Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic

• Animation
• Molecular Structure of the System
• Molecular Structure of 1 unit
• System description
• Assembly
• Tagged protein
• Mechanical description
• Workstation

Description

ClpXP is an AAA protease present in bacteria, consisting of two main components, ClpX and ClpP. The ClpX is a hexamer consisting of six identical subunits. It recognizes specific degradation tags of target substrate proteins, unfolds them in an ATP-consuming hydrolysis reaction, and uses additional cycles of ATP hydrolysis to translocate the unfolded polypeptide into an interior chamber of ClpP, where proteolysis takes place. ClpP is a multi-subunit serine peptidase, in which the proteolytic active sites reside within a barrel-shaped structure.

                      

In E. coli, the adaptor SspB tethers ssrA-tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation.

The recognition sequence

In the native organism, the SsrA tag is added to incomplete proteins whose translation has been aborted. Thus, misfunctionnal proteins do not accumulate inside the cell. The ssrA tag is a natural well-characterized recognition for ClpXP-degradation sequence in E. Coli. It is composed of the 11 amino acid sequence AANDENYALAA, localized at the C-terminal of the target protein. ClpX recognizes the last three residues LAA(Leu9, Ala10 and Ala11). Proteins with C-terminal LAA- tags are degraded rapidly in the cell, even without presence of SspB.
The publication of Tania Baker [7] based on the ClpXP protease of E. Coli which degrades substrates bearing the specific SsrA recognition sequence, has been the starting point of our reflection. In this work Baker and colleagues designed a series of modified ssrA tags which have weakened interactions with ClpXP to engineer controlled degradation. In E. coli, the adaptor SspB tethers ssrA-tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. In the absence of SspB , substrates bearing the artificially altered DAS-tag were stable, in contrast the degradation of substrates bearing these engineered peptide tags was 100-fold more efficiently when SspB was present.

To engineer controlled degradation, Baker and Sauer (2006) designed a series of modified SsrA tags that have weakened interactions with ClpXP. The DAS-tag presents one of these artificial sequence; its Kd value is significantly higher than the one of wild type SsrA, thus degradation of DAS-tagged proteins is not significant within the range of physiological concentrations. There is an N-terminal equivalent to the DAS-tag, the λO- tag. In the absence of SspB, substrates bearing the artificially altered tags are stable; however, through the action of the adaptor protein SspB, DAS- or λO –tagged proteins are significantly degraded.

Substitution of the adaptor

The role of the adaptor-protein SspB has been assumed by Pif6 in our system, only light-induced activation can lead to binding and efficient degradation of DAS bearing constructs.

 

The target protein will be fused to Pif6 and to the specific degradation tag.
To avoid problems with the accessibility or an increased activity of the target protein we provide two different variants: the C-terminal degradation tags, with the target protein construct [PIF6-linker-Protein-DAS] and the N-terminally fused λO-tag resulting in the construct [λO-Protein-linker-PIF6].

For the C-terminal degradation tags, the target protein construct will be PIF-linker-Protein-Tag. To avoid problems with the accessibility of the tag we decided to test a further contruction with a N-terminally fused degradation tag (Tag-Protein-linker-PIF).
We need then to choose an appropriate tag. It was also a critical step.

The LAA Tag

The ssrA tag is a natural well-characterized recognition for ClpXP-degradation sequence in E. Coli. It is composed of the 11 amino acid sequence AANDENYALAA, localized at the C-terminal of the target protein. At least five ClpX-recognizing motifs have been determined: three located at the N-terminus and two at the C-terminus [34]. Here we are concentrating on those located at the C-terminus where ClpX recognizes the last three residues (Leu9, Ala10 and Ala11).
ClpX alone is able to interact with the ssrA-tagged substrates and delivers them to ClpP protease. However, it has been shown that the adaptor protein, SspB, markedly enhances the recognition of the ssrA tag. So, in our case the PIF-fused target should also favor the recognition of the ssrA tag.

The left shows the molecular structure of the ClpX hexamere fused to the phytochrome B and the
schematic image in the animation

This shows the molecular structure of the trimetric unit of the ClpX fused to phytochrome B
and the schematic image in the animation.