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 Some synthetases have editing activities that clear the wrong amino acids Homology model of E.coli LeuRS Amy M. Williams. et al PNAS. 2006, 103,

Leucyl-tRNA synthetases (LeuRS) Mis-Aminoacylation of 4µM tRNA Leu 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, Isoleucyl-adenylate NvaAMS Tommie L. Lincecum, Jr. et al Molecular Cell. 2003, 11, 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

Two large flexible regions: residues and The region in red circle form the whole active site pocket for NvaAMS and regions adjacent to region are quite stable It is reasonable to consider only circled region (residues ) for complex interaction studies

Black line: Backbone of Protein, Red line: Backbone of residues , Green line: NvaAMS

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 NvaAMS Isoleucyl-adenylate

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

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