Crystal structure of cefditoren complexed with Streptococcus pneumoniae penicillin-binding protein 2X: structural basis for its high antimicrobial activity

Abstract

Cefditoren is the active form of cefditoren pivoxil, an oral cephalosporin antibiotic used for the treatment of respiratory tract infections and otitis media caused by bacteria such as Streptococcus pneumoniae, Haemophilus influenzae, Streptococcus pyogenes, Klebsiella pneumoniae, and methicillin-susceptible strains of Staphylococcus aureus. Beta-lactam antibiotics, including cefditoren, target penicillin-binding proteins (PBPs), which are membrane-associated enzymes that play essential roles in the peptidoglycan biosynthetic process. To envision the binding of cefditoren to PBPs, we determined the crystal structure of a trypsin-digested form of PBP 2X from S. pneumoniae strain R6 complexed with cefditoren. There are two PBP 2X molecules (designated molecules 1 and 2) per asymmetric unit. The structure reveals that the orientation of Trp374 in each molecule changes in a different way upon the formation of the complex, but each forms a hydrophobic pocket. The methylthiazole group of the C-3 side chain of cefditoren fits into this binding pocket, which consists of residues His394, Trp374, and Thr526 in molecule 1 and residues His394, Asp375, and Thr526 in molecule 2. The formation of the complex is also accompanied by an induced-fit conformational change of the enzyme in the pocket to which the C-7 side chain of cefditoren binds. These features likely play a role in the high level of activity of cefditoren against S. pneumoniae.

FIG. 1.

Chemical structures of cefditoren and cefditoren pivoxil with the atomic numbering scheme for the cephem skeleton.

FIG. 2.

Structural comparison between the trypsin-digested PBP 2X and the soluble PBP 2X from S. pneumoniae. The Cα models of molecule 1 (residues 74 to 232, 254 to 620, and 626 to 750) and molecule 2 (residues 73 to 101, 113 to 120, 133 to 169, 173 to 232, 254 to 620, and 626 to 750) of the trypsin-digested PBP 2X are colored red and green, respectively. The Cα model of the soluble PBP 2X (residues 71 to 92, 183 to 232, 254 to 620, and 632 to 750) is colored blue, with spheres (in magenta) representing side chains in the conserved motifs (337STMK, 395SSN, and 547KSG). The residue numbers of amino acids near the trypsin digestion sites are also shown. The loop region (residues 376 to 386), which assumes a different conformation in molecule 2 of the trypsin-digested PBP 2X, is indicated by an ellipse (yellow).

FIG. 3.

Cefditoren-PBP 2X complex structure and conformational changes. (A and B) Omit Fo-Fc difference density maps (contoured at 3 σ) covering the cefditoren moiety covalently attached to Ser337 of the trypsin-digested PBP 2X in molecules 1 and 2, respectively. Cefditoren-acylated Ser residues are shown as stick models with carbon atoms colored green, oxygens red, nitrogens blue, and sulfurs yellow. (C and D) Superposition of the active-site region from the uncomplexed (light blue) and cefditoren complex (orange) structures in molecules 1 and 2, respectively. Cefditoren and side chains of selected residues are shown as thick stick renderings. Atoms are colored as described for panel A, except that the carbon atoms of selected residues in the uncomplexed and cefditoren complex structures are colored light blue and orange, respectively. Covalent binding of cefditoren to the trypsin-digested PBP 2X requires conformational changes in the active site.

FIG. 4.

Interaction of the trypsin-digested PBP 2X with cefditoren. (A and B) Stereo view of the active sites of the cefditoren-PBP 2X complex structures in molecules 1 and 2, respectively. Atoms are colored as described in the legend to Fig. 3. Hydrogen bonds are represented by thin lines (magenta). (C and D) Schematic diagrams of cefditoren binding mode in molecules 1 and 2, respectively. Hydrogen bonds are represented by broken lines (magenta), and hydrophobic contacts are shown as arcs (green). The atomic numbering scheme of the cephem skeleton is also shown.