Subfamily-specific adaptations in the structures of two penicillin-binding proteins from Mycobacterium tuberculosis

Abstract

Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows that Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis.

Figure 1. Activity and phylogeny of Rv2911 and Rv3330.

A. The beta-lactam, meropenem, contains a D-Ala-like group (red) and irreversibly acylates penicillin-binding proteins, including Rv2911 and Rv3330. B. Enzyme acylation by peptidoglycan stem peptide, the first step in peptidoglycan carboxy- and trans-peptidation, highlighting the similarity of the terminal D-Ala leaving group (red) and the D-Ala-like acceptor moiety of DAP (purple). C. Neighbor-joining tree of Peptidase_S11 family proteins of Actinobacteria fall into several clades. Of the two mycobacterial clades, Clade 1 (orange) contains Rv2911, and Clade 2 (blue) contains Rv3330. E. coli and B. subtilis Peptidase_S11 enzymes (green) form an out-group with several actinobacterial sequences. D. Maximum-likelihood tree of Peptidase_S11 proteins from select mycobacterial species, Corynebacterium diphtheriae, E. coli, and B. subtilis. High bootstrapping values confirm the separation of mycobacterial sequences into two distinct clades.

Figure 2. Structures and domain diagrams of Rv2911opt and Rv3330 enzymes.

A. Rv2911_opt ribbon diagram showing the inhibitor, PMSF, bound to the catalytic Ser69 (sticks). Four mutations obtained during split-GFP optimization are highlighted along with two cysteines that participate in intermolecular disulfide bonds in the crystal. B. Ribbon diagram of Rv3330 structure showing meropenem bound to the catalytic Ser121 (sticks). Cys77, which forms an intermolecular disulfide bond in the crystals occurs in an N-terminal extension absent from Rv2911. In the domain diagrams, SP denotes the signal peptide, TM represents the trans-membrane helix, and numbers indicate sequence positions of domain borders. Sequences present in the crystallization constructs are underlined.

Figure 3. Conserved arrangement of catalytic-site residues of Rv2911 and Rv3330 peptidases.

A. Rv2911opt-PMSF. Electron-density (2Fo-Fc, 2.0-Å resolution, 1σ) shows the inhibitor adopts two major conformations (purple and magenta). B. Rv3330-meropenem. Electron-density (2Fo-Fc, 2.0-Å resolution, 1σ) supports a unique model with extended interactions between the inhibitor (purple) and the enzyme. Common catalytic residues are colored green and Clade 2 conserved residues in Rv3330 are colored blue.

Figure 4. Rv2911 and Rv3330 catalytic domains bind weakly to peptidoglycan in vitro.

SDS-PAGE of control (C), supernatant (S), and peptidoglycan fractions (P) following pull-down with B. subtilis peptidoglycan. Hen egg lysozyme (HEL) and bovine serum albumin (BSA) were used as positive and negative controls, respectively. The left lane contains molecular marker in kDa units.

Figure 5. Different conserved surfaces surround the catalytic site in Clade 1 and Clade 2 D,D-carboxypeptidases.

A. Surface representation of Rv2911_opt-PMSF showing regions conserved across the two clades (green, 100% identity) as well as clade-specific conserved surfaces (orange, at least 90% identity within Clade 1). Surfaces corresponding to residues in the three conserved active site motifs are labeled to establish orientation. B. Catalytic site of Rv2911 (colored as in A) showing the superposition of stem-peptide mimicking ligands from E. coli PBP6 (tan, PDB 3ITB) and B. subtilis PBP4a (light blue, PDB 2J9P). The site that distinguishes lysine (tan) from DAP (light blue) is conserved at the bottom of the figure. PMSF was omitted for clarity. C. Rv3330-meropenem surface representation showing surfaces conserved across the two clades (green) and across Clade 2 (blue, at least 90% identity within Clade 2). A large surface (300 Ų) on the lower right in the figure is conserved in Clade 2 enzymes. D. The catalytic site of Rv3330 bound to meropenem (purple) with stem-peptide mimics from the superimposed complexes of E. coli PBP6 (tan, PDB 3ITB) and B. subtilis PBP4a (light blue, PDB 2J9P). The donor-peptide mimic (tan) exits the active site to the upper left of the figure. In contrast, meropenem interacts with a surface conserved in Clade 2 (upper right).