MD simulations of wild type and mutated E.coli LeuRS CP1 domain complexed with pre-transfer editi
ng substrate analog
Haimei Zhu
June 12th, 2008
Aminoacyl-tRNA synthetases (aaRS) • aaRS are responsible for ac
curate matching each amino acid with its cognate tRNA
• Some synthetases have editing activities that clear the wrong amino acids
Homology model of E.coli LeuRSAmy M. Williams. et al PNAS. 2006, 103, 3586-3591
Leucyl-tRNA synthetases (LeuRS)
Mis-Aminoacylation of 4µM tRNALeu with isoleucine by 1µM E.coli LeuRSs
• E.coli LeuRS relies only on post-transfer editing
• Mutation T252Y prevents post-transfer editing activity
• Double mutation T252Y/A293D rescues pre-transfer editing activity Amy M. Williams. et al PNAS.
2006, 103, 3586-3591
Isoleucyl-adenylate NvaAMSTommie L. Lincecum, Jr. et al Molecular Cell. 2003, 11, 951-963
PDB 1BOH
T . Thermophilus LeuRS
Work plan
To study the rescued pre-transfer editing activity• MD simulation Systems of E. coli LeuRS CP1
domain complexed with pre-transfer substrate analog NvaAMS:
– Wild type – Single mutant T252Y – Single mutant A293D– Double mutant T252/A293D
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LeuRS CP1
RM
SF
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Residues
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0.5LeuRS CP1 T252Y
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0.5LeuRS CP1 A293D
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LeuRS CP1 T252Y/A293D
Two large flexible regions: residues 280-300 and 360-390
The region in red circle form the whole active site pocket for NvaAMS and regions adjacent to region 360-390 are quite stable
It is reasonable to consider only circled region (residues 228-352) for complex interaction studies
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RM
SD
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0.4 LeuRS CP1
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RM
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Time (ps)
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0.4 LeuRS CP1 T252Y
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RM
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Time (ps)
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LeuRS CP1 A293D
Black line: Backbone of Protein, Red line: Backbone of residues 228-352, Green line: NvaAMS
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RM
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Time (ps)
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0.4 LeuRS CP1 T252Y/A293D
Proposed hydrolysis mechanism
• No proven directly catalytic residue • Anhydride and ester linkages in pre- and post substrate
hydrolyzed by water attacking the carbonyl carbon• Nucleophilic OH- • Positive charge of the carbonyl carbon• Favorable substrate binding configuration• Active site residues positioned water molecule
NvaAMSIsoleucyl-adenylate
Wild type system
• Water analysis– Extract water molecules within 0.35 nm of carbon
yl carbon from trajectories– Find the loyal water molecules
• Hydrogen bond analysis– Extract active site residues which form hydrogen
bond to carbonyl oxygen from trajectories
• Clustering– based on the conformational states of NvaAMS
Most popular NvaAMS conformation and loyal waters in simulation 1
Cluster occupancy 36%
water occupancy 31%
Carbonyl oxygen –THR228OG hydrogen bond occupancy 65% , occurs in the last 6 ns
Most popular NvaAMS conformation and loyal waters in simulation 2
Cluster occupancy 56%
water occupancy 78%
Carbonyl oxygen –THR228OG hydrogen bond occupancy 0.3%
Most popular NvaAMS conformation and loyal waters in simulation 3
Cluster occupancy 38%
water occupancy 22%
Carbonyl oxygen –THR228OG hydrogen bond occupancy 30%, occurs in the last 3 ns
Future plan
• Water analysis and hydrogen bond analysis for other systems