Crystal structures of the transpeptidase domain of the Mycobacterium tuberculosis penicillin‐binding protein PonA1 reveal potential mechanisms of antibiotic resistance

Abstract

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.DatabaseStructural data are available in the PDB database under the accession numbers 5CRF and 5CXW.

Chemistry of Peptidoglycan in Mycobacterium tuberculosis Life Cycle: An off‐the‐wall Balance of Synthesis and Degradation

“…The crystallized PonA1 molecule contains the transpeptidase domain and one small adjacent domain at the N‐terminus of the transpeptidase domain (Figure ). This structure evidenced similarities to other PBPs: PBP1 of class A from Staphylococcus pneumoniae , PBP2 from Neisseria gonorroeae , class A PBP1 from Pseudomonas aeruginosa , and PBP4 from Listeria monocytogenes . A cleft in the middle of the TPase domain contains the active site and divides the TPase domain of PonA1 into two subdomains, one with α/β fold and the other with an α‐helical structure (Figure ).…”

“…The crystal structure of a portion of PonA1 (PDB code 5crf) was determined in the inhibitor‐free form and in complex with penicillin V . The crystallized PonA1 molecule contains the transpeptidase domain and one small adjacent domain at the N‐terminus of the transpeptidase domain (Figure ).…”

“… Domain PFAM organization and 3D structure of the PBPs PonA1 (A) and PonA2 (B). The structure of PonA1 TP domain was obtained by X‐ray crystallography and that of PonA2 by homology modelling . The PASTA domain of PonA2 (orange) is strongly involved in interactions with the TP domain (magenta) …”

50 years of The FEBS Journal: looking back as well as ahead

“…This long‐standing interest is revealed by excellent structural papers on proteins involved in neurodegenerative diseases , as well as early structural insights into cell adhesion . More recently, the journal has published structural data that might help inform drug discovery, including structures of colchicine‐binding site inhibitors in complex with tubulin , SYK in complex with new inhibitors , and the Mycobacterium tuberculosis ketol‐acid reductoisomerase and penicillin‐binding protein PonA1 .…”

Investigating β-lactam drug targets inMycobacterium tuberculosisusing chemical probes

“…LDTs and PBPs have different β-lactam susceptibilities, as evidenced by enzymatic, biochemical, and structural studies (14)(15)(16)(17)(18)(19)(20) . For example, PBPs are inactivated by covalent modification at a catalytic serine by β-lactam antibiotics (e.g., penams, monobactams, cephems, and carbapenems).…”