R) subunits, RI and RII. Unlike RI subunits, RII subunits are both substrates and inhibitors. Because RIIbeta knockout mice have important disease phenotypes, the RIIbeta holoenzyme is a target for developing isoform-specific agonists and/or antagonists. We also know little about the linker region that connects the inhibitor site to the N-terminal dimerization domain, although this linker determines the unique globular architecture of the RIIbeta holoenzyme. To understand how RIIbeta functions as both an inhibitor and a substrate and to elucidate the structural role of the linker, we engineered different RIIbeta constructs. In the absence of nucleotide, RIIbeta(108-268), which contains a single cyclic nucleotide binding domain, bound C subunit poorly, whereas with AMP-PNP, a non-hydrolyzable ATP analog, the affinity was 11 nM. The RIIbeta(108-268) holoenzyme structure (1.62 A) with AMP-PNP/Mn(2+) showed that we trapped the RIIbeta subunit in an enzyme:substrate complex with the C subunit in a closed conformation. The enhanced affinity afforded by AMP-PNP/Mn(2+) may be a useful strategy for increasing affinity and trapping other protein substrates with their cognate protein kinase. Because mutagenesis predicted that the region N-terminal to the inhibitor site might dock differently to RI and RII, we also engineered RIIbeta(102-265), which contained six additional linker residues. The additional linker residues in RIIbeta(102-265) increased the affinity to 1.6 nM, suggesting that docking to this surface may also enhance catalytic efficiency. In the corresponding holoenzyme structure, this linker docks as an extended strand onto the surface of the large lobe. This hydrophobic pocket, formed by the alphaF-alphaG loop and conserved in many protein kinases, also provides a docking site for the amphipathic helix of PKI. This novel orientation of the linker peptide provides the first clues as to how this region contributes to the unique organization of the RIIbeta holoenzyme.

Selected figure(s)

Figure 3.
Fig. 3. RIIβ holoenzyme assumes a closed confirmation: (a) shows three holoenzyme structures with the trapped transition state that is present in the RIIβ holoenzyme structure (middle). The RIIα holoenzyme (left) is in an open form whereas the RIIβ and RIα complexes (right) are in a closed conformation. The color designations are as follows: E230^C, E170^C, and E203^C are shown in red and provide the acidic docking surface for the P − 3 and P − 2 arginines; the P + 1 pocket (L198^C and P202^C) is in white and provides the hydrophobic site for the P + 1 residue; Y330^C in the C-tail is yellow; the glycine rich loop is in pink; AMP-PNP is in black. The temperature factors analysis of the C subunit in three holoenzyme structures is shown in (b). Shown on the left is RIIα(90–400):C in the absence of ATP (2QVS), with the disordered/mobile regions underlined in red; in the middle is RIIβ(108–268):C:AMP-PNP; on the right is RIα(91–244):C:AMP-PNP (1U7E).

Figure 6.
Fig. 6. Variation in positioning of the region that lies N-terminal to the inhibitor site: (a) Highlighted N-terminal binding regions of PKI (dark teal), RIα (light green), and RIIβ (red) demonstrate differential binding between inhibitors. RIIβ and PKI dock to the C-lobe, while RIα docks to the C-terminal tail and N-lobe. Surface mesh representation is shown for the highly conserved P − 3 to P + 1 region. Details of RIIβ binding peptide are highlighted. (b) The sequence and detailed structural comparison of the inhibitor peptides of PKI, RIα, and RIIβ. (c) The αF–αG loop (tan) creates a hydrophobic pocket that recognizes different substrates (red). Tyr235^C and Phe239^C are highly conserved residues in this pocket. The region of RIIβ that lies N-terminally to the inhibitor site docks as a strand to this pocket (left) while the amphipathic helix of PKI (right) docks to the same surface. In both cases, the peptides dock against Phe239^C. In RIIβ(108–268), this site is unoccupied (middle) and the side chain of Phe239^C is rotated away from Tyr235^C. In the two RIIβ holoenzymes, one can see how Tyr247^C in the G-helix interfaces with the P + 1 Val and with the Tyr226^R in the PBC of RIIβ.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2009, 393, 1070-1082) copyright 2009.

Figures were selected by an automated process.