PDBsum entry 1w1h

Transferase

PDB id: 1w1h

Contents

Protein chains

147 a.a. *

138 a.a. *

Ligands

GOL ×4

SO4 ×10

Waters ×715

* Residue conservation analysis

PDB id:

1w1h

Name:

Transferase

Title:

Crystal structure of the pdk1 pleckstrin homology (ph) domain

Structure:

3-phosphoinositide dependent protein kinase-1. Chain: a, b, c, d. Fragment: pleckstrin homology domain, residues 409-556. Synonym: hpdk1. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 511693.

Resolution:

1.45Å R-factor: 0.157 R-free: 0.206

Authors:

D.Komander,M.Deak,D.R.Alessi,D.M.F.Van Aalten

Key ref:

D.Komander et al. (2004). Structural insights into the regulation of PDK1 by phosphoinositides and inositol phosphates. EMBO J, 23, 3918-3928. PubMed id: 15457207 DOI: 10.1038/sj.emboj.7600379

Date:

21-Jun-04 Release date: 19-Nov-04

Protein chains

O15530  (PDPK1_HUMAN) -  3-phosphoinositide-dependent protein kinase 1 from Homo sapiens

Seq: 556 a.a.

Struc: 147 a.a.

Protein chains

O15530  (PDPK1_HUMAN) -  3-phosphoinositide-dependent protein kinase 1 from Homo sapiens

Seq: 556 a.a.

Struc: 138 a.a.

Enzyme reactions

• Enzyme class: Chains A, B, C, D: E.C.2.7.11.1 - non-specific serine/threonine 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: 10.1038/sj.emboj.7600379

EMBO J 23:3918-3928 (2004)

PubMed id: 15457207

Structural insights into the regulation of PDK1 by phosphoinositides and inositol phosphates.

D.Komander, A.Fairservice, M.Deak, G.S.Kular, A.R.Prescott, C.Peter Downes, S.T.Safrany, D.R.Alessi, D.M.van Aalten.

ABSTRACT

3-phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates and activates many kinases belonging to the AGC subfamily. PDK1 possesses a C-terminal pleckstrin homology (PH) domain that interacts with PtdIns(3,4,5)P3/PtdIns(3,4)P2 and with lower affinity to PtdIns(4,5)P2. We describe the crystal structure of the PDK1 PH domain, in the absence and presence of PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4. The structures reveal a 'budded' PH domain fold, possessing an N-terminal extension forming an integral part of the overall fold, and display an unusually spacious ligand-binding site. Mutagenesis and lipid-binding studies were used to define the contribution of residues involved in phosphoinositide binding. Using a novel quantitative binding assay, we found that Ins(1,3,4,5,6)P5 and InsP6, which are present at micromolar levels in the cytosol, interact with full-length PDK1 with nanomolar affinities. Utilising the isolated PDK1 PH domain, which has reduced affinity for Ins(1,3,4,5,6)P5/InsP6, we perform localisation studies that suggest that these inositol phosphates serve to anchor a portion of cellular PDK1 in the cytosol, where it could activate its substrates such as p70 S6-kinase and p90 ribosomal S6 kinase that do not interact with phosphoinositides.

Selected figure(s)

Figure 2.
Figure 2 Comparison of Ins(1,3,4,5)P[4] binding to the PH domains of PDK1, DAPP1 and PKB . Interactions of Ins(1,3,4,5)P[4] (marine) with protein residues (green in PDK1, orange in DAPP1/PKB ) in the phosphoinositide-binding site are shown in a stereo representation. Hydrogen bonds are indicated as black dotted lines, and conserved water molecules are shown as yellow spheres. (A) Protein -ligand interactions of PDK1 PH domain with Ins(1,3,4,5)P[4]. A layer of ordered water molecules (coloured in magenta) separates the ligand from VL1, only one of which is conserved in other PH domain structures (coloured in gold). (B) Ins(1,3,4,5)P[4] binding to DAPP1 (pdb-id 1fao; Ferguson et al, 2000). The D5-phosphate is closely enveloped by VL1. (C) Ins(1,3,4,5)P[4] binding to PKB (pdb-id 1h10; Thomas et al, 2001). The ligand is rotated and VL1 contacts the D1-phosphate, while the D5-phosphate is solvent exposed. (D) Overlay of the structures of the PDK1 (green), DAPP1 (orange) and PKB (blue) PH domains. The Ins(1,3,4,5)P[4] ligand and the layer of ordered water molecules of PDK1 are shown, and coloured according to (A).

Figure 4.
Figure 4 Phosphoinositide-binding properties of wild type and mutant PDK1. (A) Representation of the Ins(1,3,4,5)P[4]-binding pocket on the PDK1 PH domain. The residues that make contacts with Ins(1,3,4,5)P[4] and that are mutated in this study are labelled. (B) The ability of the indicated wild type and mutant forms of PDK1 to interact with phosphoinositides was analysed using a protein-lipid overlay assay. Serial dilutions of the indicated phosphoinositides (250, 100, 50, 25, 12.5, 6.3, 3.1 and 1.6 pmol) were spotted onto nitrocellulose membranes, which were then incubated with the purified GST-PDK1 species. The membranes were washed, and the GST-PDK1 bound to the membrane by virtue of their interaction with lipid was detected using a GST antibody (Dowler et al, 2002). A representative of at least two separate experiments is shown. (*) indicates a long exposure of the film to detect weak binding.

The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: EMBO J (2004, 23, 3918-3928) copyright 2004.

Figures were selected by an automated process.