Sandbox Reserved 822

This Sandbox is Reserved from 06/12/2018, through 30/06/2019 for use in the course "Structural Biology" taught by Bruno Kieffer at the University of Strasbourg, ESBS. This reservation includes Sandbox Reserved 1480 through Sandbox Reserved 1543.

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spinqualitylabelsall models PDB ID 1w1h Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image
1w1h, resolution 1.45Å ()
Ligands:	,
Activity:	Non-specific serine/threonine protein kinase, with EC number 2.7.11.1
Related:	1h1w, 1oky, 1okz, 1uu3, 1uu7, 1uu8, 1uu9, 1uvr, 1w1d, 1w1g
Structural annotation: Resources: CATH : 1W1Ha00, 1W1Hb00, 1W1Hc00, 1W1Hd00 InterPro : Ipr011993, Ipr015746, Ipr011009, Ipr002290, Ipr000719, Ipr001849, Ipr008271 UniProt : O15530
Functional annotation: Cellular component: membrane plasma membrane cytoplasm Biological process: insulin receptor signaling pathway intracellular signaling cascade protein amino acid phosphorylation actin cytoskeleton organization and biogenesis Molecular function: protein serine/threonine kinase activity protein kinase activity protein binding ATP binding kinase activity transferase activity nucleotide binding 3-phosphoinositide-dependent protein kinase activity
Evolutionary conservation: Toggle Conservation Colors: Rows = identical sequences: Further Information: Caveats, Explanation, Complete Results at ConSurfDB
Resources:	FirstGlance, OCA, RCSB, PDBsum
Coordinates:	save as pdb, mmCIF, xml

DescriptionDescription

1W1H is a 4 chain structure of the human pleckstrin homology (PH) domain of the 3-phosphoinositide-dependent protein kinase 1 (PDK1), which plays a various role in PI3K signaling pathways. The PH domain of PDK1 interacts with PtdIns(3,4,5)P₃/PtdIns(3,4,)P₂ and with lower affinity to PtdIns(4,5)P₂.

PDK1 contains 556 amino acids and phosphorylates and activates at least 24 Proteins of the AGC (cAMP-dependent, cGMP-dependent, protein kinase C (PKC)) family of protein kinases. It therefore consists of a N-terminal Ser/Thr kinase catalytic domain (residues 71-359) and the C-terminal pleckstrin homology (PH) domain (residues 459-550).

PDK1 is activated by binding of its PH domain to specific target molecules like phosphoinositides or phosphoatityl inositides and can then interact with its target substrates.

Even though the targeting of PDK1 to specific locations of the cell is not fully understand in detail, it has been proven that the binding of its PH domain to target molecules plays a central role in this process.

StructureStructure

The structure 1W1H has in total 4 chains. These are represented by 1 sequence-unique entity (for reasons of clarity all green links show therefore only one chain as a representation for all four chains). The PH domain of PDK1 reveals a structural variation of a standard PH domain fold with an additional 'bud' at the N-terminus.

Fig.1 Topology diagram of the PH domain of PDK1. The colors of the different sections correspond to the coloring of the Jmol structure applet.

Overall SturctureOverall Sturcture

The standard PH domain fold consists of mainly three different sections:

• One section is a formed by residues 456-530 (see Fig.1, green coloured section). This structure is formed by two, almost orthogonal, β sheets, one consisting of four (β1 - β4) and one of three (β5 - β7) stand.
• One side of the barrel is blocked by a (see Fig.1, red coloured section).
• On the other side of the barrel three variable loops (VL 1-3) form a bowl-like structure which is lined by positively charged residues, consituting the phosphoinositide-binding site.

The PH domain of PDK1 possesses an additional extension () N-terminal to the standard PH domain fold (see Fig.1 blue coloured section). This bud forms two additional β strands and one α helix and is an integral part of the overall fold. The two β strands β1' and β2'extend the β1 - β4 sheet in an antiparallel fashion through the formation of β sheet hydrogen bonds between β2'and β1. The α helix packs against this newly formed six stranded β sheet forming an additional outside of the standard PH domain fold. The bud binds to the β1 - β4 sheet by several additional hydrophobic contacts and buries more than 30% of the surface of the standard PH domain fold.

Binding SiteBinding Site

The is formed by VL 1-3 which create a shallow, positively charged pocket at the open end of the β barrel. The structure was determined from a crystal grown in the presence of Ins(1,3,4,5)P₄, which is the head group of PtdIns(3,4,5)P₃, a high affinity substrate of the PH domain of PDK1. The phosphoinositide-binding site is lined by positively charged residues which contact the phosphates of Ins(1,3,4,5)P₄ by forming up to 11 hydrogen bonds (see Fig.2). forms two hydrogen bonds to the phosphate in the D1 position (D1-phosphate). contact the D3-phosphate with three hydrogen bonds in total (two for Arg474, one for Lys465). is also in close proximity (4.4 Å) of the D3-phosphate and is able to interact with it. form up to four hydrogen bonds to the D4-phosphate. The D5-phosphate is contacted by with one hydrogen bond. The D2- and D6-hydroxy groups show no direct interactions with the protein.

Ligand InteractionLigand Interaction

Fig.2 Ins(1,3,4,5)P₄ binding to the PH domain of PDK1. Interactions of Ins(1,3,4,5)P₄ (marine) with protein residues (green) are shown in a stereo representation. Hydrogen bonds are indicated as black dotted lines. Conserved water molecules are shown as yellow spheres, not conserved water molecules are coloured magenta.

The PH domain of PDK1 binds inositol phosphates and phosphatidylinositol phosphates with different affinities depending on the phosphorylation state of the molecules. These interactions target PDK1 to particular locations inside the cell and are therefore crucial for the flawless execution of signaling pathways in which PDK1 is involved.

The binding site of the PH domain of PDK1 exhibits several structural differences compared to PH domains of other proteins like DAPP1 or PKBα. These structural exceptions were described by investigation of Protein-substrate complexes.

Interactions with Inositol PhosphatesInteractions with Inositol Phosphates

The structure of the phosphoinositide-binding site of the PDK1 PH domain is unusually spacious. Compared to other PtdIns(3,4,5)P₃-binding PH domains additional space is present around the D2- and D6-hydroxyl groups, which potentially could accomodate further phosphate groups. This indicates a special affinity of the PDK1 PH domain for inositol phosphates because, physiologically, they are known to be phosphorylated at the D2 and/or D6 position while phosphoinositides, in contrast, do not show modifications at these positions. The more spacious binding pocket could be an explanation for the ability of the PDK1 PH domain to bind different stereoisomers of phosphoinositides.

In the PDK1 PH domain Ins(1,3,4,5)P₄ complex Ins(1,3,4,5)P₄ interacts with protein side chains only. This results in a significantly reduced number of protein-ligand hydrogen bonds (a total of 11) compared to PH domain Ins(1,3,4,5)P₄ complexes of other proteins which form 15 to 16 hydrogen bonds.

In the PDK1 Ins(1,3,4,5)P₄-binding pocket a layer of five-ordered water molecules (B-factors) seperate Ins(1,3,4,5)P₄ from the protein (see Fig.2). The water molecules mediate a number of hydrogen bonds from Ins(1,3,4,5)P₄ to the protein. For example binding of the D2-hydroxyl group takes place via an ordered water molecule. But only one of these five water molecules is also conserved in the PH domains of other proteins contacting the D3-phosphate (see Fig.2, coloured yellow).

Additionally, it was shown that the binding affinity for D1-phophorylated inositol phosphates is about five-fold higher than for inositol phosphates without a phosphate group at this position. This is caused by interactions of with the delocalised negative charge on the D1 phosphate. The formed hydrogen bonds to the D1-phosphate appear to play a crucial role in mediating binding of inositol phosphates to PDK1 (see 'Interactions with Phosphatidylinositol Phosphates ').

Interactions with Phosphatidylinositol PhosphatesInteractions with Phosphatidylinositol Phosphates

Fig.3 Stereo representation of the PDK1 PH domain interacting with diC4-PtdIns(3,4,5)P₃ (marine). Under the semitransparent surface, the conserved Arg residues (green) contacting the D1- and D3- phosphates are drawn as a stick representation.

The interations of the PDK1 PH domain with phosphatidylinositol phosphates were investigated by co-crystallising the PH domain with a PtdIns(3,4,5)P₃ analogue which contains two C4 acyl chains (diC4-PtdIns(3,4,5)P₃). It was found that coordinates the free oxygen atoms on the D1-phosphate whereas the oxygen atom involved in the ester bond to the glycerol does not make any significant contact with the protein. The glycerol backbone itself projects away from the surface of the protein and does not display any other interactions (see Fig.3).

The binding arrangement of the interaction with the D1-phophodiester appears in a characteristic way, which also explains the significantly higher affinity for D1-phosphorylated inositol phosphates (see 'Interactions with Inositol Phosphates'). The two oxygen atoms, which are not involved in the two phophoester linkages, carry most of the negative charge and interact with the guanidinium group of by forming one hydrogen bond each (see Fig.3). These interactions of Arg472 with the delocalised negative charge on the D1 phosphate is a crucial factor for the binding affinity of the PDK1 PH domain for its ligands.

For binding of PtdIns(3,4,5)P₃ no significant conformational changes could be observed. Only occupies the position of the inositol ring in the absence of ligand and rotates to a position to contact the D5-phophate upon ligand binding.

Importance for Signaling PathwaysImportance for Signaling Pathways

Once activated by growth factors, various local responses, such as cell growth, cell survival and cell movement are regulated by the highly conserved PDK1 pathway.

Therefore PDK1 binds to the lipid products of PI3K, PtdIns(3,4,5)P₃ and PtdIns(3,4)P₂ (see 'Ligand Interaction'). Once localized to the plasma membrane PDK1 can phosphorylate PKB/Akt and thereby activate mTOR, which plays a major role in ageing mechanisms and Alzheimer’s disease. Other tumor supressors such as the phosphatidylinositol 3′-phosphatase PTEN act to down-regulate signaling from PI3K to PDK1 and PKB as well.

Some other substrates of PDK1 are usually activated when the lipid-binding function of the protein is inhibited. Hence, not all PDK1 mediated reactions depend necessarily on the PH domain of PDK1.

ContributorsContributors

Klesse Ramona, Gerbeth Lorenz