The CTX-M family of beta lactamases mediate broad-spectrum antibiotic resistance and present in the majority of drug-resistant gram-negative bacteria infections worldwide. Allosteric mutations that increase catalytic rates of these drug resistance enzymes have been identified in clinical isolates but are challenging to predict prospectively. We have used molecular dynamics simulations to predict allosteric mutants increasing CTX-M9 drug resistance, experimentally testing top mutants using multiple antibiotics. Purified enzymes show an increase in catalytic rate and efficiency, while mutant crystal structures show no detectable changes from wild-type CTX-M9. We hypothesize that increased drug resistance results from changes in the conformational ensemble of an acyl intermediate in hydrolysis. Machine-learning analyses on top-scoring mutants identify changes to the binding-pocket conformational ensemble by which these allosteric mutations transmit their effect. These findings show how molecular simulation can predict how allosteric mutations alter active-site conformational equilibria to increase catalytic rates and thus resistance against common clinically used antibiotics.
CTX-M9:meropenem acylenzyme run input file
Gromacs TPR for wild-type CTX-M9 in complex with meropenem
ctx_acyl.tpr
Binding pocket trajectories for CTX-M9
Gromacs trajectory files and reference PDB for drug binding pocket atoms, computed using TPR of the full solvated acylenzyme. These trajectories were used for mutual information feature selection and decision tree calculation.
ctx_pocket_trajs.tgz
Binding pocket trajectories for CTX-M9 L48A
Gromacs trajectory files and reference PDB for drug binding pocket atoms, computed using TPR of the full solvated acylenzyme. These trajectories were used for mutual information feature selection and decision tree calculation.
l48a_pocket_trajs.tgz
Binding pocket trajectories for CTX-M9 T165W
Gromacs trajectory files and reference PDB for drug binding pocket atoms, computed using TPR of the full solvated acylenzyme. These trajectories were used for mutual information feature selection and decision tree calculation.
t165w_pocket_trajs.tgz
Binding pocket trajectories for CTX-M9 S281A
Gromacs trajectory files and reference PDB for drug binding pocket atoms, computed using TPR of the full solvated acylenzyme. These trajectories were used for mutual information feature selection and decision tree calculation.
s281a_pocket_trajs.tgz
CTX-M9 L48A:meropenem acylenzyme run input file
Gromacs TPR for CTX-M9 L48A in complex with meropenem
ctx_l48a.tpr
CTX-M9 T165W:meropenem acylenzyme run input file
Gromacs TPR for CTX-M9 T165W in complex with meropenem
ctx_t165w.tpr
CTX-M9 S281A:meropenem acylenzyme run input file
Gromacs TPR for CTX-M9 S281A in complex with meropenem
ctx_s281a.tpr
PDB file for solvated CTX-M9:meropenem adduct
PDB file corresponding to simulation start state for CTX-M9:meropenem
ctx_acyl.pdb
PDB file for solvated CTX-M9 L48A:meropenem adduct
PDB file corresponding to simulation start state for L48A:meropenem acylenzyme.
ctx_l48a.pdb
PDB file for solvated CTX-M9 T165W:meropenem adduct
PDB file corresponding to simulation start state for CTX-M9 T165W meropenem acylenzyme
ctx_t165w.pdb
PDB file for solvated CTX-M9 S281A:meropenem adduct
PDB file corresponding to simulation start state for CTX-M9 S281A meropenem acylenzyme
ctx_s281a.pdb
Residue definitions for meropenem and cefotaxime acylenzymes
Modified GROMACS aminoacids.rtp file for AMBER99SB-ILDN force field containing residue definitions for serine-meropenem adducts and serine-cefotaxime adducts.
aminoacids.rtp
atom type file
aminoacids.atp in GROMACS format for modified AMBER99SB-ILDN parameters to describe acylenzymes.
atomtypes.atp
ffnonbonded.itp
Nonbonded parameter file ffnonbonded.itp for modified AMBER99SB-ILDN to define acylenzymes.