Structural Insights into Substrate Binding and Antibiotic Inhibition of Enterobacterial Penicillin-Binding Protein 6

Abstract

Shigella sonnei remains the second most common cause of shigellosis in young children and is now increasingly dominant across developing countries. The global emergence of drug resistance has become a main burden in the treatment of S. sonnei infections and β-lactam antibiotics, such as pivmecillinam and ceftriaxone, are recommended to be used as second-line treatment. They work by inhibiting the biosynthesis of the peptidoglycan layer of bacterial cell walls, in which the final transpeptidation step is facilitated by penicillin-binding proteins (PBPs). In this study, using protein homology modelling, we modelled the structure of PBP6 from S. sonnei and comprehensively examined the molecular interactions between PBP6 and its pentapeptide substrate and two antibiotic inhibitors. The docked complex of S. sonnei PBP6 with pentapeptides showed that the substrate bound to the active site groove of the DD-carboxypeptidase domain, via hydrogen bonding interactions with the residues S79, V80, Q101, G144, D146 and R240, in close proximity to the catalytic nucleophile S36 for the nucleophilic attack. Two residues, R240 and T208, were found to be important in ligand recognition and binding, where they formed strong hydrogen bonds with the substrate and β-lactams, respectively. Our results provide valuable information on the molecular interactions essential for ligand recognition and catalysis by PBP6. Understanding these interactions will be helpful in the development of effective drugs to treat S. sonnei infections.

Keywords: Shigella sonnei; antibiotic inhibition; homology modelling; molecular docking; penicillin-binding protein 6; pentapeptide binding.

Figure 1

Mass spectrometry analysis and sequence alignment of D-Ala-D-Ala carboxypeptidase from Shigella sonnei and its orthologs. Mass spectrometry analysis was performed using MALDI-ToF technique, which generated three tryptic-digested peptides (891, 2096 and 2388 Da). Multiple sequence alignment was carried out using Clustal Omega on Jalview [22]. The protein sequence is highly conserved across the bacterial species. However, D-Ala-D-Ala carboxypeptidase from S. sonnei is 7 residues longer than D-Ala-D-Ala carboxypeptidases from Escherichia coli, Shigella boydii, Shigella dysenteriae and Shigella flexneri. The 7-residue sequence is probably a non-coding region. Variations across the bacterial species are highlighted as white on black. The three tryptic-digested peptides are underlined.

Figure 2

Overall 3D structure of PBP6 from S. sonnei. (a) The S. sonnei PBP6 adopts a usual PBP 5/6-like structure, which consists of two distinctive domains, a large N-terminal DD-carboxypeptidase domain and a smaller β-sheet rich C-terminal domain of unknown function (DUF). The arrow indicates the short linker that connects the two domains. (b) The topology diagram of the S. sonnei PBP6. The two domains are linked through a short linker between the last α-helix (α6) of the DD-carboxypeptidase domain and the first β-strand (β10) of the DUF. (c) Two orthogonal ribbon plot views of the S. sonnei PBP6, colored on a gradient from the N (blue) to the C (red) terminus. (d) An electrostatic surface plot of the S. sonnei PBP6. (e) The Ramachandran plot of the protein 3D model.

Figure 3

Sequence alignment and superimpositions of PBP6 from S. sonnei and the templates used in the protein modelling. Multiple sequence alignment using Clustal Omega on Jalview [22], which showed a high degree of sequence similarity among the templates. The three sequence motifs S-L-T-K, S-G-N and K-T-G (underlined) are conserved in all proteins and the catalytic active binding sites S36, K39, S102, N104, K205 and G207, located in a groove (bottom panel) within the DD-carboxypeptidase domain, are labelled with a star (top panel).

Figure 4

PBP6 complex structures with the pentapeptide substrate. (a) Interfacial contacts between the S. sonnei PBP6 (red) with the pentapeptide substrate (green). The substrate made hydrogen bonding interactions with residues S79, V80, Q101, G144, D146 and R240 of the S. sonnei PBP6. The electrostatic surface of the complex is in the right panel. (b) Interfacial contacts between the E. coli PBP6 (PDB: 3ITB) with the pentapeptide substrate [12]. Ribbon view of the substrate (green) in the active site groove of the E. coli PBP6 (gold), which made contacts with residues S40, G81, R194, T212 and M208. R194 establishes both a salt bridge and a water-mediated hydrogen bond with the pentapeptide. The electrostatic surface of the complex is in the right panel.

Figure 5

PBP6 complex structures with the β-lactam antibiotics. (a) Interfacial contacts between the S. sonnei PBP6 (red) with ceftriaxone (green). The antibiotic made five hydrogen bonding interactions with the residues N104, D146, A163, T206 and T208 of the S. sonnei PBP6. The electrostatic surface of the complex is in the right panel. (b) Interfacial contacts between the S. sonnei PBP6 (red) with pivmecillinam (green). There are four hydrogen bonds formed between pivmecillinam and the residues S36, T208 and R240 of the S. sonnei PBP6. (c) Interfacial contacts between the S. sonnei PBP6 (red) with ampicillin (green). There are two hydrogen bonding interactions formed between ampicillin and the residues S36 and T208. (d) Interfacial contacts between the E. coli PBP6 (gold) with ampicillin (green) in the acyl–enzyme complex (PDB: 3ITA) [12].