Does changing the predicted dynamics of a phospholipase C alter activity and membrane binding?
Jiongjia Cheng1, Sashank Karri, Cédric Grauffel
1Department of Chemistry, Boston College, Chestnut Hill, Massachusetts, USA.
Biophysical Journal
|January 22, 2013
Summary
Correlated loop motions involving specific proline residues in secreted phosphatidylinositol-specific phospholipase C (PI-PLC) are crucial for bacterial virulence. Modifying these proline residues significantly impacts PI-PLC enzymatic activity and bacterial virulence.
Area of Science:
- Biochemistry
- Molecular Biology
- Microbiology
Background:
- Secreted phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes are virulence factors in bacteria.
- PI-PLC active site access is potentially regulated by correlated loop motions, involving proline residues Pro(245) and Pro(254).
- This proline-rich region is conserved in virulence-associated PI-PLCs and interacts with phosphatidylcholine (PC) to enhance activity.
Purpose of the Study:
- To investigate the role of Pro(245) and Pro(254) in the enzymatic activity and membrane binding of Bacillus thuringiensis PI-PLC.
- To elucidate the contribution of correlated loop motions to PI-PLC function using computational and experimental approaches.
Main Methods:
- In silico mutagenesis of Pro(245) and Pro(254) in Bacillus thuringiensis PI-PLC.
- Molecular dynamics simulations to analyze correlated loop motions.
- Enzymatic activity assays and membrane binding experiments to evaluate mutant PI-PLC function.
Main Results:
- In silico mutagenesis of Pro(245) disrupted correlated motions and significantly reduced PI-PLC enzymatic activity.
- Pro(245) variants showed reduced activity, with PC enhancement not reaching wild-type levels.
- Mutagenesis of Pro(254) stiffened the PI-PLC structure but had minor effects on activity and membrane binding.
- Reduced enzymatic activity in most variants was not linked to decreased membrane affinity.
Conclusions:
- Correlated motions between PI-PLC halves, influenced by Pro(245), are critical for enzymatic activity.
- While Pro(254) affects structure, its role in activity and binding appears less significant than Pro(245).
- These findings highlight the importance of protein dynamics in regulating bacterial enzyme function and virulence.
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