PDBsum entry 1syk

Transferase

PDB id: 1syk

Contents

Protein chains

350 a.a. *

325 a.a. *

Waters ×113

* Residue conservation analysis

PDB id:

1syk

Name:

Transferase

Title:

Crystal structure of e230q mutant of camp-dependent protein kinase reveals unexpected apoenzyme conformation

Structure:

Camp-dependent protein kinase, alpha-catalytic subunit. Chain: a, b. Synonym: pka c-alpha. Engineered: yes. Mutation: yes

Source:

Mus musculus. House mouse. Organism_taxid: 10090. Gene: prkaca, pkaca. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

2.80Å R-factor: 0.202 R-free: 0.249

Authors:

J.Wu,J.Yang,N.Madhusudan,N.H.Xuong,L.F.Ten Eyck,S.S.Taylor

Key ref:

J.Wu et al. (2005). Crystal structure of the E230Q mutant of cAMP-dependent protein kinase reveals an unexpected apoenzyme conformation and an extended N-terminal A helix. Protein Sci, 14, 2871-2879. PubMed id: 16253959 DOI: 10.1110/ps.051715205

Date:

01-Apr-04 Release date: 17-May-05

Protein chain

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

Seq: 351 a.a.

Struc: 350 a.a.*

Protein chain

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

Seq: 351 a.a.

Struc: 325 a.a.*

* PDB and UniProt seqs differ at 4 residue positions (black crosses)

Enzyme reactions

• Enzyme class: Chains A, B: 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⁺

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

reference

DOI no: 10.1110/ps.051715205

Protein Sci 14:2871-2879 (2005)

PubMed id: 16253959

Crystal structure of the E230Q mutant of cAMP-dependent protein kinase reveals an unexpected apoenzyme conformation and an extended N-terminal A helix.

J.Wu, J.Yang, N.Kannan, Madhusudan, N.H.Xuong, L.F.Ten Eyck, S.S.Taylor.

ABSTRACT

Glu230, one of the acidic residues that cluster around the active site of the catalytic subunit of cAMP-dependent protein kinase, plays an important role in substrate recognition. Specifically, its side chain forms a direct salt-bridge interaction with the substrate's P-2 Arg. Previous studies showed that mutation of Glu230 to Gln (E230Q) caused significant decreases not only in substrate binding but also in the rate of phosphoryl transfer. To better understand the importance of Glu230 for structure and function, we solved the crystal structure of the E230Q mutant at 2.8 A resolution. Surprisingly, the mutant preferred an open conformation with no bound ligands observed, even though the crystals were grown in the presence of MgATP and the inhibitor peptide, IP20. This is in contrast to the wild-type protein that, under the same conditions, prefers the closed conformation of a ternary complex. The structure highlights the importance of the electrostatic surface not only for substrate binding and catalysis, but also for the mechanism for closing the active site cleft. This surface mutation clearly disrupts the recognition and binding of substrate peptide so that the enzyme prefers an open conformation that cannot trap ATP. This is consistent with the reinforcing concepts of conformational dynamics and the synergistic binding of ATP and substrate peptide. Another unusual feature of the structure is the observation of the entire N terminus (Gly1-Thr32) assumes an extended alpha-helix conformation. Finally, based on temperature factors, this mutant structure is more stable than the wild-type C-subunit in the apo state.

Selected figure(s)

Figure 1.
Figure 1. Structural environment of Glu230. The D-helix, F-helix, catalytic loop, P+1 loop, and part of the inhibitory peptide IP20 are shown in ribbon diagram. Glu230 is located at the C-terminal end of the F-helix. P-2 Arg from IP20 is rendered as ball-and-sticks. Dotted lines represent interactions between Glu230 and its environment.

Figure 4.
Figure 4. Conservation of the IP20-binding site. Structures of the wild-type apoenzyme (PDB ID 1J3H [PDB] ; yellow sticks), the E230Q mutant (red sticks), and the ternary complex of wild-type protein with MgADP-aluminum fluoride and substrate peptide SP20 (PDB ID 1L3R [PDB] ; dark thin sticks) were superimposed. Some of the negatively charged residues involved in recognition of the arginines in the substrate peptide are shown in sticks. SP20 from the ternary structure (1L3R) is shown in ribbon diagram with P-site Ser, P-2, P-3, and P-6 Args rendered as ball-and-sticks. Replacement of Glu230 by Gln did not change its conformation, while its binding partner Arg133 is disordered.

The above figures are reprinted by permission from the Protein Society: Protein Sci (2005, 14, 2871-2879) copyright 2005.

Figures were selected by an automated process.