aureus PBP4 in complex with
Joshua Bloom '13 and Kushal Rao '13
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.
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
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.
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.
PBP4 is composed of α-helices
domains, an N-terminal
N-terminal domain is
composed of a β-lactamase/transpeptidase fold, which comprises
of the helical
clusters contains seven helices and a single
helix-turn-helix motif. The
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.
and other similar penicillin-interacting enzymes are characterized
by a conserved set of motifs surrounding their respective active sites.
motifs include the Ser-X-X-Lys
(SXXK) triad containing the
nucleotide, the Ser-X-Asn
(SXN) triad, and the Lys-Thr(Ser)-Gly
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
Ampicillin (β-lactam ring
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.
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.
molecule is encapsulated by
of the SXXK motif, Ser139
of the SXN motif, Thr241
of the KTG motif, and Glu297
from the EXXXN motif.
Basis for Antibiotic Resistance
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.
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.
Navratna, Vikas, Savitha Nadig, Varun
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.
Vaney, Marie Christine, Gary
L. Gilliland, James G. Harman, Alan Peterkofsky, and Irene T. Weber.
Crystal Structure of a cAMP-Independent Form of Catabolite Gene
Protein with Adenosine Substituted in One of Two cAMP-Binding Sites. Biochemistry
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