The molecular recognition and interaction of beta-lactamase II from Bacillus cereus (Bc II) with penicillin V (PV) and sulbactam (Sul) especially conformational changes of Bc II in the binding process were studied through spectroscopy analysis in combination with molecular dynamics (MD) simulation. The results show that in the binding process, a new coordination bond is observed between the Zn2 of Bc II and the carboxyl-O of PV or Sul by replacing His204. Electrostatic interaction between Zn2 and the ligand provide main driving force for the binding affinity. Compared with apo Bc II, there are mainly four loops showing significant conformational changes in ligand-bound Bc II. A weak conformational transformation from β-sheets to random coils is observed in the loop2 of ligand-bound Bc II. The conformational transformation may depend on the functional group and binding pose of the ligand, giving the binding pocket greater flexibility and accordingly allowing for an induced fit of the enzyme-ligand binding site around the newly introduced ligand. The change in the loop2 of ligand-bound Bc II may lead to the opening of the binding pocket of Bc II. Therefore, loop2 can be considered a gate for control of ligand access in Bc II, hence its dynamic response should be considered in new drug design and development.

Mycobacterium tuberculosis is a human respiratory pathogen that causes the deadly disease tuberculosis. The rapid global spread of antibiotic-resistant M. tuberculosis makes tuberculosis infections difficult to treat. To overcome this problem new effective antimicrobial strategies are urgently needed. One promising target for new therapeutic approaches is PonA1, a class A penicillin-binding protein, which is required for maintaining physiological cell wall synthesis and cell shape during growth in mycobacteria. Here, crystal structures of the transpeptidase domain, the enzymatic domain responsible for penicillin binding, of PonA1 from M. tuberculosis in the inhibitor-free form and in complex with penicillin V are reported. We used site-directed mutagenesis, antibiotic profiling experiments, and fluorescence thermal shift assays to measure PonA1's sensitivity to different classes of β-lactams. Structural comparison of the PonA1 apo-form and the antibiotic-bound form shows that binding of penicillin V induces conformational changes in the position of the loop β4'-α3 surrounding the penicillin-binding site. We have also found that binding of different antibiotics including penicillin V positively impacts protein stability, while other tested β-lactams such as clavulanate or meropenem resulted in destabilization of PonA1. Our antibiotic profiling experiments indicate that the transpeptidase activity of PonA1 in both M. tuberculosis and M. smegmatis mediates tolerance to specific cell wall-targeting antibiotics, particularly to penicillin V and meropenem. Because M. tuberculosis is an important human pathogen, these structural data provide a template to design novel transpeptidase inhibitors to treat tuberculosis infections.