title: Does changing the predicted dynamics of a phospholipase C alter activity and membrane binding?
abstract: The enzymatic activity of secreted phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes is associated with bacterial virulence. Although the PI-PLC active site has no obvious lid, molecular-dynamics simulations suggest that correlated loop motions may limit access to the active site, and two Pro residues, Pro(245) and Pro(254), are associated with these correlated motions. Whereas the region containing both Pro residues is quite variable among PI-PLCs, it shows high conservation in virulence-associated, secreted PI-PLCs that bind to the surface of cells. These regions of the protein are also associated with phosphatidylcholine binding, which enhances PI-PLC activity. In silico mutagenesis of Pro(245) disrupts correlated motions between the two halves of Bacillus thuringiensis PI-PLC, and Pro(245) variants show significantly reduced enzymatic activity in all assay systems. PC still enhanced activity, but not to the level of wild-type enzyme. Mutagenesis of Pro(254) appears to stiffen the PI-PLC structure, but experimental mutations had minor effects on activity and membrane binding. With the exception of P245Y, reduced activity was not associated with reduced membrane affinity. This combination of simulations and experiments suggests that correlated motions between the two halves of PI-PLC may be more important for enzymatic activity than for vesicle binding.
authors: Department of Chemistry, Boston College, Chestnut Hill, Massachusetts, USA.
authors: Jiongjia
authors: J
authors: Cheng
authors: Sashank
authors: S
authors: Karri
authors: Cédric
authors: C
authors: Grauffel
authors: Fang
authors: F
authors: Wang
authors: Nathalie
authors: N
authors: Reuter
authors: Mary F
authors: MF
authors: Roberts
authors: Patrick L
authors: PL
authors: Wintrode
authors: Anne
authors: A
authors: Gershenson
mesh_terms: D000595:Amino Acid Sequence; D001409:Bacillus cereus / Q000201:enzymology; D001413:Bacillus thuringiensis / Q000166:cytology* / Q000201:enzymology*; D055162:Biocatalysis; D002462:Cell Membrane / Q000201:enzymology*; D017124:Conserved Sequence; D008823:Micelles; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009038:Motion; D050505:Mutant Proteins / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C / Q000737:chemistry / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D053835:Unilamellar Liposomes / Q000737:chemistry
chemical_list: D008823:Micelles; D050505:Mutant Proteins; D053835:Unilamellar Liposomes; D011392:Proline; D010770:Phosphotransferases; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C