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Haimei Zhu June 12th, 2008

MD simulations of wild type and mutated E.coli LeuRS CP1 domain complexed with pre-transfer editing substrate analog. Haimei Zhu June 12th, 2008. Aminoacyl-tRNA synthetases (aaRS). aaRS are responsible for accurate matching each amino acid with its cognate tRNA

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Haimei Zhu June 12th, 2008

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  1. MD simulations of wild type and mutated E.coli LeuRS CP1 domain complexed with pre-transfer editing substrate analog Haimei Zhu June 12th, 2008

  2. Aminoacyl-tRNA synthetases (aaRS) • aaRS are responsible for accurate matching each amino acid with its cognate tRNA • Some synthetases have editing activities that clear the wrong amino acids Amy M. Williams. et al PNAS. 2006, 103, 3586-3591 Homology model of E.coli LeuRS

  3. Leucyl-tRNA synthetases (LeuRS) • 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 Mis-Aminoacylation of 4µM tRNALeu with isoleucine by 1µM E.coli LeuRSs Amy M. Williams. et al PNAS. 2006, 103, 3586-3591 PDB 1BOH T . Thermophilus LeuRS NvaAMS Isoleucyl-adenylate Tommie L. Lincecum, Jr. et al Molecular Cell. 2003, 11, 951-963

  4. 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

  5. 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

  6. Black line: Backbone of Protein, Red line: Backbone of residues 228-352, Green line: NvaAMS

  7. 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 Isoleucyl-adenylate NvaAMS

  8. Wild type system • Water analysis • Extract water molecules within 0.35 nm of carbonyl 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

  9. 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

  10. Most popular NvaAMS conformation and loyal waters in simulation 2 Cluster occupancy 56% water occupancy 78% Carbonyl oxygen –THR228OG hydrogen bond occupancy 0.3%

  11. 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

  12. Future plan • Water analysis and hydrogen bond analysis for other systems

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