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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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Membrane binding of PI4KIIα and its kinase activity.(a,b) Sequential membrane elution assay to evaluate the membrane binding of PI4KIIα variants. (c) The membrane-binding surface of PI4KIIα deduced from the crystallographic packing analysis and mapped with its electrostatic potentials. Blue represents positive charges and red represents negative charges. ADP and the putative PI (see Fig. 4b) are shown as stick models. Residues potentially involved in membrane binding are indicated accordingly. (d) Kinase activities of the PI4KIIα variants. The kinase activity was measured in PI/Triton X-100 (0.2%) and monitored by ADP production. The error bars represent the s.d. from three independent experiments. (e) A representative model of PI4KIIα bound to membrane, obtained from MD simulations. The crystal structure is coloured in grey and the MD structural model is shown with the same scheme of Fig. 1c. The residues evaluated in (d) are indicated accordingly. The simulated membrane is depicted in transparent dots. The left panel is perpendicular to the membrane and the right one is parallel and viewed from the membrane side.
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f3: Membrane binding of PI4KIIα and its kinase activity.(a,b) Sequential membrane elution assay to evaluate the membrane binding of PI4KIIα variants. (c) The membrane-binding surface of PI4KIIα deduced from the crystallographic packing analysis and mapped with its electrostatic potentials. Blue represents positive charges and red represents negative charges. ADP and the putative PI (see Fig. 4b) are shown as stick models. Residues potentially involved in membrane binding are indicated accordingly. (d) Kinase activities of the PI4KIIα variants. The kinase activity was measured in PI/Triton X-100 (0.2%) and monitored by ADP production. The error bars represent the s.d. from three independent experiments. (e) A representative model of PI4KIIα bound to membrane, obtained from MD simulations. The crystal structure is coloured in grey and the MD structural model is shown with the same scheme of Fig. 1c. The residues evaluated in (d) are indicated accordingly. The simulated membrane is depicted in transparent dots. The left panel is perpendicular to the membrane and the right one is parallel and viewed from the membrane side.

Mentions: PI4KIIα behaves as an ‘integral’ membrane protein without a transmembrane domain29. We utilized a membrane sequential elution assay29 to study membrane binding of PI4KIIα (Fig. 3a,b). Wild-type construct PI4KIIαCCPCC could not be eluted from the membrane by 1M NaCl, but was partially eluted by 0.1 M Na2CO3 and completely eluted by 1.0% Triton X-100, indicating the presence of strong hydrophobic interactions between PI4KIIα and membrane (Fig. 3a). Increases in pH or NaCl concentration increased the elution of construct PI4KIIαSSPSSΔC, revealing the contribution of electrostatic interactions (Fig. 3a). In addition to the palmitoylation motif, the C-terminal region (454–479) of PI4KIIα is also reported to contribute to membrane binding by hydrophobic interactions33. We confirmed this observation (Fig. 3b) by comparing the elution behaviours among the constructs PI4KIIαSSPSS, PI4KIIαFFPFF, PI4KIIαSSPSSΔC and PI4KIIαFFPFFΔC (Supplementary Table 1). Deletion of the C-terminal region decreased membrane-binding affinity. The decreased interaction was restored, however, by mutating SSPSS to FFPFF, suggesting that the palmitoylation motif plays a role in membrane binding (Fig. 3b).


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)

Membrane binding of PI4KIIα and its kinase activity.(a,b) Sequential membrane elution assay to evaluate the membrane binding of PI4KIIα variants. (c) The membrane-binding surface of PI4KIIα deduced from the crystallographic packing analysis and mapped with its electrostatic potentials. Blue represents positive charges and red represents negative charges. ADP and the putative PI (see Fig. 4b) are shown as stick models. Residues potentially involved in membrane binding are indicated accordingly. (d) Kinase activities of the PI4KIIα variants. The kinase activity was measured in PI/Triton X-100 (0.2%) and monitored by ADP production. The error bars represent the s.d. from three independent experiments. (e) A representative model of PI4KIIα bound to membrane, obtained from MD simulations. The crystal structure is coloured in grey and the MD structural model is shown with the same scheme of Fig. 1c. The residues evaluated in (d) are indicated accordingly. The simulated membrane is depicted in transparent dots. The left panel is perpendicular to the membrane and the right one is parallel and viewed from the membrane side.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f3: Membrane binding of PI4KIIα and its kinase activity.(a,b) Sequential membrane elution assay to evaluate the membrane binding of PI4KIIα variants. (c) The membrane-binding surface of PI4KIIα deduced from the crystallographic packing analysis and mapped with its electrostatic potentials. Blue represents positive charges and red represents negative charges. ADP and the putative PI (see Fig. 4b) are shown as stick models. Residues potentially involved in membrane binding are indicated accordingly. (d) Kinase activities of the PI4KIIα variants. The kinase activity was measured in PI/Triton X-100 (0.2%) and monitored by ADP production. The error bars represent the s.d. from three independent experiments. (e) A representative model of PI4KIIα bound to membrane, obtained from MD simulations. The crystal structure is coloured in grey and the MD structural model is shown with the same scheme of Fig. 1c. The residues evaluated in (d) are indicated accordingly. The simulated membrane is depicted in transparent dots. The left panel is perpendicular to the membrane and the right one is parallel and viewed from the membrane side.
Mentions: PI4KIIα behaves as an ‘integral’ membrane protein without a transmembrane domain29. We utilized a membrane sequential elution assay29 to study membrane binding of PI4KIIα (Fig. 3a,b). Wild-type construct PI4KIIαCCPCC could not be eluted from the membrane by 1M NaCl, but was partially eluted by 0.1 M Na2CO3 and completely eluted by 1.0% Triton X-100, indicating the presence of strong hydrophobic interactions between PI4KIIα and membrane (Fig. 3a). Increases in pH or NaCl concentration increased the elution of construct PI4KIIαSSPSSΔC, revealing the contribution of electrostatic interactions (Fig. 3a). In addition to the palmitoylation motif, the C-terminal region (454–479) of PI4KIIα is also reported to contribute to membrane binding by hydrophobic interactions33. We confirmed this observation (Fig. 3b) by comparing the elution behaviours among the constructs PI4KIIαSSPSS, PI4KIIαFFPFF, PI4KIIαSSPSSΔC and PI4KIIαFFPFFΔC (Supplementary Table 1). Deletion of the C-terminal region decreased membrane-binding affinity. The decreased interaction was restored, however, by mutating SSPSS to FFPFF, suggesting that the palmitoylation motif plays a role in membrane binding (Fig. 3b).

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