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=='''Physical Model of the Staphylococcus aureus Transpeptidase PBP2a in Complex with an Anti-Methicillin-Resistant Staphylococcus aureus Cephalosporin'''==
<StructureSection load= size=550 side='right' scene='37/372724/Tp_1_in_grey/1'>
Fatima Assad1, Kavita Bhikhi1, Annie Briglall1, Diana Eusebio1, Edwin Flores1, Andrew Ramirez1, Hillary Ramirez1, Tashina Valentin1, Mohammed Zaman1, Joel L. Sussman2, Andrew L. Lovering3, Lars F. Westblade4, and Allison Granberry1
1Hostos-Lincoln Academy, 600 St. Ann’s Avenue, Bronx, NY 10455, USA; 2Department of Structural Biology, The Weizmann Institute of Science, Rehovot 76100, Israel; 3School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK; 4Department of Pathology and Laboratory Medicine, Hofstra
North Shore-LIJ School of Medicine, Hempstead, NY 11549, USA


   
==='''Introduction'''===
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Peptidoglycan transpeptidases, also known as penicillin-binding proteins
(PBP), catalyze the cross-linking of peptidoglycan polymers during
bacterial cell wall synthesis. Beta-lactam (β-lactam) antibiotics,
which include penicillins, cephalosporins and carbapenems, bind and
irreversibly inhibit transpeptidases. The overuse and misuse of β-lactam
antibiotics has led to strains of Staphylococcus aureus that are resistant
to all β-lactams and are often only susceptible to “last resort antibiotics”,
such as vancomycin.
 
 
 
==='''Cell Wall Structure'''===
The cell wall, which is composed of peptidoglycans, is crucial for maintaining
the structural integrity of the bacterium. Peptidoglycans consists of
N-acetylmuramic Acid (NAM) and N-acetylglucosamine (NAG) polymers. Rows of
peptidoglycan are cross-linked together with pentaglycine chains. The NAM residues
have a five amino acid side chain that terminates with two D-Alanine (D-Ala)
residues. [[Image:Cell Wall 7 30 2013.jpg|thumb|alt= Alt text| Figure 1.(A)Rows of Peptidoglycans forming a Bacterial Cell Wall (B)Peptidoglycan with D-Ala-D-Ala substrate |550px]]
 
 
==='''Structure of a Resistant Transpeptidase'''===
Methicillin resistant Staphylococcus aureus (MRSA) is resistant to all β-lactams because it acquires an alternative PBP, PBP2a, that is not bound or inhibited by any β-lactams. PBP2a is composed of two domains:
<scene name='37/372724/Non_penicillin_binding/1'>non penicillin binding(NPB)</scene>  domain and a <scene name='37/372724/Transpeptidase_domain/1'>Transpeptidase(TP)</scene> domain. The NBP domain of PBP2a is anchored in the cell membrane, while the TP domain “sits” in the periplasm with its active site facing the inner surface of the cell wall. The active site contains a serine residue at position 403 (<scene name='37/372724/Serine403label/2'>Ser403</scene>)which catalyzes the cross-linking of the peptidoglycan rows with pentaglycine cross-links.


==='''Introduction'''===
 
<Structure load='4dki' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' />
==='''Catalytic Mechanism of PBP2a'''===
Transpeptidases ('''TP'''), also known as penicillin-binding proteins ('''PBP'''),
[[Image:Schematic TP 3steps.jpg|thumb|alt= Alt text|Figure 2. Schematic showing Catalytic Mechanism of PBP2a |550px]]
catalyze the cross-linking of peptidoglycan polymers during bacterial cell wall
(a) The D-Ala-D-Ala side-chain substrate of the peptidoglycan accesses
synthesis. The natural transpeptidase substrate is the D-Ala-D-Ala
the active site of the PBP2a.
peptidoglycan side chain terminus. Beta-lactam ('''β-lactam''') antibiotics, which
 
include penicillins, cephalosporins and carbapenems, bind and irreversibly
(b) Ser403 nucleophilically attacks the peptide bond of the terminal
inhibit transpeptidases by mimicking the D-Ala-D-Ala substrate, resulting in
D-Ala residues of the substrate. The terminal D-Ala residue then exits
the inhibition of cell wall synthesis and ultimately bacterial cell growth.
the active site. The now terminal D-Ala residue forms a covalent bond to Ser403,
Overuse and misuse of β-lactams has led to the generation of methicillin resistant
while a crosslinking pentaglycine chain enters the active site.
Staphylococcus aureus ('''MRSA''') isolates that have acquired an
 
alternative transpeptidase, PBP2a, which is neither bound nor inhibited by β-
(c) A covalent bond forms between the pentaglycine chain and the
lactams. MRSA isolates are resistant to all β-lactams, can be hospital- or
terminal D-Ala residue, regenerating the active site serine residue.
community-acquired, and are often the cause of significant morbidity and
 
mortality. Furthermore, they are often only susceptible to “last resort
The entire process takes 4 milliseconds.
antibiotics”, such as vancomycin. Recently, two cephalosporins - ceftobiprole
 
and ceftaroline - that bind and inhibit PBP2a have been developed. The
==='''How Do Antibiotics Work?'''===
Hostos-Lincoln Academy Students Modeling A Research Topic (SMART)
The β-lactam antibiotics inhibit bacterial growth by inhibiting PBPs and ultimately cell wall
Team generated a model of the PBP2a/ceftobiprole complex (PDB [[4dki]])
synthesis. Specifically, β-lactams are molecular mimics of D-Ala-D-Ala, which is the normal
using 3D printing technology to illustrate the mechanism of action of
substrate of PBPs. Nucleophillic attack of the β-lactam results in the PBP being irreversibly
ceftobiprole. Supported by a grant from the Camille and Henry Dreyfus Foundation.
inhibited by the β-lactam. As a result, the synthesis of the cell wall is inhibited which leads
to cell lysis.
[[Image:Structures on penicillin and b lactam.jpg|thumb|alt= Alt text|Figure 3. Mechanism of action of β-lactams. A. Structure of a β-lactam (penicillin) showing the amide, carboxyl, and β-lactam ring groups β-lactam ring groups. B. Structure of the D-Ala-D-Ala substrate. C. Overlay of the D-Ala-D-Ala substrate in red with penicillin demonstrating molecular mimicry.|550 px]]
 
 
==='''PBP2a and Ceftobiprole'''===
MRSA becomes resistant to β-lactams by acquiring an alternative PBP, PBP2a, that is
neither bound nor inhibited by β-lactams. Recently, two cephalosporins –
<scene name='37/372724/Ceftobiprole/1'>ceftobiprole</scene> and
ceftaroline – that have anti-MRSA activity have been developed. Ceftobiprole is able to
inhibit PBP2a because additional chemical groups at the
<scene name='37/372724/Ceftobiprole/7'>R2</scene>
position of the cephalosporin backbone are able to interact with additional amino acid
residues in PBP2a; specifically
<scene name='37/372724/Tyr446_and_met641_label/2'>Tyr446 and Met641</scene>.
As a result of ceftobiprole <scene name='37/372724/R2_interaction/4'>tighter binding</scene> to PBP2a as highlighted in green , <scene name='37/372724/Ceftobiprole_in_cpk/1'>the medicine</scene>, shown as colors of the atom types ([[CPK]]), is able to more efficiently react with the serine active site residue and therefore inhibit the activity of PBP2a.
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