Part:BBa_K2356001
14-3-3 tetramer with GFP
Proteins belonging to the 14-3-3 family are dimers, where each monomer consists out of nine anti-parallel α-helices. This causes the dimer to obtain a cup-like shape with two amphipathic binding grooves. The structure forms a rigid scaffold that is capable of anchoring proteins. 14-3-3 proteins are involved in multiple cellular processes and are mostly known to bind phosphorylated peptide motifs, especially those containing phosphoserine and phosphothreonine sequences. Most regions are conserved among different 14-3-3 isoforms, but the C-terminus appears to show more variability and is important in binding different target proteins.[1]
In this project, the specific tobacco isoform 14-3-3c is used and stripped of its last 18 C-terminal amino acids, called T14-3cΔC. This allows higher affinity towards the CT33 peptide, more specifically towards the YDI tail (see Figure 1), in the presence of small molecule fusicoccin.[2] Next to the shortening of 14-3-3Δc, this project also connects two T14-3cΔC dimers, forming a tetramer scaffold. Mutation of one or more monomers consecutively allows varying the amount of available binding pockets. Tunability of the number of binding pockets can be useful to create a valency that is ideal for phase separation. It is expected that a valency of 3 for 14-3-3 is optimal in combination with a valency of 4 for the CT33/52 construct with Strep-tactin. These three binding pockets can then be blocked using covalently attached ExoS domains, which can be cleaved by beforementioned MMPs. This would mean that gelation is induced only in the presence of these MMPs. ExoS is not yet included in this part. The 4th monomer has been mutated so that it will most likely not result in binding.
CT33 One motif that is known to bind to 14-3-3 is the phosphorylated C-terminus of H+-ATPase, an enzyme that catalyzes the hydrolysis of ATP to ADP.[3] In this project we use peptides compromising the final 33 amino acids of this C-terminus, which is referred to as CT33. The binding of 14-3-3 with CT33 has been optimized by previous research, yielding a Kd of 0.85 nM.[2] Due to this low value and tunability of fusicoccin this binding is interesting for contributing to a PPI network based on 14-3-3 scaffolds.
GFP is added to allow visualization for different purposes, e.g. Protein-Protein Interactions (PPIs).
[1] Obsilova V, Kopecka M, Kosek D, Kacirova M, Kylarova S. Mechanisms of the 14-3-3 Protein Function : Regulation of Protein Function Through Conformational Modulation. 2014;63. [2] Ottmann C, Marco S, Jaspert N, et al. Article Structure of a 14-3-3 Coordinated Hexamer of the Plant Plasma Membrane H + -ATPase by Combining X-Ray Crystallography and Electron Cryomicroscopy. 2007:427-440. doi:10.1016/j.molcel.2006.12.017. [3] Morsomme P, Boutry M. The plant plasma membrane H ‡ -ATPase : structure , function and regulation. 2000;1465.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1306
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Illegal PstI site found at 3967 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1306
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Illegal PstI site found at 3967 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1306
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Illegal BamHI site found at 3229 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1306
Illegal EcoRI site found at 1461
Illegal PstI site found at 521
Illegal PstI site found at 3967 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1306
Illegal EcoRI site found at 1461
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Illegal PstI site found at 3967
Illegal AgeI site found at 3958 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1009
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