Penicillin-binding proteins 2 (PBP2) are critically important enzymes in the formation of the bacterial cell wall. Inhibition of PBP2 is utilized in the treatment of various diseases, including gonorrhea. Ceftriaxone is the only drug used to treat gonorrhea currently, and recent growth in PBP2 resistance to this antibiotic is a serious threat to human health. Our study reveals mechanistic aspects of the inhibition reaction of PBP2 from the wild-type FA19 strain and mutant 35/02 and H041 strains of Neisseria Gonorrhoeae by ceftriaxone. QM(PBE0-D3/6-31G**)/MM MD simulations show that the reaction mechanism for the wild-type PBP2 consists of three elementary steps including nucleophilic attack, C-N bond cleavage in the β-lactam ring and elimination of the leaving group in ceftriaxone. In PBP2 from the mutant strains, the second and third steps occur simultaneously. For all considered systems, the acylation rate is determined by the energy barrier of the first step that increases in the order of PBP2 from FA19, 35/02 and H041 strains. Dynamic behavior of ES complexes is analyzed using geometry and electron density features including Fukui electrophilicity index and Laplacian of electron density maps. It reveals that more efficient activation of the carbonyl group of the antibiotic leads to the lower energy barrier of nucleophilic attack and larger stabilization of the first reaction intermediate. Dynamical network analysis of MD trajectories explains the differences in ceftriaxone binding affinity: in PBP2 from the wild-type strain, the β3-β4 loop conformation facilitates substrate binding, whereas in PBP2 from the mutant strains, it exists in the conformation that is unfavorable for complex formation. Thus, we clarify that the experimentally observed decrease in the second-order rate constant of acylation (k2/KS) in PBP2 from the mutant strains is due to both a decrease in the acylation rate constant k2 and an increase in the dissociation constant KS.
Keywords: QM/MM; bacterial antibiotic resistance; ceftriaxone; molecular dynamics; reaction mechanism.