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Molecular insights into the membrane-associated phosphatidylinositol 4-kinase IIα.

Zhou Q, Li J, Yu H, Zhai Y, Gao Z, Liu Y, Pang X, Zhang L, Schulten K, Sun F, Chen C - Nat Commun (2014)

Bottom Line: Phosphatidylinositol 4-kinase IIα (PI4KIIα), a membrane-associated PI kinase, plays a central role in cell signalling and trafficking.The structure identifies the nucleotide-binding pocket that differs notably from that found in PI3Ks.We conclude from our results that PI4KIIα's activity is regulated indirectly through changes in the membrane environment.

View Article: PubMed Central - PubMed

Affiliation: 1] National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China [2] University of Chinese Academy of Sciences, Beijing 100049, China [3].

ABSTRACT
Phosphatidylinositol 4-kinase IIα (PI4KIIα), a membrane-associated PI kinase, plays a central role in cell signalling and trafficking. Its kinase activity critically depends on palmitoylation of its cysteine-rich motif (-CCPCC-) and is modulated by the membrane environment. Lack of atomic structure impairs our understanding of the mechanism regulating kinase activity. Here we present the crystal structure of human PI4KIIα in ADP-bound form. The structure identifies the nucleotide-binding pocket that differs notably from that found in PI3Ks. Two structural insertions, a palmitoylation insertion and an RK-rich insertion, endow PI4KIIα with the 'integral' membrane-binding feature. Molecular dynamics simulations, biochemical and mutagenesis studies reveal that the palmitoylation insertion, containing an amphipathic helix, contributes to the PI-binding pocket and anchors PI4KIIα to the membrane, suggesting that fluctuation of the palmitoylation insertion affects PI4KIIα's activity. We conclude from our results that PI4KIIα's activity is regulated indirectly through changes in the membrane environment.

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Overall structure of the human PI4KIIα catalytic domain.(a) Primary structure of PI4KIIα. I1, I2 and I3 represent three insertions in PI4KIIα not found in PI3Ks. Palmitoylation and RK-rich insertions are referred as I1 and I2, respectively. The crystallized fragment PI4KIIαSSPSSΔC is indicated by a black arrow. (b) Purification and kinase activity assay of PI4KIIαSSPSSΔC. The kinase activity of PI4KIIαSSPSSΔC in PI/Triton X-100 (0.2%)(▴) or PI-containing liposome/Triton X-100 (0.2%)(▪) was measured by monitoring ADP production. The error bars represent the s.d. from three independent experiments. (c) Overall structure of the ADP-bound PI4KIIα catalytic domain. Untraced segments are depicted with dashed lines. The G-loop, catalytic loop and activation loop are coloured in pink, bright orange and magenta, respectively. The palmitoylation insertion (PAL insertion, I1) and RK-rich insertion (I2) are coloured in red and gold, respectively. ADP is shown in a sphere model and is coloured according to its atoms (carbon, yellow; oxygen, red; nitrogen, blue; phosphorus, orange). The colour scheme here is used in all figures unless otherwise mentioned. (d) Secondary structure topology of the PI4KIIα catalytic domain. All structural figures were prepared with PyMol (DeLano, 2002, http://www.pymol.org) or UCSF Chimera ( http://www.cgl.ucsf.edu/chimera/).
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f1: Overall structure of the human PI4KIIα catalytic domain.(a) Primary structure of PI4KIIα. I1, I2 and I3 represent three insertions in PI4KIIα not found in PI3Ks. Palmitoylation and RK-rich insertions are referred as I1 and I2, respectively. The crystallized fragment PI4KIIαSSPSSΔC is indicated by a black arrow. (b) Purification and kinase activity assay of PI4KIIαSSPSSΔC. The kinase activity of PI4KIIαSSPSSΔC in PI/Triton X-100 (0.2%)(▴) or PI-containing liposome/Triton X-100 (0.2%)(▪) was measured by monitoring ADP production. The error bars represent the s.d. from three independent experiments. (c) Overall structure of the ADP-bound PI4KIIα catalytic domain. Untraced segments are depicted with dashed lines. The G-loop, catalytic loop and activation loop are coloured in pink, bright orange and magenta, respectively. The palmitoylation insertion (PAL insertion, I1) and RK-rich insertion (I2) are coloured in red and gold, respectively. ADP is shown in a sphere model and is coloured according to its atoms (carbon, yellow; oxygen, red; nitrogen, blue; phosphorus, orange). The colour scheme here is used in all figures unless otherwise mentioned. (d) Secondary structure topology of the PI4KIIα catalytic domain. All structural figures were prepared with PyMol (DeLano, 2002, http://www.pymol.org) or UCSF Chimera ( http://www.cgl.ucsf.edu/chimera/).

Mentions: Full-length human PI4KIIα contains a Pro-rich amino-terminal domain (1–93) and a carboxy-terminal catalytic domain (94–479)33, the latter including a palmitoylation motif -174CCPCC178- (Fig. 1a). As an integral membrane protein, PI4KIIα is prone to aggregate. We therefore screened a fragment PI4KIIαSSPSSΔC (Supplementary Table 1), which could be purified to high homogeneity and crystallized into high-quality crystals. Although the purified PI4KIIαSSPSSΔC showed low kinase activity in solution, once it was reconstituted in liposomes, its kinase activity increased approximately fivefold to a level nearly close to that of the wild type (Fig. 1b); moreover, this construct still associated with membranes when expressed in E. coli. These observations suggest that PI4KIIαSSPSSΔC preserves the active conformation of the wild-type PI4KIIα catalytic domain. All PI4KIIα constructs (including PI4KIIαSSPSSΔC) used in the present study are described and summarized in Supplementary Table 1.


Molecular insights into the membrane-associated phosphatidylinositol 4-kinase IIα.

Zhou Q, Li J, Yu H, Zhai Y, Gao Z, Liu Y, Pang X, Zhang L, Schulten K, Sun F, Chen C - Nat Commun (2014)

Overall structure of the human PI4KIIα catalytic domain.(a) Primary structure of PI4KIIα. I1, I2 and I3 represent three insertions in PI4KIIα not found in PI3Ks. Palmitoylation and RK-rich insertions are referred as I1 and I2, respectively. The crystallized fragment PI4KIIαSSPSSΔC is indicated by a black arrow. (b) Purification and kinase activity assay of PI4KIIαSSPSSΔC. The kinase activity of PI4KIIαSSPSSΔC in PI/Triton X-100 (0.2%)(▴) or PI-containing liposome/Triton X-100 (0.2%)(▪) was measured by monitoring ADP production. The error bars represent the s.d. from three independent experiments. (c) Overall structure of the ADP-bound PI4KIIα catalytic domain. Untraced segments are depicted with dashed lines. The G-loop, catalytic loop and activation loop are coloured in pink, bright orange and magenta, respectively. The palmitoylation insertion (PAL insertion, I1) and RK-rich insertion (I2) are coloured in red and gold, respectively. ADP is shown in a sphere model and is coloured according to its atoms (carbon, yellow; oxygen, red; nitrogen, blue; phosphorus, orange). The colour scheme here is used in all figures unless otherwise mentioned. (d) Secondary structure topology of the PI4KIIα catalytic domain. All structural figures were prepared with PyMol (DeLano, 2002, http://www.pymol.org) or UCSF Chimera ( http://www.cgl.ucsf.edu/chimera/).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3974213&req=5

f1: Overall structure of the human PI4KIIα catalytic domain.(a) Primary structure of PI4KIIα. I1, I2 and I3 represent three insertions in PI4KIIα not found in PI3Ks. Palmitoylation and RK-rich insertions are referred as I1 and I2, respectively. The crystallized fragment PI4KIIαSSPSSΔC is indicated by a black arrow. (b) Purification and kinase activity assay of PI4KIIαSSPSSΔC. The kinase activity of PI4KIIαSSPSSΔC in PI/Triton X-100 (0.2%)(▴) or PI-containing liposome/Triton X-100 (0.2%)(▪) was measured by monitoring ADP production. The error bars represent the s.d. from three independent experiments. (c) Overall structure of the ADP-bound PI4KIIα catalytic domain. Untraced segments are depicted with dashed lines. The G-loop, catalytic loop and activation loop are coloured in pink, bright orange and magenta, respectively. The palmitoylation insertion (PAL insertion, I1) and RK-rich insertion (I2) are coloured in red and gold, respectively. ADP is shown in a sphere model and is coloured according to its atoms (carbon, yellow; oxygen, red; nitrogen, blue; phosphorus, orange). The colour scheme here is used in all figures unless otherwise mentioned. (d) Secondary structure topology of the PI4KIIα catalytic domain. All structural figures were prepared with PyMol (DeLano, 2002, http://www.pymol.org) or UCSF Chimera ( http://www.cgl.ucsf.edu/chimera/).
Mentions: Full-length human PI4KIIα contains a Pro-rich amino-terminal domain (1–93) and a carboxy-terminal catalytic domain (94–479)33, the latter including a palmitoylation motif -174CCPCC178- (Fig. 1a). As an integral membrane protein, PI4KIIα is prone to aggregate. We therefore screened a fragment PI4KIIαSSPSSΔC (Supplementary Table 1), which could be purified to high homogeneity and crystallized into high-quality crystals. Although the purified PI4KIIαSSPSSΔC showed low kinase activity in solution, once it was reconstituted in liposomes, its kinase activity increased approximately fivefold to a level nearly close to that of the wild type (Fig. 1b); moreover, this construct still associated with membranes when expressed in E. coli. These observations suggest that PI4KIIαSSPSSΔC preserves the active conformation of the wild-type PI4KIIα catalytic domain. All PI4KIIα constructs (including PI4KIIαSSPSSΔC) used in the present study are described and summarized in Supplementary Table 1.

Bottom Line: Phosphatidylinositol 4-kinase IIα (PI4KIIα), a membrane-associated PI kinase, plays a central role in cell signalling and trafficking.The structure identifies the nucleotide-binding pocket that differs notably from that found in PI3Ks.We conclude from our results that PI4KIIα's activity is regulated indirectly through changes in the membrane environment.

View Article: PubMed Central - PubMed

Affiliation: 1] National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China [2] University of Chinese Academy of Sciences, Beijing 100049, China [3].

ABSTRACT
Phosphatidylinositol 4-kinase IIα (PI4KIIα), a membrane-associated PI kinase, plays a central role in cell signalling and trafficking. Its kinase activity critically depends on palmitoylation of its cysteine-rich motif (-CCPCC-) and is modulated by the membrane environment. Lack of atomic structure impairs our understanding of the mechanism regulating kinase activity. Here we present the crystal structure of human PI4KIIα in ADP-bound form. The structure identifies the nucleotide-binding pocket that differs notably from that found in PI3Ks. Two structural insertions, a palmitoylation insertion and an RK-rich insertion, endow PI4KIIα with the 'integral' membrane-binding feature. Molecular dynamics simulations, biochemical and mutagenesis studies reveal that the palmitoylation insertion, containing an amphipathic helix, contributes to the PI-binding pocket and anchors PI4KIIα to the membrane, suggesting that fluctuation of the palmitoylation insertion affects PI4KIIα's activity. We conclude from our results that PI4KIIα's activity is regulated indirectly through changes in the membrane environment.

Show MeSH
Related in: MedlinePlus