skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Dimer Structure of an Interfacially Impaired Phosphatidylinositol-Specific Pholpholipase C

Abstract

The crystal structure of the W47A/W242A mutant of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis has been solved to 1.8{angstrom} resolution. The W47A/W242A mutant is an interfacially challenged enzyme, and it has been proposed that one or both tryptophan side chains serve as membrane interfacial anchors (Feng, J., Wehbi, H., and Roberts, M. F. (2002) J. Biol. Chem. 277, 19867-19875). The crystal structure supports this hypothesis. Relative to the crystal structure of the closely related (97% identity) wild-type PI-PLC from Bacillus cereus, significant conformational differences occur at the membrane-binding interfacial region rather than the active site. The Trp {yields} Ala mutations not only remove the membrane-partitioning aromatic side chains but also perturb the conformations of the so-called helix B and rim loop regions, both of which are implicated in interfacial binding. The crystal structure also reveals a homodimer, the first such observation for a bacterial PI-PLC, with pseudo-2-fold symmetry. The symmetric dimer interface is stabilized by hydrophobic and hydrogen-bonding interactions, contributed primarily by a central swath of aromatic residues arranged in a quasiherringbone pattern. Evidence that interfacially active wild-type PI-PLC enzymes may dimerize in the presence of phosphatidylcholine vesicles is provided by fluorescence quenching of PI-PLC mutants with pyrene-labeled cysteinemore » residues. The combined data suggest that wild-type PI-PLC can form similar homodimers, anchored to the interface by the tryptophan and neighboring membrane-partitioning residues.« less

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
929911
Report Number(s):
BNL-80496-2008-JA
Journal ID: ISSN 0021-9258; JBCHA3; TRN: US200822%%1086
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Biological Chemistry; Journal Volume: 282
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; AROMATICS; BACILLUS; BACILLUS CEREUS; CRYSTAL STRUCTURE; CYSTEINE; DATA; DIMERS; ENZYMES; FLUORESCENCE; HYPOTHESIS; INTERACTIONS; INTERFACES; LECITHINS; MEMBRANES; MUTANTS; MUTATIONS; QUENCHING; RESIDUES; RESOLUTION; SYMMETRY; TRYPTOPHAN; national synchrotron light source

Citation Formats

Shao,C., Shi, X., Wehbi, H., Zambonelli, C., Head, J., Seaton, B., and Roberts, M,.. Dimer Structure of an Interfacially Impaired Phosphatidylinositol-Specific Pholpholipase C. United States: N. p., 2007. Web. doi:10.1074/jbc.M610918200.
Shao,C., Shi, X., Wehbi, H., Zambonelli, C., Head, J., Seaton, B., & Roberts, M,.. Dimer Structure of an Interfacially Impaired Phosphatidylinositol-Specific Pholpholipase C. United States. doi:10.1074/jbc.M610918200.
Shao,C., Shi, X., Wehbi, H., Zambonelli, C., Head, J., Seaton, B., and Roberts, M,.. Mon . "Dimer Structure of an Interfacially Impaired Phosphatidylinositol-Specific Pholpholipase C". United States. doi:10.1074/jbc.M610918200.
@article{osti_929911,
title = {Dimer Structure of an Interfacially Impaired Phosphatidylinositol-Specific Pholpholipase C},
author = {Shao,C. and Shi, X. and Wehbi, H. and Zambonelli, C. and Head, J. and Seaton, B. and Roberts, M,.},
abstractNote = {The crystal structure of the W47A/W242A mutant of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis has been solved to 1.8{angstrom} resolution. The W47A/W242A mutant is an interfacially challenged enzyme, and it has been proposed that one or both tryptophan side chains serve as membrane interfacial anchors (Feng, J., Wehbi, H., and Roberts, M. F. (2002) J. Biol. Chem. 277, 19867-19875). The crystal structure supports this hypothesis. Relative to the crystal structure of the closely related (97% identity) wild-type PI-PLC from Bacillus cereus, significant conformational differences occur at the membrane-binding interfacial region rather than the active site. The Trp {yields} Ala mutations not only remove the membrane-partitioning aromatic side chains but also perturb the conformations of the so-called helix B and rim loop regions, both of which are implicated in interfacial binding. The crystal structure also reveals a homodimer, the first such observation for a bacterial PI-PLC, with pseudo-2-fold symmetry. The symmetric dimer interface is stabilized by hydrophobic and hydrogen-bonding interactions, contributed primarily by a central swath of aromatic residues arranged in a quasiherringbone pattern. Evidence that interfacially active wild-type PI-PLC enzymes may dimerize in the presence of phosphatidylcholine vesicles is provided by fluorescence quenching of PI-PLC mutants with pyrene-labeled cysteine residues. The combined data suggest that wild-type PI-PLC can form similar homodimers, anchored to the interface by the tryptophan and neighboring membrane-partitioning residues.},
doi = {10.1074/jbc.M610918200},
journal = {Journal of Biological Chemistry},
number = ,
volume = 282,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • The inositol phosphate products formed during the cleavage of phosphatidylinositol by phosphatidylinositol-specific phospholipase C from Bacillus cereus were analyzed by {sup 31}P NMR. {sup 31}P NMR spectroscopy can distinguish between the inositol phosphate species and phosphatidylinositol. Chemical shift values (with reference to phosphoric acid) observed are {minus}0.41, 3.62, 4.45, and 16.30 ppm for phosphatidylinositol, myo-inositol 1-monophosphate, myo-inositol 2-monophosphate, and myo-inositol 1,2-cyclic monophosphate, respectively. It is shown that under a variety of experimental conditions this phospholipase C cleaves phosphatidylinositol via an intramolecular phosphotransfer reaction producing diacylglycerol and D-myo-inositol 1,2-cyclic monophosphate. The authors also report the new and unexpected observation that themore » phosphatidylinositol-specific phospholipase C from B. cereus is able to hydrolyze the inositol cyclic phosphate to form D-myo-inositol 1-monophosphate. The enzyme, therefore, possesses phosphotransferase and cyclic phosphodiesterase activities. The second reaction requires thousandfold higher enzyme concentrations to be observed by {sup 31}P NMR. This reaction was shown to be regiospecific in that only the 1-phosphate was produced and stereospecific in that only D-myo-inositol 1,2-cyclic monophosphate was hydrolyzed. Inhibition with a monoclonal antibody specific for the B.cereus phospholipase C showed that the cyclic phosphodiesterase activity is intrinsic to the bacterial enzyme. They propose a two-step mechanism for the phosphatidyl-inositol-specific phospholipase C from B. cereus involving sequential phosphotransferase and cyclic phosphodiesterase activities. This mechanism bears a resemblance to the well-known two-step mechanism of pancreatic ribonuclease, RNase A.« less
  • In course of the molecular characterization of a human extragonadal germ cell tumor (EGCT)-associated chromosomal translocation, we identified YACs and cosmids from the 11q13 region. The endclone of one of these YACs appeared to contain a stretch of DNA homologous to part of the human phosphatidylinositol-specific phospholipase C {Beta}{sub 3} gene (PLCB3). Since we considered PLCB3 a candidate gene for these EGCTs, we set out to clone the PLCB3 cDNA, from which the 5{prime} end was still missing, and performed Northern and Southern blot analyses. The localization of PLCB3 to 11q13 was confirmed. In addition, we were able to excludemore » the gene from involvement in EGCT development. 10 refs., 2 figs.« less
  • The authors have mapped the PLCG2 gene, which encodes the enzyme phosphatidyl inositol-specific phospholipase C-{gamma}2. This is one of the phospolipases responsible for catalyzing the hydrolysis of phosphatidyl inositol in response to a great many mitogenic stimuli. PL C-{gamma}2 is an essential component of the signal transduction pathway between tyrosine kinases and downstream events such as protein kinase C activation and intracellular calcium release. The authors assigned PLCG2 to human chromosome 16 by amplification within a somatic cell hybrid mapping panel. To position the locus at a much finer resolution, PLCG2 sequences were amplified from a chromosome 16-specific somatic cellmore » hybrid panel, which placed the gene on the long arm of the chromosome in band 16q24.1, a region that has few known genes. The authors have hybridized a mouse Plcg2 open reading frame probe to mouse DNAs from the European interspecific Backcross. The segregation pattern reveals the mouse Plcg2 locus maps to distal chromosome 8. 16 refs., 3 figs., 1 tab.« less
  • Cleavage of phosphatidylinositol (PI) to inositol 1,2-(cyclic)-phosphate (cIP) and cIP hydrolysis to inositol 1-phosphate by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C are activated by the enzyme binding to phosphatidylcholine (PC) surfaces. Part of this reflects improved binding of the protein to interfaces. However, crystallographic analysis of an interfacially impaired phosphatidylinositol-specific phospholipase (W47A/W242A) suggested protein dimerization might occur on the membrane. In the W47A/W242A dimer, four tyrosine residues from one monomer interact with the same tyrosine cluster of the other, forming a tight dimer interface close to the membrane binding regions. We have constructed mutant proteins in which two or more ofmore » these tyrosine residues have been replaced with serine. Phospholipid binding and enzymatic activity of these mutants have been examined to assess the importance of these residues to enzyme function. Replacing two tyrosines had small effects on enzyme activity. However, removal of three or four tyrosine residues weakened PC binding and reduced PI cleavage by the enzyme as well as PC activation of cIP hydrolysis. Crystal structures of Y247S/Y251S in the absence and presence of myo-inositol as well as Y246S/Y247S/Y248S/Y251S indicate that both mutant proteins crystallized as monomers, were very similar to one another, and had no change in the active site region. Kinetic assays, lipid binding, and structural results indicate that either (i) a specific PC binding site, critical for vesicle activities and cIP activation, has been impaired, or (ii) the reduced dimerization potential for Y246S/Y247S/Y248S and Y246S/Y247S/Y248S/Y251S is responsible for their reduced catalytic activity in all assay systems.« less
  • No abstract available.