Mechanism of membrane binding of the phospholipase D1 PX domain

Robert Stahelin, Bharath Ananthanarayanan, Nichole E. Blatner, Shaneen Singh, Karol S. Bruzik, Diana Murray, Wonhwa Cho

Research output: Contribution to journalArticle

77 Citations (Scopus)

Abstract

Mammalian phospholipases D (PLD), which catalyze the hydrolysis of phosphatidylcholine to phosphatidic acid (PA), have been implicated in various cell signaling and vesicle trafficking processes. Mammalian PLD1 contains two different membrane-targeting domains, pleckstrin homology and Phox homology (PX) domains, but the precise roles of these domains in the membrane binding and activation of PLD1 are still unclear. To elucidate the role of the PX domain in PLD1 activation, we constructed a structural model of the PX domain by homology modeling and measured the membrane binding of this domain and selected mutants by surface plasmon resonance analysis. The PLD1 PX domain was found to have high phosphoinositide specificity, i.e. phosphatidylinositol 3,4,5-trisphospliate (PtdIns-(3,4,5)P3) ≫ phosphatidylinositol 3-phosphate > phosphatidylinositol 5-phosphate ≫ other phosphoinositides. The PtdIns(3,4,5)P3 binding was facilitated by the cationic residues (Lys119, Lys121, and Arg179) in the putative binding pocket. Consistent with the model structure that suggests the presence of a second lipid-binding pocket, vesicle binding studies indicated that the PLD1 PX domain could also bind with moderate affinity to PA, phosphatidylserine, and other anionic lipids, which were mediated by a cluster of cationic residues in the secondary binding site. Simultaneous occupancy of both binding pockets synergistically increases membrane affinity of the PX domain. Electrostatic potential calculations suggest that a highly positive potential near the secondary binding site may facilitate the initial adsorption of the domain to the anionic membrane, which is followed by the binding of PtdIns(3,4,5)P 3 to its binding pocket. Collectively, our results suggest that the interaction of the PLD1 PX domain with PtdIns(3,4,5)P3 and/or PA (or phosphatidylserine) may be an important factor in the spatiotemporal regulation and activation of PLD1.

Original languageEnglish (US)
Pages (from-to)54918-54926
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number52
DOIs
StatePublished - Dec 24 2004
Externally publishedYes

Fingerprint

Membranes
Phosphatidylinositols
Phosphatidic Acids
Chemical activation
Phosphatidylserines
Binding Sites
Cell signaling
Lipids
Phospholipase D
Surface Plasmon Resonance
Structural Models
Surface plasmon resonance
Model structures
4-ethoxymethylene-2-phenyl-2-oxazoline-5-one
phospholipase D1
Static Electricity
Phosphatidylcholines
Adsorption
Electrostatics
Hydrolysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Stahelin, R., Ananthanarayanan, B., Blatner, N. E., Singh, S., Bruzik, K. S., Murray, D., & Cho, W. (2004). Mechanism of membrane binding of the phospholipase D1 PX domain. Journal of Biological Chemistry, 279(52), 54918-54926. https://doi.org/10.1074/jbc.M407798200

Mechanism of membrane binding of the phospholipase D1 PX domain. / Stahelin, Robert; Ananthanarayanan, Bharath; Blatner, Nichole E.; Singh, Shaneen; Bruzik, Karol S.; Murray, Diana; Cho, Wonhwa.

In: Journal of Biological Chemistry, Vol. 279, No. 52, 24.12.2004, p. 54918-54926.

Research output: Contribution to journalArticle

Stahelin, R, Ananthanarayanan, B, Blatner, NE, Singh, S, Bruzik, KS, Murray, D & Cho, W 2004, 'Mechanism of membrane binding of the phospholipase D1 PX domain', Journal of Biological Chemistry, vol. 279, no. 52, pp. 54918-54926. https://doi.org/10.1074/jbc.M407798200
Stahelin R, Ananthanarayanan B, Blatner NE, Singh S, Bruzik KS, Murray D et al. Mechanism of membrane binding of the phospholipase D1 PX domain. Journal of Biological Chemistry. 2004 Dec 24;279(52):54918-54926. https://doi.org/10.1074/jbc.M407798200
Stahelin, Robert ; Ananthanarayanan, Bharath ; Blatner, Nichole E. ; Singh, Shaneen ; Bruzik, Karol S. ; Murray, Diana ; Cho, Wonhwa. / Mechanism of membrane binding of the phospholipase D1 PX domain. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 52. pp. 54918-54926.
@article{1fc06f5fa2594c3a9dd5a3247e67f96e,
title = "Mechanism of membrane binding of the phospholipase D1 PX domain",
abstract = "Mammalian phospholipases D (PLD), which catalyze the hydrolysis of phosphatidylcholine to phosphatidic acid (PA), have been implicated in various cell signaling and vesicle trafficking processes. Mammalian PLD1 contains two different membrane-targeting domains, pleckstrin homology and Phox homology (PX) domains, but the precise roles of these domains in the membrane binding and activation of PLD1 are still unclear. To elucidate the role of the PX domain in PLD1 activation, we constructed a structural model of the PX domain by homology modeling and measured the membrane binding of this domain and selected mutants by surface plasmon resonance analysis. The PLD1 PX domain was found to have high phosphoinositide specificity, i.e. phosphatidylinositol 3,4,5-trisphospliate (PtdIns-(3,4,5)P3) ≫ phosphatidylinositol 3-phosphate > phosphatidylinositol 5-phosphate ≫ other phosphoinositides. The PtdIns(3,4,5)P3 binding was facilitated by the cationic residues (Lys119, Lys121, and Arg179) in the putative binding pocket. Consistent with the model structure that suggests the presence of a second lipid-binding pocket, vesicle binding studies indicated that the PLD1 PX domain could also bind with moderate affinity to PA, phosphatidylserine, and other anionic lipids, which were mediated by a cluster of cationic residues in the secondary binding site. Simultaneous occupancy of both binding pockets synergistically increases membrane affinity of the PX domain. Electrostatic potential calculations suggest that a highly positive potential near the secondary binding site may facilitate the initial adsorption of the domain to the anionic membrane, which is followed by the binding of PtdIns(3,4,5)P 3 to its binding pocket. Collectively, our results suggest that the interaction of the PLD1 PX domain with PtdIns(3,4,5)P3 and/or PA (or phosphatidylserine) may be an important factor in the spatiotemporal regulation and activation of PLD1.",
author = "Robert Stahelin and Bharath Ananthanarayanan and Blatner, {Nichole E.} and Shaneen Singh and Bruzik, {Karol S.} and Diana Murray and Wonhwa Cho",
year = "2004",
month = "12",
day = "24",
doi = "10.1074/jbc.M407798200",
language = "English (US)",
volume = "279",
pages = "54918--54926",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "52",

}

TY - JOUR

T1 - Mechanism of membrane binding of the phospholipase D1 PX domain

AU - Stahelin, Robert

AU - Ananthanarayanan, Bharath

AU - Blatner, Nichole E.

AU - Singh, Shaneen

AU - Bruzik, Karol S.

AU - Murray, Diana

AU - Cho, Wonhwa

PY - 2004/12/24

Y1 - 2004/12/24

N2 - Mammalian phospholipases D (PLD), which catalyze the hydrolysis of phosphatidylcholine to phosphatidic acid (PA), have been implicated in various cell signaling and vesicle trafficking processes. Mammalian PLD1 contains two different membrane-targeting domains, pleckstrin homology and Phox homology (PX) domains, but the precise roles of these domains in the membrane binding and activation of PLD1 are still unclear. To elucidate the role of the PX domain in PLD1 activation, we constructed a structural model of the PX domain by homology modeling and measured the membrane binding of this domain and selected mutants by surface plasmon resonance analysis. The PLD1 PX domain was found to have high phosphoinositide specificity, i.e. phosphatidylinositol 3,4,5-trisphospliate (PtdIns-(3,4,5)P3) ≫ phosphatidylinositol 3-phosphate > phosphatidylinositol 5-phosphate ≫ other phosphoinositides. The PtdIns(3,4,5)P3 binding was facilitated by the cationic residues (Lys119, Lys121, and Arg179) in the putative binding pocket. Consistent with the model structure that suggests the presence of a second lipid-binding pocket, vesicle binding studies indicated that the PLD1 PX domain could also bind with moderate affinity to PA, phosphatidylserine, and other anionic lipids, which were mediated by a cluster of cationic residues in the secondary binding site. Simultaneous occupancy of both binding pockets synergistically increases membrane affinity of the PX domain. Electrostatic potential calculations suggest that a highly positive potential near the secondary binding site may facilitate the initial adsorption of the domain to the anionic membrane, which is followed by the binding of PtdIns(3,4,5)P 3 to its binding pocket. Collectively, our results suggest that the interaction of the PLD1 PX domain with PtdIns(3,4,5)P3 and/or PA (or phosphatidylserine) may be an important factor in the spatiotemporal regulation and activation of PLD1.

AB - Mammalian phospholipases D (PLD), which catalyze the hydrolysis of phosphatidylcholine to phosphatidic acid (PA), have been implicated in various cell signaling and vesicle trafficking processes. Mammalian PLD1 contains two different membrane-targeting domains, pleckstrin homology and Phox homology (PX) domains, but the precise roles of these domains in the membrane binding and activation of PLD1 are still unclear. To elucidate the role of the PX domain in PLD1 activation, we constructed a structural model of the PX domain by homology modeling and measured the membrane binding of this domain and selected mutants by surface plasmon resonance analysis. The PLD1 PX domain was found to have high phosphoinositide specificity, i.e. phosphatidylinositol 3,4,5-trisphospliate (PtdIns-(3,4,5)P3) ≫ phosphatidylinositol 3-phosphate > phosphatidylinositol 5-phosphate ≫ other phosphoinositides. The PtdIns(3,4,5)P3 binding was facilitated by the cationic residues (Lys119, Lys121, and Arg179) in the putative binding pocket. Consistent with the model structure that suggests the presence of a second lipid-binding pocket, vesicle binding studies indicated that the PLD1 PX domain could also bind with moderate affinity to PA, phosphatidylserine, and other anionic lipids, which were mediated by a cluster of cationic residues in the secondary binding site. Simultaneous occupancy of both binding pockets synergistically increases membrane affinity of the PX domain. Electrostatic potential calculations suggest that a highly positive potential near the secondary binding site may facilitate the initial adsorption of the domain to the anionic membrane, which is followed by the binding of PtdIns(3,4,5)P 3 to its binding pocket. Collectively, our results suggest that the interaction of the PLD1 PX domain with PtdIns(3,4,5)P3 and/or PA (or phosphatidylserine) may be an important factor in the spatiotemporal regulation and activation of PLD1.

UR - http://www.scopus.com/inward/record.url?scp=11144237619&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=11144237619&partnerID=8YFLogxK

U2 - 10.1074/jbc.M407798200

DO - 10.1074/jbc.M407798200

M3 - Article

VL - 279

SP - 54918

EP - 54926

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 52

ER -