Role of helix B residues in interfacial activation of a bacterial phosphatidylinositol-specific phospholipase C.
The Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), an interfacial enzyme associated with prokaryotic infectivity, is activated by binding to zwitterionic surfaces, particularly phosphatidycholine (PC). Two tryptophan residues (Trp47 in the two-turn helix B and Trp242 in a disordered loop) at the rim of the barrel structure are critical for this interaction. The helix B region (Ile43 to Gly48) in wild-type PI-PLC orients the side chains of Ile43 and Trp47 so that they pack together and form a hydrophobic protrusion from the protein surface that likely facilitates initial membrane binding. In previous studies we reported that in the crystal structure of the dimeric W47A/W242A mutant, which is unable to bind to PC, the helix B region has been reorganized by the mutation into an extended loop. Here we report the construction and characterization (catalytic activity, fluorescence, and NMR studies) of a series of PI-PLC mutants targeting helix B residues and surrounding regions to explore what is needed to stabilize the "membrane-active" conformation of the helix B region. Results strongly suggest that, while hydrophobic groups and presumably an intact helix B are critical for the initial binding of PI-PLC to membranes, disruption of helix B to allow enzyme dimerization is what leads to the activated PI-PLC conformation.
Guo
Su
S
Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA.
Zhang
Xin
X
Seaton
Barbara A
BA
Roberts
Mary F
MF
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D001413:Bacillus thuringiensis / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D054801:Phosphoinositide Phospholipase C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D010770:Phosphotransferases; D054801:Phosphoinositide Phospholipase C; D000409:Alanine