Interaction between a novel antimicrobial compound HL-J6 and Staphylococcus aureus PBP1

To investigate the interaction between a novel antimicrobial compound, HL-J6, and penicillin-binding protein 1 (PBP1) of Staphylococcus aureus.

With MRSA252 genomic DNA as the template and PBP1F and PBP1R as primers, the expression plasmid pET30a-pbp1-39-608 was constructed by amplifying the target gene fragment followed by cloning into the Nde Ⅰ/Xho Ⅰ restriction sites of the pET30a vector. Then the obtained plasmids were transformed into Escherichia coli for the expression of PBP1-39-608 protein, and the product was purified by affinity chromatography. The inhibitory effect of HL-J6 on the transpeptidase activity of PBP1-39-608 was measured using peptidoglycan side chain backbone peptide, with thiol ester analog S2d as the substrate. The affinity between HL-J6 and PBP1-39-608 was detected using microscale thermophoresis (MST), and the binding interaction was confirmed by cellular thermal shift assay (CETSA). Molecular docking and dynamics simulation were performed using AutoDock Vina and Desmond software, respectively, to elucidate the binding mode of HL-J6 with the PBP1-39-608 protein and the key amino acid residues involved.

The recombinant plasmid pET30a-pbp1-39-608 was successfully constructed, and PBP1-39-608 protein was produced after induction and purified, yielding a protein with an approximate molecular mass of 65×10³. HL-J6 inhibited the transpeptidase activity of PBP1-39-608 in a time-dependent manner (P<0.001). The dissociation constant Kd of the binding between HL-J6 and PBP1-39-608 was 64.92 μ mol/L. Molecular docking results showed that HL-J6 bound to the active pocket of PBP1-39-608 by interacting with key residues such as ILE-348, ASN-370, THR-516 and PHE-423, with a binding score of-8.38 kcal/mol (<-5.00 kcal/mol). Dynamics simulation results indicated that the complex became stable after 50 ns.

HL-J6 effectively inhibits the transpeptidase activity of Staphylococcus aureus PBP1, and shows stable interaction with the protein.