Sandbox Reserved 1488

Penicillin-binding proteins (PBPs) have been scrutinized for over 40 years. Recent structural information on PBPs together with the ongoing long-term biochemical experimental investigations, and results from more recent techniques such as protein localization by green fluorescent protein-fusion immunofluorescence or double-hybrid assay, have brought our understanding of the last stages of the peptidoglycan biosynthesis to an outstanding level that allows a broad outlook on the properties of these enzymes. Details are emerging regarding the interaction between the peptidoglycan-synthesizing PBPs and the peptidoglycan, their mesh net-like product that surrounds and protects bacteria^[1].

Function

The bacterial cell wall is essential for cell survival. It is composed of layers of peptidoglycan modified with proteins and polymers. In bacteria, this peptidoglycan layer is formed by the coordinated action of multiple proteins, including penicillin-binding proteins (PBPs). PBPs are transpeptidases, carboxypeptidases and endopeptidases that synthesize new and remodel existing peptidoglycan^[2] .

PBPs are classified by their enzymatic activity:

(1) class A, bifunctional PBPs with both glycosyltransferase and transpeptidase activities;

(2) class B, transpeptidases; and

(3) class C, carboxy-peptidases and endopeptidases.

Disease

Enterococci exhibits tolerance to the bactericidal activity of β-lactams ^[3] , a phenomenon that compromises the use of β-lactam antibiotics as single agents in the treatment of enterococcal endocarditis ^[4]. As a consequence, multi-resistant E. faecium and E. faecalis represent one of the most dangerous threats in infectious diseases therapeutics.

Rare strains of E. faecalis and most nosocomial strains of E. faecium exhibit even higher levels of resistance to penicillins, effectively eliminating β-lactams as a treatment option ^[5][6]. Of greater concern is the observation that prolonged β-lactam therapy can lead to the emergence of highly resistant strains.

Structure

The structure of PBP4 was determined to 1.8 Å resolution. Strong electron density was observed for residues 172-680; interpretable electron density was not observed for the N1 domain. The structure show that both PBP4 is composed of three distinct structural domains: N-terminal domain (N2), a non-penicillin binding domain (nPB) and a C-terminal catalytic transpeptidase (TPase) domain, which contains the nucleophilic serine .

The PBP active site is located in the TPase domain and is defined by three conserved motifs: motif I, which includes the catalytic serine (SxxK: 424STFK427); motif II, which is involved in the protonation of the β-lactam leaving group (S/YxN; 482SDN484); and motif III which facilitates substrate binding and defines the oxyanion hole (K[T/S]GT; 619KTGT622)^[7]. The nucleophilic serine (Ser424) is located at the N-terminus of helix a2, while the oxyanion hole is defined by the backbone nitrogen atoms of the nucleophilic serine and the motif III threonine (Thr622). These motifs are bordered above by the ‘lid’ (aa 445-473) and below by the C-terminal helix (aa 657-680), which together enclose the active site in a deep cleft.

In class B PBP transpeptidases, the catalytic serine attacks the carbonyl of the penultimate D-Ala residue of a ‘donor’ stem peptide, releasing the C-terminal D-Ala and forming a covalent acyl-enzyme adduct with the donor peptide. In a second step, the carbonyl of D-Ala adduct undergoes nucleophilic attack from a primary amine located at the extremity of a side chain of an acceptor stem peptide. This creates a bridge between the peptides and, in turn, links the glycan strands to one another.

Structural insights into β-lactam resistance ^[2]

Penicillins, carbapenems and cephalosporins mimic the D-Ala-D-Ala sequence in the donor substrate and function as suicide inhibitors. Due to the fact that these PBPs have unusually low affinities for β-lactams, the β-lactam acylation rates are negligible compared with bacterial generation times, allowing the pathogens to survive antibiotic treatment. Of greater concern is the observation that prolonged β-lactam therapy can lead to the emergence of highly resistant strains. β-lactam resistance of PBP4 has also been shown to be due to the inefficient formation of the acyl-PBP intermediate. The molecular basis of PBP4 resistance to β-lactams are related to domain movements about the active site and changes in the structures of the active site motifs. However, they depend upon the particular acyl-adduct formed.

Penicillins: Benzylpenicillin acylation induces a rotation of the nucleophilic serine and a twist of strand β3


The benzylpenicillin forms a covalent adduct with PBP4 via its catalytic serine, Ser424. The electron density is well-defined for the entire molecule, with the benzylpenicillin carbonyl oxygen pointing towards the oxyanion hole defined by the backbone nitrogen atoms of Ser424 and Thr622. The benzylpenicillin is further stabilized by polar contacts with both PBP4 backbone atoms and the sidechains of Ser482, Asn484, Lys619, Thr620 and Thr622 and the formation of intraprotein contacts between Lys427 a Asn484 and Ser482 .

The PBP4 apo and benzylpenicillin-acyl-PBP complex structure reveals that has a distorted active site that undergoes local and distributed conformational changes upon β–lactam acylation. Upon acylation with benzylpenicillin, the nucleophilic Ser424 hydroxyl is oriented away from the oxyanion hole, instead of pointing down towards the oxyanion catalytic pocket.

The conformation of benzylpenicillin in the PBP4:benzylpenicillin complex is similar to other benzylpenicillin-bound PBP structures, with the exception that the phenyl-acetamidol group is observed in some structures to be rotated upwards away from motif III. Finally, because the catalytic site of PBP4 is located in a deep, narrow cleft, the structural elements that enclose the catalytic site open in order to accommodate benzylpenicillin acylation. This results in a displacement of the ‘lid’ moiety by 1.9 Å for benzylpenicillin-acyl-PBP4 compared to its apo conformation.

Carbapenems: imipenem acylation does not alter the twist of strand β3

Imipenem also forms a covalent adduct with Ser424 and its electron density is well ordered for the entire molecule with the exception of its iminomethyl-amino tail. In general, imipenem binds PBP4 similarly to the benzylpenicillin-acyl-PBP complex, however with some distinct differences. Unlike in benzylpenicillin where the β-lactam carbonyl points downward into the oxyanion hole, the imipenem carbonyl points upward, away from the oxyanion hole where it hydrogen bonds with Lys427 and Asn484 . As a result, the carbonyl of motif III Thr622 does not rotate out of the oxyanion hole, but instead retains the twisted conformation of β3 observed in the apostate.

Additional polar contacts are observed between imipenem and the backbone and/or sidechain atoms of Ser482, Thr620 and Thr622. The conformations of imipenem bound to PBP4 and PBP5 are identical to one another and other imipenem-transpeptidase complexes. The single exception is that the iminomethyl-amino tail of imipenem adopts a wide range of conformations, an observation consistent with a lack of strong electron density for this element. Finally, as observed for benzylpenicillin, both the lid and central β-sheet of PBP4 open to accommodate imipenem acylation. However, they do so to a far lesser extent than for benzylpenicillin, with the lid moving by only 0.7 Å.

Cephalosporins: Ceftaroline acylation results in the widest opening of the catalytic cleft


Density is observed for only a single ceftaroline molecule in PBP4. The ceftaroline carbonyl oxygen points towards the oxyanion hole defined by the backbone nitrogen atoms of Ser424 and Thr622. Ceftaroline is further stabilized by multiple polar interactions with the sidechains of Ser482, Asn484, Lys619 and Thr620 and the backbone atoms of Gly541 and Thr622 and intraprotein polar interactions between Lys427, Ser482, Asn484 and Lys619 . Acylation results in a rotation of the nucleophilic serine upwards away from the oxyanion hole. However, acylation of PBP4 by ceftaroline displaces the Thr622 carbonyl out of the oxyanion hole, causing strand β3 to twist outward. This new orientation of the Thr622 carbonyl is stabilized by a hydrogen bond with from ceftaroline. Ceftaroline binding results in the greatest opening of the catalytic cleft, with the lid and central β-sheet both moving by ~2.7 Å to accommodate ceftaroline binding.