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Staphylococcus aureus PBP4 in complex with ampicillin

Joshua Bloom '13 and Kushal Rao '13


I. Introduction

Penicillin Binding Proteins (PBPs) are a class of membrane-bound proteins which catalyze the final step of peptidoglycan synthesis in bacteria. Peptidoglycan, an extensive polymer consisting of sugars and amino acids, forms the cell wall outside the plasma membrane in bacteria. The sugar component of peptidoglycan consists of alternating residues of N-acteylglucosamine and N-acetyl-muramic acid. Ampicillin acts as a competitive inhibitor with respect to the binding of these substrates to the PBPs active sites necessary for peptidoglycan synthesis.

Staphylococcus aureus is a gram-positive bacteria that is responsible for many hospital-associated infections as well as skin infections in humans and is characterized by its thick peptidoglycan cell wall layer common to gram-positive bacteria. Penicillin binding protein 4 (PBP4), isolated from Staphylococcus aureus, is a carboxypeptidase necessary for the secondary cross-linking of peptidoglycan. Despite its crucial role in cross-linking of peptidoglycan, PBP4 is not necessary for cell growth and proliferation in laboratory conditions. Furthermore, based on previous in vitro and genetic studies, S. aureus PBP4 functions mainly as a transpeptidase with little carboxypeptidase activity. It is similar in structure and function to other low molecular mass PBPs and is included in the family of penicillin-susceptible and penicillin-interacting enzymes.

PBP4 possesses β-lactamase activity that can hydrolyze β-lactam antibiotics such as ampicillin and render them inactive. In fact, PBP4 expression in S. aureus is associated with low-level resistance to penicillin antibiotics. Increased expression of PBP4 has been shown to lend increased β-lactam resistance to S. aureus.

II. General Structure

PBP4 was crystallized in unit cells containing two copies of the protein. However, the active form of PBP4 that is present in S. aureus is a monomer.

S. aureus PBP4 is composed of α-helices and β-sheets separated into two domains, an N-terminal β-lactamase domain and a C-terminal all-β domain The N-terminal domain is composed of a β-lactamase/transpeptidase fold, which comprises a five-stranded antiparallel β-sheet between two helical clusters. One of the helical clusters contains seven helices and a single helix-turn-helix motif. The second helical cluster contains two helices. Two antiparallel β-sheets make up the C terminus of PBP4. The functional role of this all β-sheet domain has not yet been determined.

PBPs and other similar penicillin-interacting enzymes are characterized by a conserved set of motifs surrounding their respective active sites. These motifs include the Ser-X-X-Lys (SXXK) triad containing the serine nucleotide, the Ser-X-Asn (SXN) triad, and the Lys-Thr(Ser)-Gly (KTG) triad. An additional motif, present only in Class A β-lactamases, is the Glu-X-X-X-Asn (EXXXN). The active site of S. aureus’s PBP4 is formed by the arrangement of these SXXK, SXN, and KTG motifs at the junction of an antiparallel β-sheet and a larger α-helical cluster within the N-terminal domain; catalytic residue include Ser75, Ser139 and Lys259.  

III. Ampicillin (β-lactam ring backbone)

Ampicillin is a beta-lactam antibiotic belonging to the aminopenicillin class of antibiotics. It functions as a competitive inhibitor of the enzyme transpeptidase, used by bacteria to assemble their cell walls. Ampicillin inhibits the third and final step of bacterial cell wall synthesis in binary synthesis, which leads to cell lysis and thus death.

In the case of PBP4 in complex with ampicillin, no bound or unbound forms were found by mass spectroscopy despite prolonged incubation. Therefore, in this model, ampicillin is modeled in its hydrolyzed form. The hydrolyzed ampicillin molecule is encapsulated by residues Ser75 of the SXXK motif, Ser139 of the SXN motif, Thr241 and Gly261 of the KTG motif, and Glu297 from the EXXXN motif.

IV. Basis for Antibiotic Resistance

Increased levels of PBP4 have been demonstrated to correspond with β-lactam resistance, while decreased levels of PBP4 corresponds to vancomycin resistance. As PBP4 functions as a β-lactamase, it is readily able to cleave the β-lactam ring of penicillin antibiotics, rendering them ineffective. Furthermore, as it is now known through previous studies that inhibition of PBP4 leads to vancomycin resistance, researchers can strive to promote activation of this enzyme through rational drug-design to increase the efficiency of vancomycin and other gylcopeptide antimicrobials. Through this method, it is researchers hope that they may be able to combat the growing challenge of treating MRSA and other antibiotic resistant strains of pathogenic bacteria.

V. References

Fani, Fereshteh, Philippe Leprohon, Danielle Legare, and Marc Ouellette. "Whole Genome Sequencing of Penicillin Resistant Streptococcus Pneumoniae Reveals Mutations in Penicillin-binding Proteins and in a Putative Iron Permease." Genome Biology 12.11 (2011): R115. Print.

Navratna, Vikas, Savitha Nadig, Varun Sood, K. Prasad, Gayathri Arakere, and B. Gopal. "Molecular Basis for the Role of Staphylococcus Aureus Penicillin Binding Protein 4 in Antimicrobial Resistance." Journal of Bacteriology (2010): 134-44. Print.

Vaney, Marie Christine, Gary L. Gilliland, James G. Harman, Alan Peterkofsky, and Irene T. Weber. 1989. Crystal Structure of a cAMP-Independent Form of Catabolite Gene Activator Protein with Adenosine Substituted in One of Two cAMP-Binding Sites. Biochemistry 28:4568-4574.

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