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Crystal Structure of the Y247S/Y251S Mutant of Phosphatidylinositol-Specific Phospholipase C from Bacillus ThuringiensisCrystal Structure of the Y247S/Y251S Mutant of Phosphatidylinositol-Specific Phospholipase C from Bacillus Thuringiensis
Structural highlights
Function[PLC_BACTU] Cleaves glycosylphosphatidylinositol (GPI) and phosphatidylinositol (PI) anchors but not PI phosphates. Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedCleavage 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 of 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. Modulation of Bacillus thuringiensis phosphatidylinositol-specific phospholipase C activity by mutations in the putative dimerization interface.,Shi X, Shao C, Zhang X, Zambonelli C, Redfield AG, Head JF, Seaton BA, Roberts MF J Biol Chem. 2009 Jun 5;284(23):15607-18. Epub 2009 Apr 15. PMID:19369255[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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