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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}
}