Periplasmic peptide alignment
RpfC transmembrane protein is the first component of our two-component detection system. Xanthomononas campestris pv. campestris (Xcc) RpfC protein functions by DSF binding at the periplasmic sensor as shown in figure 1, where a 22 amino acid sensing sequence participates in DSF interaction. To investigate whether Xcc signaling cascade can be the basis of our design, we conducted protein alignment of various Xanthomonas species (figure 2), which shows sequence conservation at key DSF binding residues between Xcc and Xcm (Xanthomononas campestris pv. musacearum). Gene cassette organization of Rpf proteins in Xcm from genome sequences further supports their close relatedness, supporting our rationale of using well-studied Xcc DSF signaling pathway as our system design.
Kinase domain alignment
Further analysis of Xanthomononas campestris pv. musacearum RpfC sequence data from recently published genome sequencing data reveals the conserved HisKA and HPt domains and their histidine residues involved in phosphorelay (figure 3). RpfG and Clp proteins in the downstream two-component signaling pathway also reveals sequence homology and thus we conclude the compatibility of Xcc as the basis of our system’s design.
3D models of RpfC periplasmic sensor peptide from Xcc and Xcm
We conducted de novo peptide prediction of N-terminal receptor using PEP-FOLD 3.5 RPBS Mobyle platform. Figure 4 shows the prediction of RpfC periplasmic sensor peptide from Xcc and figure 5 shows that of Xcm.
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| Figure 4. De novo peptide prediction of Xcc RpfC (BBa_K3071000) N-terminal receptor (1-22) using PEP-FOLD 3.5. R15, D17, S18, E19 and Q22 residues are shown in licorice showing the DSF common backbone interactions consensus sequence. Model was displayed with Pymol. | Figure 5. De novo peptide prediction of Xcm RpfC (BBa_K3071001) N-terminal receptor (1-22) using PEP-FOLD 3.5. R15, D17, S18, E19 and Q22 residues are shown in licorice showing the DSF common backbone interactions consensus sequence. Model was displayed with Pymol. |