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content.text= "<b> Introduction: </b> </br>The ClpXP protein complex is an AAA+ protease, which means that it uses the energy of ATP hydrolysis to unfold and degenerate marked proteins. The genetic code of this complex is highly conserved and can be found in human cells as well as in the bacteria Escherichia coli. The degradation system was discovered in the early 1990's and is now well established ^{<a href='#13.1'>[13.1]</a>}.In our project, we used ClpXP to degrade specific proteins in order to control their amount and effect. Therefor, we utilized the common adaptor sspB.This protein recognizes substrates tagged with ssrA . In order to have a better control, we actually made use of a sspB split system. For more detailed information about the ClpXP degradation system in our project go to ClpXP general. </br> </br> <b> Structure:  </b> </br> The ClpXP complex consists of two functional and structural different parts. The ClpX protein, an ATPase, is a hexameric ring (Fig.1) with a pore in the center<div align='left'><img src='https://static.igem.org/mediawiki/2013/9/98/Bonn_Clp_Fig1.jpg' height='348' width='320'>Fig. 1: the hexameric ring of ClpX, each color represents a subunit, from  'ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophys Acta, 2012, PMCID: PMC3209554'</div> </br> Each subunit contains a N-terminal domain (Fig.2, B), which assumes the adaptor recognition and is stabilized by coordinated zinc atoms.However, the important part of a subunit is the AAA+ module (Fig.2, C), divided in a large and a small domain. <div align='left'><img src='https://static.igem.org/mediawiki/2013/d/d0/BonnClp_Fig2.jpg' height='262' width='499'>Fig. 2: structure of a ClpX subunit, B: the N-terminal domain with brown zinc atoms, C: the AAA+ module, from  'ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophys Acta, 2012, PMCID: PMC3209554'</div> </br> Between these domains the ATP binding site can be found, but not every subunit is able to bind the nucleotide. The arrangement of ATP binding and not-binding subunits in the hexameric ring is essential for the tertiary structure and the conformation changes after hydrolysis. The ClpP protein is a tetradecameric peptidase (Fig. 3, A and B).The subunits are arranged as two heptameric rings, one ring stacked on top of the other, with also a narrow pore in the center. This pore leads into the proteolytic chamber, which is barrel-shaped. Every subunit accommodates a classical Ser-His-Asp catalytic triad and oxyanion hole inside the chamber. Those proteolytic acitve  sites (Fig. 3, C) can form several hydrogen bonds to the substrate^{<a href='#13.2'>[13.2]</a>}. <div align='left'><img src='https://static.igem.org/mediawiki/2013/0/06/BonnClp_Fig3.jpg' height='401' width='382'>Fig. 3: structure of ClpP, A: side view with stabilizing residues (blue), B: top view with the pore (red), C: active site of a subunit with a bonded substrate, from 'ClpXP, an ATP-powered unfolding and protein-degradation machine, Bakeret al, Biochim Biophys Acta, 2012, PMCID: PMC3209554'</div> </br> </br> </br><b> Functions: </b> </br>The ClpXP complex has three tasks to fulfill: </br> </br>1. Binding: The substrate binding process at the ClpX unit is normally conducted with the aid of an adaptor protein.This protein identifies tagged substrates and delivers them to the complex (Fig. 4, left). In order to transfer the protein,the adaptor also binds to the ClpX unit (Fig. 4, right), so that parts of the tag get approximated to a special binding site on the complex. After the linking between the tag and the binding site has been performed, the unfolding starts.The binding process also works without an adaptor protein, but an adaptor enhances the degratation by improving enzyme-substrate affinity. </br> </br> 2. Unfolding and translocation: The translocation of polypeptids through the ClpX unit to the ClpP chamber is an active process using energy from ATP-binding and -hydrolysing cycles. Therefor are several ATP molecules linked to the ClpXprotein. The separation of one phosphate molecule results in conformation changes, which pulls the linked protein more inside the pore located in the center of ClpX. The remaining ADP has to be replaced by a new ATP molecule before the cycle can start again. Meanwhile the unfolding is driven automatically, because the large tagged protein has to fit into the narrow pore, which forces the three-dimensional structure to become linear. </br> </br> 3. Degradation: The axial pore of the ClpP unit is also very narrow, allowing the entry of only small unfolded peptides into the proteolytic chamber. Inside the chamber, the substrate binds to an active site over several hydrogen bonds. It also can be linked to multiple active sites. In this position, proteins are cleaved in a maximum speed of around 10,000 proteins per minute by ClpP alone. If the ClpX unit is added, the rate is with ~0.2 proteins per minute and 0.3 &my;M substrate much lower, because the unfolding process takes longer time^{<a href='#13.3'>[13.3]</a>}^{<a href='13.4'>[13.4]</a>}. </br> </br> <div align='left'><img src='https://static.igem.org/mediawiki/2013/b/b8/BonnClp_Fig4.jpg' height='311' width='628'>Fig. 4: Model of the degradation process with the sspB adaptor, from 'Engineering controllable protein degradation, McGinness et al, Molecular cell, 2006, PMID:16762842'</div> <h2><b> References </b> </h2></br> </br> <p><a id='13.1'>[13.1]</a> ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophys Acta, 2012, PMCID: PMC3209554</br> <a id='13.2'>[13.2]</a> See above </br> <a id='13.3'>[13.3]</a> See above </br> <a id='13.4'>[13.4]</a> Flexible Linkers Leash the Substrate Binding Domain of SspB to a Peptide Module that Stabilizes DeliveryComplexes with the AAA ClpXP Protease, Wah et al, 2003, Molecular cell, PMID: 14536075</p></br>';