Difference between revisions of "Part:BBa K2356001"

Revision as of 10:16, 27 October 2017

14-3-3 tetramer with GFP

About 14-3-3
Proteins belonging to the 14-3-3 family are dimers, where each monomer consists out of nine anti-parallel alpha-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]

The 14-3-3 protein in this part is the specific tobacco isoform 14-3-3c and it is stripped of its last 18 C-terminal amino acids, called T14-3c-delta-C. 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 14-3-3, this part also connects two 14-3-3 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 or other Protein-Protein Interactions (PPIs). In this part, the 4th monomer is mutated, preventing it from binding CT33, yielding a trimeric scaffold. These three binding pockets can then be blocked using covalently attached ExoS domains, which can be cleaved by beforementioned MMPs. This would mean that PPIs will be 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.

Connection to 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] The CT33 peptide comprises the final 33 amino acids of this C-terminus, which is referred. The binding of 14-3-3 with CT33 in the presence of fusicoccin 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). The His-tag allows protein purification with Nickel columns.

The protein mass is 146 kDa.

[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