Molecular Basis of Influence of A501X Mutations in Penicillin-Binding Protein 2 of Neisseria gonorrhoeae Strain 35/02 on Ceftriaxone Resistance.

The increase in the resistance of mutant strains of Neisseria gonorrhoeae to the antibiotic ceftriaxone is pronounced in the decrease in the second-order acylation rate constant, k 2 /K S , by penicillin-binding protein 2 (PBP2). These changes can be caused by both the decrease in the acylation rate constant, k 2 , and the weakening of the binding affinity, i.e., an increase in the substrate constant, K S . A501X mutations in PBP2 affect second-order acylation rate constants. The PBP2 A501V variant exhibits a higher k 2 /K S value, whereas for PBP2 A501R and PBP2 A501P variants, these values are lower. We performed molecular dynamic simulations with both classical and QM/MM potentials to model both acylation energy profiles and conformational dynamics of four PBP2 variants to explain the origin of k 2 /K S changes. The acylation reaction occurs in two elementary steps, specifically, a nucleophilic attack by the oxygen atom of the Ser310 residue and C-N bond cleavage in the β-lactam ring accompanied by the elimination of the leaving group of ceftriaxone. The energy barrier of the first step increases for PBP2 variants with a decrease in the observed k 2 /K S value. Submicrosecond classic molecular dynamic trajectories with subsequent cluster analysis reveal that the conformation of the β 3 -β 4 loop switches from open to closed and its flexibility decreases for PBP2 variants with a lower k 2 /K S value. Thus, the experimentally observed decrease in the k 2 /K S in A501X variants of PBP2 occurs due to both the decrease in the acylation rate constant, k 2 , and the increase in K S .