Part:BBa_K1909002

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.

Fig. 1 **Comparison of the structures of *meso*-2,6-Diaminopimelic acid (mDAP) and aspartate. A:** mDAP, B: aspartate. In molecular count fingerprint analysis, aspartate and mDAP share a Tanimoto coefficient of T=0.8.

Design considerations

The Sequence

As 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 results

We 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
Receptor Model	mDAP	aspartate
4Z9H	-4.595	-7.413
mTaz	-6.576	-5.995

Fig. 2: *meso*-Diaminopimelic Acid (mDAP) and Aspartate docked to the structure of the Tar ligand binding domain (PDB: 4Z9H) and mTaz. A: Aspartate in its native conformation at the receptor site of Tar. B: mDAP docked to the receptor site of Tar. C: mDAP docked to mTaz. Y149 was changed to serine to make room for mDAP to enter the ligand binding site. R64 was changed to lysine to weaken the receptor's affinity to aspartate.

Usage and Biology

The pathway

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.

Characterization

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).

Fig. 3: **mTaz pathway in *E. coli*.** Upon mDAP binding mTaz is autophosphorylated, transphosphorylating OmpR, which acts as a transcription factor, activating GFP expression.

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).

Fig. 4: **Validation of mTaz using flow cytometry. A:** GFP fluorescence histogram. **Yellow:** *E. coli* incubated in LB medium with 0.1 mM mDAP for 30 min at 37°C. **Black:** Negative control, *E. coli* incubated in LB medium without mDAP for 30 min at 37°C. B: K-S-statistics indicating statistical significance of fluorescence difference between negative control and experiment, p<0.001.

References

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 111