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Figure 1: Frequency of Amino Acid residues around the 17 TACE cleavage sites of 14 TACE substrates (Source: created by Kevin Collins and Sydney George).
Figure 1: Frequency of Amino Acid residues around the 17 TACE cleavage sites of 14 TACE substrates (Source: created by Kevin Collins and Sydney George).
We analyzed 17 TACE cleavage sites from 14 of TACE's known substrates to identify the amino acid residues found around each cleavage site, from the P5 to the P4' positions (with the inclusion of a known P5' site on one of TACE's substrates called PRNP). The highlighted numbers in each column represent the residue most commonly found at each position.
Using this table, Kevin and I identified a possible cleavage site sequence of PLAEQVVSTA. This pattern could then be aligned with the ErbB4 amino acid sequence to find any similar series of residues, which may represent a possible TACE cleavage site.
Figure 2: Most common amino acid residues located around cleavage sites of substrates cleaved by TACE/ADAM17.
Figure 2: Most common amino acid residues located around cleavage sites of substrates cleaved by TACE/ADAM17.
This graph was retrieved from a paper on the protein Klotho, one of TACE's many substrates. In the graph, TACE/ADAM17's substrates were analyzed in a manner similar to that of Figure 1. The most common residues at each cleavage site position are represented in the graph toward the top of each column. We used the findings from this graph to ascertain another possible cleavage site sequence, PEAEAVVST. This pattern was also used in sequence alignment procedures to determine possible matches in the ErbB4 sequence.
Source: “Identification of the Cleavage Sites Leading to the Shed Forms of Human and Mouse Anti-Aging and Cognition-Enhancing Protein Klotho.”
Figure 3: Frequency of predicted secondary structures at 17 known TACE/ADAM17 cleavage sites. Secondary structures are reported whether or not they covered the entire cleavage site (Source: created by Kevin Collins and Sydney George on Microsoft Excel).
Figure 3: Frequency of predicted secondary structures at 17 known TACE/ADAM17 cleavage sites. Secondary structures are reported whether or not they covered the entire cleavage site (Source: created by Kevin Collins and Sydney George on Microsoft Excel).
This graph represents the secondary structure found in the known cleavage sites of each of the 14 TACE substrates. "None" refers to the cleavage sites which contain no secondary structure at all, whereas a cleavage site containing any length of beta sheet or alpha helix was recorded to include one. This graph shows that although presence of beta sheet of lack of secondary structure are most common at the known TACE cleavage sites, the presence of alpha helices can also occur. So we looked for cleavage sites that contained the more common options of the three, but did not rule out the possible cleavage sites which contained alpha helices.
Figure 4: Location of possible ErbB4 cleavage site (amino acids 651-659) at edge of Transmembrane Domain (Source: modeled by Kevin Collins and Sydney George in PyMOL Software).
Figure 4: Location of possible ErbB4 cleavage site (amino acids 651-659) at edge of Transmembrane Domain (Source: modeled by Kevin Collins and Sydney George in PyMOL Software).
This visualization from PyMOL represents the middle section, or transmembrane domain, of the ErbB4 protein in surface-view. One of the two possible TACE cleavage sites we identified, PLIAAGVIG (amino acid positions 651-659), is colored in orange. This possible cleavage site aligns with our previous research, as it is located just barely into the transmembrane domain, similar to the known TACE cleavage sites which often occurred just before or slightly inside the TM domain.
Figure 5: Location of ALS mutation 927 (R→Q) in relation to catalytic domain ligand binding site within active and inactive structure of ErbB4. Mutation may restrict access to binding site. (Source: modeled by Kevin Collins and Sydney George in PyMOL Software)
Figure 5: Location of ALS mutation 927 (R→Q) in relation to catalytic domain ligand binding site within active and inactive structure of ErbB4. Mutation may restrict access to binding site. (Source: modeled by Kevin Collins and Sydney George in PyMOL Software)
In this image from PyMOL, we visualized the active and inactive surface-view structure of ErbB4 containing the 1st ALS-associated point mutation at codon 927, which is colored in red. This visualization showed us that the 1st mutation is significantly far away from our two possible TACE cleavage sites, and therefore likely has no impact on TACE's cleavage of ErbB4. For the mutation to have an impact on proteolytic cleavage, it would need to be in much closer proximity to either one of the possible cleavage sites.
However, in comparing the inactive and active structures, we noticed that the mutation may block or inhibit access to the unrelated ligand docking site shown by the pink arrows. This ligand is associated with the kinase domain, which the 1st mutation is also found within. Therefore, these results have led us to predict that the mutation, although likely not impacting the proteolytic activity of ErbB4, may have an effect on the protein's catalytic activity.
Figure 6: Secondary structure of ALS mutation 927 (R→Q) within active and inactive structure of ErbB4. Indicates an area of crucial conformational change in cytoplasmic catalytic domain structure. (Source: modeled by Kevin Collins and Sydney George in PyMOL Software)
Figure 6: Secondary structure of ALS mutation 927 (R→Q) within active and inactive structure of ErbB4. Indicates an area of crucial conformational change in cytoplasmic catalytic domain structure. (Source: modeled by Kevin Collins and Sydney George in PyMOL Software)
This comparison of the active and inactive ErbB4 structure provides some interesting results which could be a place for further research regarding the 1st mutation's impact on ErbB4. The structures shown here are the same as that of Figure 5, except they are shown in secondary structure view rather than surface view (i.e., it illustrates the presence of alpha helices and beta sheets within the protein's secondary structure). Once again, the 1st mutation at codon 927 is colored in red. We discovered that the mutation lacks secondary structure when ErbB4 is in inactive conformation, but contains an alpha helix once the protein is activated. Such a difference in secondary structure could be an interesting subject of future research.
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