Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Daniel Wedemeyer, Alp Mirdoğan
Date created: 2016-10-13 11:00:00
Date modified: 2016-10-14 10:47:39
mTaz (Taz-derived mDAP receptor)
| Types | DnaRegion |
| Roles | CDS Coding |
| Sequences | BBa_K1909002_sequence (Version 1) |
Description
Coming as soon as we validated mTaz.Notes
meso-2,6-Diaminopimelic acid (mDAP) is a non-proteinogenic amino acid that is synthesized and se-creted by Chlamydia trachomatis. 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 struc-tures; T=1: identity]. 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 crys-tal structure of the ligand binding domain of Tar bound to aspartate (PDB: 4Z9H).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 de-sign in RFC 12, 21, 23 and 25 assembly standards.
We used the Glide algorithm, Maestro and the Schrodinger Suite for molecular docking of aspartate and mDAP to the Tar ligand binding domain (PDB: 4Z9H). 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. 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 we performed a constitutive substitution and substituted it with lysine, creating the R64K Y149S double mutant called mTaz hereafter. Using mTaz, a higher docking score was achieved for mDAP than for aspartate (Table 1).