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<StructureSection load= size=475 side='right' scene='37/372725/Kapb2/4'>
<StructureSection load= size=475 side='right' scene='36/365380/4dki_cartoon/19'>
==='''Introduction'''===
'''Karyopherin beta 2''' (Kapβ2) is an importin that transports various cargo proteins into the nucleus through interactions with nucleoporins, which are proteins of the nuclear pore complex (NPC). One might overlook the significance of this protein but it actually plays a crucial role in the human body by mediating transport of RNA-binding proteins involved in transcription, RNA processing, RNA transport and translation. The structure of Kapβ2 is composed of 20 antiparallel helices called HEAT repeats. These HEAT repeats contribute to Kapβ2’s large superhelical shape. The protein is shown to form two arches: one at the N-terminal and the other at the C-terminal. Through recognition of a nuclear localization signal (NLS) located on its cargo, Kapβ2 binds to its cargo via its C-terminal arch. Release of the cargo is mediated by RanGTP, which once bound, leads to a large movement of the 62-residue heat repeat 8 loop into the C-terminal arch. This conformational change results in the dissociation of the cargo as the b2 loop binds the NLS binding site.


Transpeptidase (TP), 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.


==='''Identification of the cargo and Transport through the NPC'''===
In eukaryotes, proteins must be transported in and out of the nucleus. This nucleocytoplasmic transport of proteins across the nuclear envelope must occur through the gateway of the NPC. The NPC is a large structure consisting of 456 constituent binding proteins called nucleoporins (Nups).1  Movement through the NPC is facilitated transport that relies on interaction with specific Nups.  Importins and exportins are proteins that aid this facilitated transport by both binding to a specific cargo to be transported and interacting with specific Nups located in the central channel of the NPC.2


<font color='dodgerblue'><b> Karyopherin Beta 2 </b></font><scene name='37/372725/Kapb2/7'>KapB2</scene> is a group of proteins that is composed of both importins and exportins. Importins are proteins that carry cargos into the nucleus while exportins serve the opposite function. As of today, twenty different Kapβs have been identified. Each of these Kapβs is capable of recognizing and transporting a specific group of cargos. In order to bind to its cargo a Kapβ has to recognize a <font color='magenta'><b> Nuclear Localizaton or Export Signal </b></font><scene name='37/372725/Kapb2_with_nls/3'>NLS or NES</scene>is located in the polypeptide chain of the cargo. These signals can vary from 7 amino acids to longer than 100 amino acids in length.
== Cell Wall Structure ==


An importin, such as Kapβ2, binds to a specific cargo by recognition of an NLS and carries the cargo through the NPC by interacting with intrinsically disordered Nups called FG-Nups. FG-Nups line the passageway of the NPC and contain repeats of phenylalanine and glycine. These unstructured FG-Nups form a low-density cloud within the central channel extending from the cytoplasm to the nucleoplasm. The cloud acts as an effective exclusion filter for those particles that do not contain FG repeat binding sites. This is referred to as the zone of selectivity.
The cell wall, which is composed of peptidoglycan, is crucial for maintaining the structural integrity of the bacterium. Peptidoglycan 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.Bacterial Cell Wall  B.Peptidoglycan with D-Ala-D-Ala substrate |550px]]


'''Structure of Kapβ2'''
== Structure of a Resistant Transpeptidase ==


<scene name='37/372725/Kapb2/3'>KapB2</scene>
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: a <font color='orange'><b>non-penicillin binding domain </b><scene name='36/365380/4dki_cartoon/20'>(NPB) </scene></font> and a <font color='dodgerblue'><b>transpeptidase <scene name='36/365380/4dki_cartoon/21'>(TP)</scene> binding domain </b></font>. 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 <scene name='36/365380/Ser403/19'>a serine residue at position 403 (ser403)</scene> which catalyzes the cross-linking of the peptidoglycan rows with pentaglycine cross-links.


<!--
-->


[[cpk]]
== Catalytic Mechanism of PBP2a ==
[[Image:Schematic TP 3steps.jpg|thumb|alt= Alt text|Figure 2. Schematic diagram illustrating the mechanism of action of PBP2a |550px]]
 
(a)The D-Ala-D-Ala side-chain substrate of the peptidoglycan accesses the active site of the PBP2a.
 
(b)Ser403 nucleophilically attacks the peptide bond of the terminal D-Ala residues of the substrate. The terminal D-Ala residue then exits the active site. The now terminal D-Ala residue forms a covalent bond to Ser403, while a crosslinking pentaglycine chain enters the active site.
 
(c)A covalent bond forms between the pentaglycine chain and the  terminal D-Ala residue, regenerating the active site serine residue.
 
The entire process takes 4 milliseconds.
 
== How Do Antibiotics Work? ==
 
The β-lactam antibiotics inhibit bacterial growth by inhibiting PBPs and ultimately cell wall
synthesis. Specifically, β-lactams are molecular mimics of D-Ala-D-Ala, which is the normal
substrate of PBPs. Nucleophillic attack of the β-lactam results in the PBP being irreversibly
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='36/365380/Ceftobiprole/23'>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='36/365380/Ceftobiprole/12'>R2</scene> position of the cephalosporin backbone are able to interact with additional amino acid residues in PBP2a; specifically
<scene name='36/365380/Ceftobiprole/22'>Tyr446 and Met641</scene>. As a result of its tighter binding to PBP2a, ceftobiprole is able to more
efficiently react with the serine active site residue and therefore inhibit the activity of
PBP2a.
 
 
 
 
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/4⅝'>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.
 
 
<scene name='37/372724/Medicine_interaction/3'>R2</scene>
<scene name='37/372724/Medicine_interaction/2'>Tyr446 and Met641</scene>
<scene name='37/372724/Medicine_interaction/1'>ceftobiprole</scene>
<scene name='37/372724/R2_interaction/6'>tighter binding</scene>

Revision as of 17:38, 15 August 2013

<StructureSection load= size=475 side='right' scene='36/365380/4dki_cartoon/19'>

Transpeptidase (TP), 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 peptidoglycan, is crucial for maintaining the structural integrity of the bacterium. Peptidoglycan 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.

Alt text
Figure 1. A.Bacterial Cell Wall B.Peptidoglycan with D-Ala-D-Ala substrate

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: a non-penicillin binding domain (NPB) and a transpeptidase (TP) binding 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 (ser403) which catalyzes the cross-linking of the peptidoglycan rows with pentaglycine cross-links.


Catalytic Mechanism of PBP2a

Alt text
Figure 2. Schematic diagram illustrating the mechanism of action of PBP2a

(a)The D-Ala-D-Ala side-chain substrate of the peptidoglycan accesses the active site of the PBP2a.

(b)Ser403 nucleophilically attacks the peptide bond of the terminal D-Ala residues of the substrate. The terminal D-Ala residue then exits the active site. The now terminal D-Ala residue forms a covalent bond to Ser403, while a crosslinking pentaglycine chain enters the active site.

(c)A covalent bond forms between the pentaglycine chain and the terminal D-Ala residue, regenerating the active site serine residue.

The entire process takes 4 milliseconds.

How Do Antibiotics Work?

The β-lactam antibiotics inhibit bacterial growth by inhibiting PBPs and ultimately cell wall synthesis. Specifically, β-lactams are molecular mimics of D-Ala-D-Ala, which is the normal substrate of PBPs. Nucleophillic attack of the β-lactam results in the PBP being irreversibly inhibited by the β-lactam. As a result, the synthesis of the cell wall is inhibited which leads to cell lysis.

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.


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 – ceftobiprole and ceftaroline – that have anti-MRSA activity have been developed. Ceftobiprole is able to inhibit PBP2a because additional chemical groups at the R2 position of the cephalosporin backbone are able to interact with additional amino acid residues in PBP2a; specifically Tyr446 and Met641. As a result of its tighter binding to PBP2a, ceftobiprole is able to more efficiently react with the serine active site residue and therefore inhibit the activity of PBP2a.



MRSA becomes resistant to β-lactams by acquiring an alternative PBP, PBP2a, that is neither bound nor inhibited by β-lactams. Recently, two cephalosporins – ceftobiprole and ceftaroline – that have anti-MRSA activity have been developed. Ceftobiprole is able to inhibit PBP2a because additional chemical groups at the R2 position of the cephalosporin backbone are able to interact with additional amino acid residues in PBP2a; specifically Tyr446 and Met641. As a result of ceftobiprole tighter binding to PBP2a as highlighted in green , the medicine, 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.


R2 Tyr446 and Met641 ceftobiprole tighter binding

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