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The binding site of the PH domain of PDK1 exhibits several structural differences compared to PH domains of other proteins like [http://www.rcsb.org/pdb/protein/Q9UN19?evtc=Suggest&evta=UniProtGene DAPP1] or [http://www.rcsb.org/pdb/protein/P31749?evtc=Suggest&evta=UniProtGene PKBα]. These structural exceptions were described by investigation of Protein-substrate complexes.<ref name="Structural" /> | The binding site of the PH domain of PDK1 exhibits several structural differences compared to PH domains of other proteins like [http://www.rcsb.org/pdb/protein/Q9UN19?evtc=Suggest&evta=UniProtGene DAPP1] or [http://www.rcsb.org/pdb/protein/P31749?evtc=Suggest&evta=UniProtGene PKBα]. These structural exceptions were described by investigation of Protein-substrate complexes.<ref name="Structural" /> | ||
=== | === Interactions 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)P<sub>3</sub>-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 structure of the phosphoinositide-binding site of the PDK1 PH domain is unusually spacious. Compared to other PtdIns(3,4,5)P<sub>3</sub>-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.<ref> PMID: 9445477 </ref> | ||
In the PDK1 PH domain Ins(1,3,4,5)P<sub>4</sub> complex Ins(1,3,4,5)P<sub>4</sub> 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)P<sub>4</sub> complexes of other proteins which form 15 to 16 hydrogen bonds.<ref name="Structural" /> | In the PDK1 PH domain Ins(1,3,4,5)P<sub>4</sub> complex Ins(1,3,4,5)P<sub>4</sub> 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)P<sub>4</sub> complexes of other proteins which form 15 to 16 hydrogen bonds.<ref name="Structural" /> | ||
In the PDK1 Ins(1,3,4,5)P<sub>4</sub>-binding pocket a layer of five-ordered water molecules (B-factors) seperate Ins(1,3,4,5)P<sub>4</sub> from the protein (see Fig.2). The water molecules mediate a number of hydrogen bonds from Ins(1,3,4,5)P<sub>4</sub> 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).<ref name="Structural" /> | In the PDK1 Ins(1,3,4,5)P<sub>4</sub>-binding pocket a layer of five-ordered water molecules (B-factors) seperate Ins(1,3,4,5)P<sub>4</sub> from the protein (see Fig.2). The water molecules mediate a number of hydrogen bonds from Ins(1,3,4,5)P<sub>4</sub> 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).<ref name="Structural" /> | ||
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 <scene name='56/568020/Arg472/1'>Arg472</scene> 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 ''''). <ref name="Structural" /> | |||
=== Interactions with Phosphatidylinositol Phosphates === | |||
[[Image:PDK1 PH Domain Interacting With diC4-PtdIns(3,4,5)P3.jpg|right|320px|thumb|Fig.3 Stereo representation of the PDK1 PH domain interacting with diC4-PtdIns(3,4,5)P<sub>3</sub> (marine). Under the semitransparent surface, the conserved Arg residues (green) contacting the D1- and D3- phosphates are drawn as a stick representation.<ref name="Structural" />]] | |||
The interations of the PDK1 PH domain with phosphatidylinositol phosphates were investigated by co-crystallising the PH domain with a PtdIns(3,4,5)P<sub>3</sub> analogue which contains two C4 acyl chains (diC4-PtdIns(3,4,5)P<sub>3</sub>). It was found that <scene name='56/568020/Arg472/1'>Arg472</scene> 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). <ref name="Structural" /> | |||
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 <scene name='56/568020/Arg472/1'>Arg472</scene> 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. <ref name="Structural" /> | |||
For binding of PtdIns(3,4,5)P<sub>3</sub> no significant conformational changes could be observed. Only <scene name='56/568020/Lys467/1'>Lys467</scene> 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. <ref name="Structural" /> | |||
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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. | 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)P<sub>3</sub> and PtdIns(3,4)P<sub>2</sub> (see 'Ligand Interaction'). Once localized to the plasma membrane PDK1 can phosphorylate PKB/Akt and thereby activate [http://en.wikipedia.org/wiki/Mammalian_target_of_rapamycin 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. <ref>Hemmings, Brian A., and David F. Restuccia. "PI3K-PKB/Akt Pathway." Cold Spring Harbor Perspectives in Biology 4.9 (2012) [http://cshperspectives.cshlp.org/content/4/9/a011189.full DOI:10.1101/cshperspect.a011189]</ref> | Therefore PDK1 binds to the lipid products of [[PI3K]], PtdIns(3,4,5)P<sub>3</sub> and PtdIns(3,4)P<sub>2</sub> (see ''''Ligand Interaction''''). Once localized to the plasma membrane PDK1 can phosphorylate PKB/Akt and thereby activate [http://en.wikipedia.org/wiki/Mammalian_target_of_rapamycin 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. <ref>Hemmings, Brian A., and David F. Restuccia. "PI3K-PKB/Akt Pathway." Cold Spring Harbor Perspectives in Biology 4.9 (2012) [http://cshperspectives.cshlp.org/content/4/9/a011189.full DOI:10.1101/cshperspect.a011189]</ref> | ||
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. <ref>Scheid, Michael P., Michael Parsons, and James R. Woodgett. "Phosphoinositide-dependent phosphorylation of PDK1 regulates nuclear translocation." Molecular and cellular biology 25.6 (2005): 2347-2363. [http://mcb.asm.org/content/25/6/2347.full DOI:10.1128/MCB.25.6.2347-2363.2005]</ref> | 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. <ref>Scheid, Michael P., Michael Parsons, and James R. Woodgett. "Phosphoinositide-dependent phosphorylation of PDK1 regulates nuclear translocation." Molecular and cellular biology 25.6 (2005): 2347-2363. [http://mcb.asm.org/content/25/6/2347.full DOI:10.1128/MCB.25.6.2347-2363.2005]</ref> | ||