PDBsum entry 2phk

Complex (transferase/peptide)

PDB id: 2phk

Contents

Protein chain

277 a.a. *

Ligands

ARG-GLN-MET-SER-PHE-ARG-LEU

ATP

GOL

Metals

_MN ×2

Waters ×86

* Residue conservation analysis

References listed in PDB file

Key reference

Title

The crystal structure of a phosphorylase kinase peptide substrate complex: kinase substrate recognition.

Authors

E.D.Lowe, M.E.Noble, V.T.Skamnaki, N.G.Oikonomakos, D.J.Owen, L.N.Johnson.

Ref.

EMBO J, 1997, 16, 6646-6658. [DOI no: 10.1093/emboj/16.22.6646]

PubMed id

9362479

Abstract

The structure of a truncated form of the gamma-subunit of phosphorylase kinase (PHKgammat) has been solved in a ternary complex with a non-hydrolysable ATP analogue (adenylyl imidodiphosphate, AMPPNP) and a heptapeptide substrate related in sequence to both the natural substrate and to the optimal peptide substrate. Kinetic characterization of the phosphotransfer reaction confirms the peptide to be a good substrate, and the structure allows identification of key features responsible for its high affinity. Unexpectedly, the substrate peptide forms a short anti-parallel beta-sheet with the kinase activation segment, the region which in other kinases plays an important role in regulation of enzyme activity. This anchoring of the main chain of the substrate peptide at a fixed distance from the gamma-phosphate of ATP explains the selectivity of PHK for serine/threonine over tyrosine as a substrate. The catalytic core of PHK exists as a dimer in crystals of the ternary complex, and the relevance of this phenomenon to its in vivo recognition of dimeric glycogen phosphorylase b is considered.

Figure 3.
Figure 3 Conformations of kinase inhibitor and substrate peptides. (A) Conformation of residues 11 -17 of GPa. (B) Conformation of residues 11 -17 of GPb. (C) Conformation of the MC-peptide, as observed in complex with PHK [t]. (D) Conformation of the equivalent part of the protein kinase inhibitor peptide, as observed in complex with cAPK.

Figure 6.
Figure 6 Interactions of nucleophile, base and phosphate group. Interactions which might occur in a general base-catalysed reaction mechanism via: (A) the observed structure of the ternary complex and (B) an alternative conformation modelled by giving the attacking serine residue a 1 angle of -60°.

The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: EMBO J (1997, 16, 6646-6658) copyright 1997.

Secondary reference #1

Title

Two structures of the catalytic domain of phosphorylase kinase: an active protein kinase complexed with substrate analogue and product.

Authors

D.J.Owen, M.E.Noble, E.F.Garman, A.C.Papageorgiou, L.N.Johnson.

Ref.

Structure, 1995, 3, 467-482. [DOI no: 10.1016/S0969-2126(01)00180-0]

PubMed id

7663944

Figure 1.
Figure 1. Schematic representation of the Phk γ-subunit. The catalytic domain is coloured red and the calmodulin-binding regulatory domain is coloured blue. Figure 1. Schematic representation of the Phk γ-subunit. The catalytic domain is coloured red and the calmodulin-binding regulatory domain is coloured blue.

Figure 7.
Figure 7. Comparison of the Cα traces of Phkγtrnc (white) and cAPK (grey). The view is along the axis about which relative domain motion is maximum. The Phkγtrnc structure is in a more ‘open’ conformation by approximately 5.0°. The figure was prepared using XOBJECTS (MEM Noble, unpublished program). Figure 7. Comparison of the Cα traces of Phkγtrnc (white) and cAPK (grey). The view is along the axis about which relative domain motion is maximum. The Phkγtrnc structure is in a more ‘open’ conformation by approximately 5.0°. The figure was prepared using XOBJECTS (MEM Noble, unpublished program).

The above figures are reproduced from the cited reference with permission from Cell Press