DexA belongs to glycoside hydrolase family 66, consisting of five regions from the N- to the C-termini: the N-terminal signal peptide sequence (N-terminal 24 amino acids), variable region (25–99), catalytic region (100–615), glucan-binding site (616–732), and C-terminal region (733–850)[1]. DexA70 (residues 100–732) plays very important role in catalytic activity and also is the glucan-binding site.The structure of DexA70 protein was resolved in previous research work[1].
Figure 1. Crystal structure of dextranase (DexA) from Streptococcus mutans
Figure 2. Sequence of DexA70 (residues 100–732)
We aim to find out whether there are more conservative and critical sequences among the residues 100-732 that plays a decisive role.We verify dextranase from different organism sources, including Streptococcus downei, Streptococcus macacae, Streptococcus ratti. By comparing the amino acid sequences of the above enzymes, the sequence similarity among them is analyzed.
1. Based on Sequence Alignment method, 59 proteins in the database were detected to have varying degrees of sequence similarity with and DexA70. During the first studies, we selected proteins with an identity value of 0.40 or higher for analysis as shown in Figure 3 and Table 1.
DEXT_STRDO: May play a role in sucrose-independent adherence to the pellicle-coated tooth surface.
G5JWW3: Catalysis of the endohydrolysis of 1,6-alpha-D-glucosidic linkages in dextran.
J3A4S2: May remedy S. sobrinus’ deficiency in citrate utilization pathway.
Figure 3. A :Table of aligned sequences with UniProt and PDB identifiers. B: Pairwise identity 2D map
| No. | Name | Organism | Identity |
|---|---|---|---|
| 1 | DEXT_STRDO | Dextranase-Streptococcus downei (Streptococcus sobrinus) | 0.57 |
| 2 | G5JWW3 | Dextranase-Streptococcus macacae NCTC 11558 | 0.74 |
| 3 | J3A4S2 | Dextranase-Streptococcus ratti FA-1 = DSM 20564 | 0.78 |
Figure 4. A: 3D Structure of DEXT_STRDO, B:3D Structure of G5JWW3, C:3D Structure of J3A4S2
⚫ Model Confidence:
Blue: Very high (pLDDT > 90)
Aqua: Confident (90 > pLDDT > 70)
Yellow: Low (70 > pLDDT > 50)
Orange:Very low (pLDDT < 50)
AlphaFold produces a per-residue confidence score (pLDDT) between 0 and 100. Some regions with low pLDDT may
be unstructured in isolation.
2. As well-known, conserved domains of protein play vital role in protein-protein interactions. By analyzing the respective domains of DexA70 and the three proteins (DEXT_STRDO/G5JWW3/J3A4S2), we tried to find more conserved and critical sequences that had a greater inhibitory effect against the Streptococcus mutan. These dextranases belong to glycoside hydrolase family 66 protein (GH66), which hydrolyzes dextran alpha-1,6 linkages.
| Protein Name | Domain Sequence |
|---|---|
| DEXT_STRDO | LPKTG |
| G5JWW3 | LPQTGDKKRSSLLTAVGAGVILIGLAGFLSLRHKRK |
| J3A4S2 | LPETGEGKVSKIAAIVGAGILAAGAIGLLALKRRKN |
Most proteins in cells are composed of multiple folding units (or domains) to perform complex functions in a cooperative manner. 4 domains of DexA70 were predicted through the I-TASSER-MTD model (the prediction structure is shown in the figure below)[2,3,4], so what is the specificity of these 4 domains? Which domain plays a more important role for the activity of DexA70.
Figure 5. Final Full-length Models of DexA70 Predicted by I-TASSER-MTD. Colored by domain: domain 1 in red;
domain 2 in blue; domain 3 in green; domain 4 in cyan.
Figure 6．Predicted Individual Domain（1/2/3/4） Structures
Figure 7. a: Predicted domain boundary on contact map; b: FU-score curve of continuous domain; c：FU-score
heatmap of discontinuous domain;
3. Antimicrobial Peptide (AMPs) have been discovered in most life forms, including bacteriocins, fungal peptide antibiotics, plant thionins and defensins, insect defensins and cecropins, amphibian magainins and temporis, as well as defensins and cathelicidins from higher vertebrates[5,6,7]. We should compare the core domain sequences(Table 2) of DEXT_STRDO, G5JWW3, J3A4S2 and four domains of DexA70 with the reported peptides(C16G2, Sequence: TFFRLFNRSFTQALGKGGGKNLRIIRKGIHIIKKY)，which enhanced antimicrobial activity and specificity against planktonic S. mutans cells[8].
3.1 Methods as follows:
1) Training set;
2) Test set;
3) Cutoff threshold for sequence identity;
4) Supposing a query peptide P and the training set｛P1,P2,...,Pn｝,then the high-scoring segment pairs (HSPs)
score between the query peptide and each peptide in the training set are calculated by BLASTP with default
parameters. The peptide is predicted to share the same category as the peptide Pk if the HSP score
between P
and Pk is higher than other scores.
Expressed in a formula, Pk subjects to:
HSPs Score (P, Pk)=max｛HSPs Score(P, P f i)|i~1, 2, ...., n｝
If more than one Pk fulfils the Eq. (1), one of them is chosen at random and its category was assigned to the query peptide P.
The comparative genome analyses revealed diversities in the Streptococci mutans group. The results are helpful for better understanding the evolution and adaptive mechanisms of these oral pathogen microorganisms and for combating them. In order to verify the biological inhibition effect against Streptococcus mutans of the three dextran (DEXT_STRDO, G5JWW3, J3A4S2), we will conduct experiments in the laboratory in the future. To further validate the active core region of DexA70, we can construct 4 domains individually into the vector and transform them into E. coli to induce the expression of proteins for in vitro bacteriostatic experimental testing. If positive results, antimicrobial peptides may be designed, so as to provide new research insight for subsequent research or other iGEM teams.
