mTaz (Taz-derived mDAP receptor)
mTaz – An EnvZ Fusion Protein designed to detect mDAP secreted by Chlamydia trachomatis.
To detect Chlamydia trachomatis we designed and assembled a receptor based on the Tar-EnvZ chimeric receptor capable of detecting aspartate in the medium as described by Utsumi et al. 19891. Tar, or Methyl-accepting chemotaxis protein II, is a signal transducer in E. coli chemotaxis, being activated upon the recognition of aspartate2. It has been shown that Tar is sensible to mutations altering its ligand specificity, sometimes resulting in strong activity upon recognition of other amino acids3. EnvZ is part of the well-studied and commonly used two-component system EnvZ/OmpR in E coli, offering a simple way of signal transduction4 5 6 7. Its periplasmic domain was shown to be exchangeable with different sensor domains as in the Tar/EnvZ chimeric receptor capable of detecting aspartate in the medium8.
Two fairly alike ligands
meso-2,6-Diaminopimelic acid (mDAP) is a non-proteinogenic amino acid that is synthesized and secreted by Chlamydia trachomatis9. mDAP has a relatively high chemical similarity to aspartate, sharing a Tanimoto coefficient of T=0.8 in a molecular fingerprint analysis [T <0: chemically very different structures; T=1: identity] (Fig. 1). As a proof of concept, a molecular docking model proposes few mutations which would change the Tar specificity towards mDAP. As input for molecular docking we used the crystal structure of the ligand binding domain of Tar bound to aspartate (PDB: 4Z9H)10.
The SequenceAs a base for the part sequence, the BioBrick representation of the original Taz, BBa_C0082, was used. The BBa_C0082 BioBrick features a small part of the vector (64 nucleotides) it was derived from on its 3’ end, which we have removed along with the stop codon inside the Part for further fusion protein design in RFC 12, 21, 23 and 25 assembly standards.
Mutagenesis based on docking resultsWe used the Glide11 algorithm, Maestro and the Schrodinger Suite for molecular docking of aspartate and mDAP to the Tar ligand binding domain. Because of its structural similarity to aspartate, mDAP showed some affinity to the native Tar aspartate binding site (Table 1). Since mDAP is larger than aspartate, Y149 led to structural interference, not allowing mDAP to fully enter the binding site (Fig. 2). To create the additional space needed for mDAP binding, we substituted Y149 with smaller amino acids. The best results were obtained with serine. Because of the additional size of mDAP, the bigger challenge was to reduce the affinity of aspartate rather than to raise the affinity of mDAP. We identified R64 as a key residue for aspartate binding which hovewer was not needed to bind mDAP. To minimally interfere with folding and function performed a constitutive substitution and substituted it with lysine, creating the R64K Y149S double mutant (Fig 2 C), called mTaz hereafter. Using mTaz, a higher docking score was achieved for mDAP than for aspartate (Table 1).
Table 1: Glide Docking Scores of mDAP and aspartate docked to Tar (4Z9H) and mTaz
|Receptor Model||Glide Docking Score|
Usage and Biology
mTaz uses the EnvZ/OmpR pathway in E. coli (Fig. 3). For optimal results, BBa_K1909002 is to be used in a ΔEnvZ E. coli cell line. Expression may be induced using one of the Anderson Promoter / RBS / mTaz constructs BBa_K1909007 or BBa_K1909009. mDAP concentration and receptor activation may be monitored using one of the Omp-Promoter / GFP or eYFP reporter systems BBa_K1909013 or BBa_K1909014.
mTaz was characterized by growing E. coli transformed with pSB1C3/BBa_K1909013 and pSB1A3/BBa_K1909002 in LB medium for an hour. Cells were inoculated with 0.1 mM mDAP as well as no mDAP as negative control and incubated for 30 min at 37°C. GFP expression induced by mTaz activation was measured by flow cytometry (Fig. 4A).
Statistical analysis using K-S-test was used to evaluate significance of the results. The shift in fluorescence induced by 0.1 mM mDAP was statistically very significant (p<0.001, Fig. 4B).
- ↑ Utsumi, R. et al. Activation of Bacterial Porin Gene Expression by a Chimeric Signal Transducer in Response to Aspartate. Science 245, 1246-1249 (1989).
- ↑ Reader, R. W., Tso, W. W., Springer, M. S., Goy, M. F., Adler, J. Pleiotropic aspartate taxis and serine taxis mu-tants of Escherichia coli. J. Gen. Microbiol 111, 363-374 (1979).
- ↑ Derr, P., Boder, E., Goulian, M. Changing the Specificity of a Bacterial Chemoreceptor. . Mol. Biol.J 355, 923-932 (2006).
- ↑ Lan, C. Y., Igo, M. M. Differential expression of the OmpF and OmpC porin proteins in Escherichia coli K-12 depends upon the level of active OmpR. J. Bacteriol. 180, 171.174 (1998).
- ↑ Mizuno, T., Mizushima, S. Signal transduction and gene regulation through the phosphorylation of two regu-latory components: The molecular basis for the osmotic regulation of the porin genes. Mol. Microbiol. 4, 1077-1082 (1990).
- ↑ Russo, F. D., Silhavy, T.J. EnvZ controls the concentration of phosphorylated OmpR to mediate osmoregula-tion of the porin genes. J. Mol. Biol. 222, 567-580 (1991).
- ↑ Slauch, J. M., et al. EnvZ functions through OmpR to control porin gene expression in Escherichia coli K-12. J. Bacteriol. 170, 439-441 (1988).
- ↑ Yoshida, T., Phadtare, S., Inouye, M. The design and development of Tar-EnvZ chimeric receptors. Methods Enzymol. 423, 166-183 (2007).
- ↑ Henrichfreise, B. et al. Functional conservation of the lipid II biosynthesis pathway in the cell wall-less bacte-ria Chlamydia and Wolbachia: why is lipid II needed? Mol. Microbiol. 73, 913-923 (2009).
- ↑ Mise, T. Structural Analysis of the Ligand-Binding Domain of the Aspartate Receptor Tar from Escherichia coli. Biochemistry 55, 3708-3713 (2016).
- ↑ Small-Molecule Drug Discovery Suite 2016-3: Glide, version 7.2, Schrödinger, LLC, New York, NY (2016).
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000INCOMPATIBLE WITH RFCIllegal SapI.rc site found at 111
|chassis||E. coli JW 3367-3|