 |
|
|
 |
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
296 a.a.
_ZN
 1-phosphatidyl-1D-myo-inositol |
= |
 1D-myo-inositol 1,2-cyclic phosphate |
+ |
 diacylglycerol |
|---|
Key reference
DOI no: 10.1074/jbc.M901601200 J Biol Chem 284:15607-15618 (2009) PubMed id: 19369255
Modulation of bacillus thuringiensis phosphatidylinositol-specific phospholipase C activity by mutations in the putative dimerization interface. X.Shi, C.Shao, X.Zhang, C.Zambonelli, A.G.Redfield, J.F.Head, B.A.Seaton, M.F.Roberts. ABSTRACT
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 (PI-PLC) 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 PI-PLC (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 in order 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.
Selected figure(s)
Figure 1.
Stereo structure of the mutation sites in B. thuringiensis Y247S/Y251S PI-PLC. Shown in the gray network is the electron density (from the simulated-annealing composite omit map and contoured at 1.2σ) for the residues in mutation sites. The helix hydrogen-bond network is shown as orange dotted lines.Figure 2.
Stereoview showing tyrosine mutation sites at the crystallographic dimer interface of B. thuringiensis PI-PLC mutant, W47A/W242A (PDB code 2OR2). Subunit A is shown with Corey-Pauling-Koltun coloration, labels are in bold. subunit B is shown in cyan, and labels are in italics. Hydrogen bonds shown as green dashed lines.The above figures are reprinted by permission from the ASBMB: J Biol Chem (2009, 284, 15607-15618) copyright 2009. Figures were selected by an automated process.
 |